Effect of surgical approach on bone vascularisation, fracture ......Effect of surgical approach on...

$: Effect of surgical approach on bone vascularisation, fracture ......Effect of surgical approach on bone vascularisation, fracture and soft tissue healing: comparison of less invasive$
EEffffeecctt ooff ssuurrggiiccaall aapppprrooaacchh oonn bboonnee vvaassccuullaarriissaattiioonn,,

ffrraaccttuurree aanndd ssoofftt ttiissssuuee hheeaalliinngg::

ccoommppaarriissoonn ooff lleessss iinnvvaassiivvee ttoo ooppeenn aapppprrooaacchh

Dr Martin Eduard Wullschleger

MBBS University of Zurich MD University of Zurich

FMH Surgery EBSQ Trauma

Submitted for the award of degree of Doctor of Philosophy in

The Faculty of Built Environment and Engineering, Queensland University of Technology

Brisbane, Australia

2010

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KKeeyywwoorrddss Animal model Fracture healing Internal Fixator Minimally invasive osteosynthesis Minimally invasive plate osteosynthesis Multi-fragmentary fracture Ovine model Percutaneous plating Plate Osteosynthesis Soft tissue trauma Trauma model

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AAbbssttrraacctt Over the past ten years, minimally invasive plate osteosynthesis (MIPO) for the

fixation of long bone fractures has become a clinically accepted method with good

outcomes, when compared to the conventional open surgical approach (open reduction

internal fixation, ORIF). However, while MIPO offers some advantages over ORIF, it

also has some significant drawbacks, such as a more demanding surgical technique and

increased radiation exposure. No clinical or experimental study to date has shown a

difference between the healing outcomes in fractures treated with the two surgical

approaches. Therefore, a novel, standardised severe trauma model in sheep has been

developed and validated in this project to examine the effect of the two surgical

approaches on soft tissue and fracture healing.

Twenty four sheep were subjected to severe soft tissue damage and a complex distal

femur fracture. The fractures were initially stabilised with an external fixator. After five

days of soft tissue recovery, internal fixation with a plate was applied, randomised to

either MIPO or ORIF. Within the first fourteen days, the soft tissue damage was

monitored locally with a compartment pressure sensor and systemically by blood tests.

The fracture progress was assessed fortnightly by x-rays. The sheep were sacrificed in

two groups after four and eight weeks, and CT scans and mechanical testing performed.

Soft tissue monitoring showed significantly higher postoperative Creatine Kinase

and Lactate Dehydrogenase values in the ORIF group compared to MIPO.

After four weeks, the torsional stiffness was significantly higher in the MIPO group

(p=0.018) compared to the ORIF group. The torsional strength also showed increased

values for the MIPO technique (p=0.11). The measured total mineralised callus

volumes were slightly higher in the ORIF group. However, a newly developed

morphological callus bridging score showed significantly higher values for the MIPO

technique (p=0.007), with a high correlation to the mechanical properties (R2

After eight weeks, the same trends continued, but without statistical significance.

=0.79).

In summary, this clinically relevant study, using the newly developed severe trauma

model in sheep, clearly demonstrates that the minimally invasive technique minimises

additional soft tissue damage and improves fracture healing in the early stage compared

to the open surgical approach method.

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TTaabbllee ooff CCoonntteennttss Keywords………………………………………………………………………...……ii Abstract……………………………………………………………………………….iii Table of Contents……………………………………………………………………..iv List of Figures………………………………………………………………………..viii List of Tables……………………………………………………………………..…xviii List of Abbreviations………………………………………………………………..xviii Statement of Original Authorship………………………………….…………………xx Acknowledgments……………………………………………………………………xxi CHAPTER 1: INTRODUCTION……………………………………………………1 1.1 Background………………………………………………………………….....1 1.2 Aims and Objectives…………………………………………………………..2 CHAPTER 2: LITERATURE REVIEW……………………………………………3 2.1 High energy injuries…………………………………………………….…......3 2.2 Fracture healing……………………………………………………….……….5 2.2.1 Types and phases of bone healing…………………………………………....…5 2.2.2 Role of the soft tissues in the process of fracture healing………………….…...8 2.3 Fixation strategies ..............…………………………………………………...9 2.3.1 Plate osteosynthesis……………………………………………………...…….10 2.3.1.1 Open reduction and internal fixation (ORIF)………………………………….12 2.3.1.2 Minimally invasive plate osteosynthesis (MIPO)……………………………...15 2.3.1.3 Clinical evidence of MIPO………………………………………………...…..17 2.3.1.4 Experimental evidence of MIPO…………………………………...………….18

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2.4 Animal trauma models………………………………………………………19 2.4.1 Soft tissue trauma models.…………………………………………………….20 2.4.2 Fracture models ……………………………………………………………….21 2.4.3 Combined animal trauma models……………………………………………...25 2.5 Methods to monitoring soft tissue damage………………………………….28 2.6 Methods to assess fracture healing…………………………………………..30 CHAPTER 3: RESEARCH DESIGN………………………………………………34 3.1 General Research Plan……………………………………………………….34 3.2 Development of trauma model……………………………………………….35 3.2.1 Development and validation of soft tissue trauma model……………...………36 3.2.1.1 Requirements on the degree and extent of the soft tissue damage………….....36 3.2.1.2 Development of the device………………………………………...…………..36 3.2.1.3 Description of the soft tissue trauma (STT) device……………………...…….39 3.2.1.4 Soft tissue trauma device: tests and validation…………………...……………40 3.2.2 Development and validation of fracture creation model…...…...……….…….50 3.2.2.1 Requirements of the fracture model………………………...…………………50 3.2.2.2 Development of the fracture model……………………………………………51 3.2.3 Validation of combined trauma model…..…………………………………….62 3.2.4 Development and validation of ‘Supporting Trolley System’…...…………….63 3.3 Study design……………………………………………….…………..….…...66 3.3.1 Establishment of fracture fixation model…………………………………...….66 3.3.2 Experimental design and description of operative techniques…...…………….71 3.3.3 Instruments and implants…………..……………………………………….….79

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3.4 Animal care and welfare…………………………………….……………….81 3.4.1 Sheep data……………………………………………..……………………….81 3.4.2 Perioperative management ………………………………………………..…...82 3.4.3 Postoperative management and care…………………………………...………83 3.4.4 Euthanasia…………………………………..………………………….....……84 3.5 Methods to monitor the soft tissue damage……………………………..…..84 3.5.1 Local soft tissue assessments…………………………………………………..84 3.5.2 Systemic investigation (blood serum tests)…………………………………….86 3.6 Methods to assess fracture healing…………………………………………..87 3.6.1 Conventional radiographs………..……………………………………...……..87 3.6.2 Computer tomography (CT)…………………………………...………………90 3.6.3 Mechanical testing……………………………………...……………………...96 3.7 Statistical analysis………………………………………...…………………..98 3.7.1 Estimation of sample size and power analysis…………………………......…..98 3.8 Animal ethics documentation…………………………...……………………98 3.9 Procedures and Timeline……………………………………...……………...99 3.9.1 Pilot study…………………………………………..………………...…...….100 3.9.1.1 Methodology and timeline………………………………………........………100 3.9.1.2 Results……………………………………………..………………………….100 3.9.1.3 Discussion and Conclusion………………………………………………..….108 3.9.2 Main test series…………………………………………………..………...…109 CHAPTER 4: RESULTS…………………………………………..………………110 4.1 Characterisation of Trauma………….………………...………..…………110

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4.1.1 Soft tissue trauma recovery...…………………………………………..…….110 4.1.2 Fracture model………….…………………………………………..….…….112 4.2 Minimally invasive versus open plate osteosynthesis………………….…114 4.2.1 General results………………………………….………………………...….114 4.2.2 Soft tissue recovery………………………………………………………….116 4.2.3 Fracture healing………..………………………………………………...….118 CHAPTER 5: DISCUSSION……………………………………………………..124 5.1 Trauma model evaluation……………………………………………….…124 5.2 Minimally invasive versus open plate osteosynthesis……………………128 CHAPTER 6: CONCLUSION AND FUTURE DIRECTIONS……...………...134 BIBLIOGRAPHY………………………………………………………………….138 APPENDICES………………………………………………………………...……149 Appendix A: List of publications (2005 – 2009)

Appendix B: List of conference papers (2005 – 2009)

Appendix C: AO Research Grant approval document 05-W17 (20 June 2005)

Appendix D: QUT Animal project approval certificate 4222A (26 October 2005)

Appendix E: Flowchart of trauma model algorithm (June 2005)

Appendix F: Detailed results of macroscopic dissection from trials of the soft tissue

trauma model with the pendulum device

Appendix G: Trial of fracture creation model

Appendix H: Timetable of pilot study (September 2006 – November 2006)

Appendix I: Timetable of main series study (November 2006 – October 2007)

Appendix J: Table with detailed list of fracture length evaluation

Appendix K: Table of list of physical data from all 24 animals

Appendix L: Two representative X-ray series of the fracture healing progress

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LLiisstt ooff FFiigguurreess Figure 1: Clinical picture of a severe lower leg injury sustained by a motorbike

crash: large wound with a substantial, soft tissue damage (Gustilo type

IIIB) and multiple bone fragments of the complex tibial shaft fracture.

Figure 2: C. Hansmann worked in the middle of the 19th century in Hamburg

(Germany) as a surgeon and is nowadays considered as the pioneer of

plate osteosynthesis. In his publication 27

Figure 3: Albin Lambotte (1866-1955)

he presented his developed

method for fracture fixation by plate. 28

Figure 4: The design of the Locking Compression Plate (LCP)

was a Belgian surgeon, who defined the

term: osteosynthesis. To the right side: A drawing of the treatment of a

non reduced tibial shaft fracture by using a plate. 54

Figure 5: First sketch (drawing) of the soft tissue trauma device.

with the ‘eight-

shaped combi’ hole, the left part for conventional, compression screw

insertion and the right part for head locking screw insertion.

Figure 6: Picture of the newly developed soft tissue trauma device.

Figure 7: Top view from the impactor and its counterpart.

Figure 8: Impactor from the outside.

Figure 9: Picturial overview of the high-speed camera setup.

Figure 10: Ruler fixed perpendicular to the impactor and counterpart. The foam

noodle is positioned to dampen the hit.

Figure 11: Illustration of frame-by-frame evaluation with x-vision software.

Figure 12: Time vs displacement curve 1: Linear portion of the curve representing the

velocity before the impact.

Figure 13: Time vs displacement curve 2: Determination of the velocity of the

impactor. Slope of the linear portion = Impact velocity.

Figure 14: Outer side of impactor showing 2 kg additional weight.

Figure 15: Final position of the counterpart block for blow against the right thigh.

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Figure 16: (Left) In vivo trial of soft tissue trauma device: The pendulum is moving

towards the point of impact on the distal thigh. The ‚marked cross’ on the

right picture shows the point of contact of the pendulum with the sheep.

Figure 17: Surgical dissection to visualise the damage incurred. This figure also

shows incidental femur shaft fracture caused due to the mal-positioning of

the sheep’s leg.

Figure 18: The team involved in the first in vivo trial of the soft tissue trauma device

in the animal operating theatre of The Prince Charles Hospital, Brisbane.

Figure 19: This macroscopic dissection of the left picture shows a moderate muscle

contusion to the lateral vastus muscle and the right picture shows an intact

sciatic nerve with superficial perineural haematomas. Both the pictures

represent the damage resulted from a pendulum hit without any additional

weights.

Figure 20: These pictures represent the extensive damage after the pendulum hit with

2 kg additional weights. In the left picture, the lateral vastus muscle is

severely damaged, partially disruptured, the subcutanous layers degloved

with small bruises and haematomas. The right picture shows the sciatic

nerve substantially contused with epineural haematomas, but

longitudinally intact. The deep soft tissues are bruised with underlining

haematomas and edematous.

Figure 21: High-resolution CT scan of a right sheep leg after soft tissue trauma trial

showing sagittal plane of the entire leg (Sheep 336).

Figure 22: CT scan with mid-sagittal reconstruction of the right thigh. Yellow spots

show: intramuscular bruises and haematomas.

Figure 23: Axial view of distal thigh region.

Figure 24: Multi-fragmentary distal femur fracture – a butterfly type, AO C-type

fracture 3

Figure 25: Custom made blade bars with two, three and four blades.

of a cadaver sheep bone.

Figure 26: Right femur bone tested with the Instron machine for three-point bending

properties.

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Figure 27: Test curve showing breakage of an ovine femur on application of three-

point bending forces by the Instron machine (Figure 26).

Figure 28: Fractured femur resulted after the application of three-point bending

forces. The image shows a random fracture configuration with a posterior

intermediate split fragment.

Figure 29 (left): Cadaver femur bone with drill holes in H shape. Two external fixator

frames, proximal and distal to the fracture, are inserted for fracture

initiation.

Figure 29 (right): Fractured femur with random fracture lines. There is no evident

fracture seen through some of the drill holes.

Figure 30: All the instruments used on the fracture model. From left, saw blade, drill

bit 3.5 mm, drill bit 2.5 mm, blade bar with two wings and chisel

respectively.

Figure 31: C-type fracture with long lateral fragment of 66 mm.

Figure 32: Proportion of partial proximal osteotomy to entire femur circumference.

Figure 33: Proportion of partial distal osteotomy to entire femur circumference.

Figure 34: Fracture configuration with intact periosteum and fragments in place.

Figure 35: Fracture pattern (periosteum removed and fragments laid to sides).

Figure 36: Ovine cadaver leg fixed in the vice of the bench: After the fracture model

was performed, the outcome was checked by a standard lateral approach to

the femur shaft.

Figure 37 (left): 2.5 cm skin incision required to perform the fracture model.

Figure 37 (right): Standard lateral surgical approach to visualise and evaluate the

fracture outcome.

Figure 38: Picture with external fixator frame in situ and the fracture model

performed by blade bar and chisel insertion through 3 cm skin incision.

Figure 39: Fracture creation sequence: 1) Two partial transverse anterior osteotomies

performed at 30 mm apart from each other. 2) In between the osteotomies,

an antero-posterior (a-p), bicortical, 3.5 mm drill hole was placed. 3)

Through the anterior osteotomies, four bicortical, 2.5 mm, oblique holes

were drilled at 30 degrees to each other. 4) In the latero-medial direction:

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four bicortical, 2.5mm diameter holes were drilled at 30 degrees to each

other. 5) To split the middle segment longitudinally into two fragments, a

sharpened blade bar was inserted into the a-p drill hole by soft hits with a

hammer. 6) Finally, two chisels were used to initiate the two transverse

fractures by a strong hit with the hammer.

Figure 40: Supporting Trolley System with the first sheep in trial based at the animal

housing on The Prince Charles Hospital campus.

Figure 41: Shade cloth with five holes.

Figure 42: Trolley trial with another ‘free’ sheep in the same cage.

Figure 43: First step: The four Schanz’ screws were placed in relation to the approach

and application of the fracture model. In this case, the figure illustrates a

small anterior approach marked in bold blue in longitudinal direction and

fracture zone is marked with transverse blue lines. The proximal two pins

were positioned parallel and in longitudinal direction and the distal two

pins were positioned in a perpendicular plane, slightly convergent to each

other.

Figure 44: Second step: After attaching the ‘unilateral external fixator’ to the pins,

the fracture creation model was performed via the small anterior approach.

The figure also shows the chisel and blade bar being inserted.

Figure 45: Third step: The percutaneous plate insertion was tested with the support of

the locked drill guide at the end of the plate. This external fixator

configuration clearly shows, that without removal of the frame, the plate

can not be fully inserted and that the longitudinal rod crosses the

percutaneous screw insertion. Therefore, the distal condylar block pins

have to be re-arranged and the rod must be moved to the anterior region of

the pins.

Figure 46: Fourth step: The last part of the trial concludes with the performance of

the open, lateral surgical approach, but as stated in the previous step, the

rods and clamps of the external fixator have to be re-positioned or even

removed to proceed with the open approach. Distally, this pin

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configuration is too close to each other and must also be altered, otherwise

the plate can not be fixed properly.

Figure 47: Lateral view (left) and axial distal view (right) of the external fixator

frame (‚V-shape’ type) and the locking compression plate (LCP) fixed

onto the antero-lateral distal femur of a plastic ovine bone model.

Figure 48: The internal fixator configuration with a 7-hole narrow 4.5mm, stainless

steel LCP with four 5.0mm head locking screws.

Figure 49: Figure showing the insertion of the four external fixator pins. The two

proximal pins at the left are positioned longitudinal to the femur shaft and

the two distal pins at the right are placed perpendicular to the proximal

once.

Figure 50: Figure showing the small antero-lateral approach employed for the

application of the fracture creation model. The oscillating saw was used to

carried out the partial anterior osteotomies.

Figure 51: External fixator frame (two carbon rods) on the upper part of the right

hind limb of a sheep. The three stitches of the fracture model approach are

seen in front of the longitudinal rod. The fixation tool of the probe to

monitor the muscle condition such as compartment pressure and partial

oxygen tension is placed on top of the proximal clamps.

Figure 52: Conventional lateral open surgical approach: The dissection of the

subcutaneous layers is being performed with the ultrasound knife. The

pins of the external fixator are still in situ and might be supportive during

the fracture reduction process.

Figure 53: The lateral aspect of the femur shaft is being prepared for visualisation by

retracting the lateral vastus muscle anteriorly using a metal retractor

‘Langenbeck hook’.

Figure 54: LCP is being placed on the lateral aspect of the femur and fixed with

screws. The two proximally applied drill guides act as a guide to drill the

holes in exact, perpendicular direction to the plate.

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Figure 55: The LCP was fixed with four screws (HLS), two proximal and two distal.

The two intermediate fracture fragments are shown to be anatomically

reduced, but not fixed as supposed to with the bridging plate technique.

Figure 56: The final result of the open surgical approach with many skin sutures in

place. The soft tissue monitoring probe shown as the orange cable fixed

with the blue caped device is still in situ for post-operative measurements.

Figure 57: The LCP with a threaded drill guide fixed at the most distal plate hole is

being slid into the small incision.

Figure 58: LCP is percutaneously advanced passing the fracture region.

Figure 59: LCP is positioned with the distal plate end in the ‘condylar groove’

between the two distal pins of the external fixator.

Figure 60: The image intensifier (Philips, BV 25, Netherlands) is used to check and

control the fracture reduction as well as the implant position during the

whole procedure.

Figure 61: The postoperative clinical picture showing few skin stitches and the soft

tissue monitoring probe in situ.

Figure 62: General surgical instruments used for the fracture creation model and

external fixator application except the implants, the sagittal saw and the

drill.

Figure 63: Sheep flock in the yard at the Biological Research Facility of the Prince

Charles Hospital; nowadays, Medical Engineering Research Facility

(MERF), Chermside.

Figure 64: This graph shows the localisation and direction of the surgical approaches

and the inserted probe in a cross-section through the middle part of the

ovine thigh. The red lines indicate the planes of the two surgical

approaches and the bold black line presents the direction of the inserted

probe.

Figure 65: Clinical post-operative site of the proximal right hind limb with the

external fixator frame. The figure also shows the sutures, the inserted soft

tissue monitoring probe as well as the cables secured in a circle.

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Figure 66: Latero-medial x-ray taken using the image intensifier in the animal

facility. The sheep positioned in lateral recumbent position on the special

large animal trolley. The ‚black’ radiographic plate is placed on the

medial, inner side of the femur and the x-ray taken from the top.

Figure 67: Antero-posterior x-ray taken with the BV 25. The radiographic plate is

placed on the back side (posterior) of the leg and the x-ray shot taken from

the front.

Figure 68: This screenshot of the AMIRA programme that illustrates the

measurements of the fracture length at three areas (left to right: lateral,

middle, medial) from the antero-posterior view of the post-operative x-ray

after the fracture creation and external fixation procedure.

Figure 69: This cropped AMIRA screenshot from the lateral view of a post-operative

x-ray shows posterior, middle and anterior (left to right) fracture length

measurements.

Figure 70: X-rays taken at 4 weeks of MIPO group with medially and posteriorly

complete callus bridges (2 bridges).

Figure 71: X-rays taken at 4 weeks of ORIF group with medial and posterior callus,

but not completely bridged (0).

Figure 72: Right explanted femur without soft tissues, but implant still attached.

Figure 73: Right femur after the implant removal.

Figure 74: With the AMIRA software, (left) a three-dimensional histogram is

generated and the proximal and distal callus planes are defined. After

cropping this middle callus field, (right) the total mineralised callus

volume is visualised and measured (mm3

Figure 75: This picture, gained with the AMIRA software, shows the areas of the

callus formation in the proximal and the distal, transverse fracture planes.

Example from the eight week ORIF group.

). The longitudinal length of that

callus volume defines the total callus length.

Figure 76: This three-dimensional reconstruction of the cortical bone fragments

shows the two intermediate fragments (lateral (left) aligned, medial (right)

distally displaced). The anterior fracture length (23.27mm), the maximal

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fracture length (25.77mm) and the maximal distance of fragment

displacement (6.15mm) is measured and displayed.

Figure 77: With the AMIRA software, two perpendicular planes cut the 3D CT model

(red - callus, blue - cortical bone).

Figure 78: (left) Cross-sectional surface in medio-lateral direction and (right) cross-

sectional cut in antero-posterior direction. Numbers and quality of callus

bridges are assessed according to the morphologic scoring system. This

example belongs to one of the four weeks ORIF group with medial and

posterior callus formation bridging the fracture gap distally and

proximally, respectively.

Figure 79: Clockwise from left top: lateral, anterior, medial and posterior view of a

3D CT-reconstruction. A representative picture of the four weeks MIPO

group.

Figure 80: Dissected femur proximally and distally embedded and fixed into metallic

cups. It was attached to the Instron testing machine to perform the

torsional test of stiffness and strength.

Figure 81: This picture shows the spiral fracture in the distal part of the femur shaft

(right femur of sheep 418) after performing the mechanical test run to

assess the ultimate torque.

Figure 82: This graph presents the compartment pressure values of the two pilot

sheep from the rectus femoris muscle compartment measured twice a day

over a period of 14 days post trauma.

Figure 83: This graph shows the measurements of the partial oxygen pressures of two

pilot sheep in the anterior muscle compartment of the thigh.

Figure 84: This graph shows the serum Creatine Kinase levels of both pilot sheep

over the first two weeks of the experiments.

Figure 85: This graph presents the serum Lactate Dehydrogenase levels of both pilot

sheep over the first 14 days of the experiments.

Figure 86: Conventional radiograph series of recovering fracture in Pilot sheep 1

(antero-posterior view).

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Figure 87: Conventional radiograph series of recovering fracture in Pilot sheep 1

(lateral view).

Figure 88: Conventional radiograph series of Pilot sheep 2 (antero-posterior view).

Figure 89: Conventional radiograph (x-ray) series of Pilot sheep 2 (lateral view).

Figure 90: Coronal reconstruction of CT scan of Pilot sheep 1.

Figure 91: Sagittal reconstruction of CT scan of Pilot sheep 2. The accidental, second

displaced fracture is clearly visible proximal to the most proximal screw

hole.

Figure 92: Mean values of the intra-compartmental pressures of 16 animals over a

time span of five days. Critical compartment pressures (> 20 mmHg) were

not reached.

Figure 93: Partial oxygen pressure monitoring within the rectus femoris muscle of the

right thigh following operations. Due to damage to measuring equipment,

only seven animals remained for evaluation. The mean values (n=7) and

the standard deviations are listed.

Figure 94: Mean values (n=24) and their standard deviations of the CK and LDH

serum levels over a period of five days.

Figure 95: These post-operative x-rays show the fracture configuration (AO C-type

with two intermediate fragments), the aligned fracture reduction as well as

the appropriate positioning of the external fixator.

Figure 96: The three-dimensional CT reconstructions of the cortical fragments

illustrates the multi-fragmentary fracture pattern (AO C-type) with three

intermediate fragments, the smallest fragment located in the proximal

posterior region. The images are from the antero-lateral and medial

perspectives respectively.

Figure 97: These two post-operative x-ray radiographs processed with AMIRA show

the angles of alignment in both axes: On the left, a varus position of 7

degrees, and the x-ray on the right shows a slight retro curved position of

3 degrees.

Figure 98: X-rays of a sheep which sustained a fall on the first post-operative day

confirmed a suspected fracture extension with proximal implant failure.

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Figure 99: Intra-compartmental pressures over the entire soft tissue monitoring time

of 14 days. After the second, randomised operation (MIPO and ORIF), the

peak of the ORIF group is higher (p=0.08) than the MIPO group.

Figure 100: The Creatine Kinase serum levels (n=12) show two peaks, after the trauma

and external fixator application as well as after the second operation for

definitive plate fixation. The second peak is significantly higher with the

ORIF group compared to the MIPO group.

Figure 101: Serum LDH concentrations (n=12) over a period of the first 14 days. The

first peak indicates the soft tissue damage after the first procedure (trauma

and external fixator application). The second peak follows the second

operation of the randomised plate fixation: the first three days post-

operatively (day 6, 7 and 8) the LDH levels are significantly higher in the

ORIF group than in the MIPO group.

Figure 102: Callus bridges at each of the fortnightly time points from the conventional

radiographs in both planes. At four weeks recovery time, MIPO group

presents significantly higher callus bridges than the ORIF group

(p=0.006).

Figure 103: The mechanical properties of torsional rigidity and ultimate torque in

relation (percentages) to the intact, contra-lateral femur are higher in both

timelines with MIPO than in ORIF technique. After four weeks, the MIPO

group shows significantly stiffer mechanical properties than the ORIF

group (p=0.018).

Figure 104: This graph shows the good correlation (R² = 0.43) between the absolute

torsional stiffness (Nm/deg) and the cross-sectional callus area of the

proximal fracture zone of all eleven animals at four weeks.

Figure 105: Correlation between the relative torsional stiffness and the 3D callus

bridging score of all eleven sheep at four weeks recovery time.

Figure 106: Correlation between the relative ultimate torque at failure and the 3D

callus bridging score of all twelve animals at eight weeks observation

time.

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LLiisstt ooff TTaabblleess Table 1: General anaesthesia and operation times

Table 2: Mechanical test results of Pilot sheep 1 and 2.

Table 3: Total mineralised callus volumes and callus lengths determined by 3D-

reconstructions of the images from the CT scans at four and eight weeks

post-operatively.

Table 4: Cross-sectional callus area in the proximal and distal fracture plane of

both groups and at both time points.

LLiisstt ooff AAbbbbrreevviiaattiioonnss ADP adenosine diphosphate

AIHW Australian Institute of Health and Welfare

AO/ASIF ‘Arbeitsgemeinschaft fuer Osteosynthesefragen’ (german) / Association

for the study of internal fixation

a-p antero-posterior

ATP adenosine triphosphate

BMP bone morphogenetic protein

CK Creatine Kinase

CT computer tomography

DCP dynamic compression plate

DPI Department of Primary Industries

FGF fibroblast growth factor

GDF growth differentiation factor

HIV Human immunodeficiency virus

IGF insulin-like growth factor

IL-1 Interleukin-1

IL-6 Interleukin-6

LC-DCP low contact dynamic compression plate

LCP locking compression plate

DCS dynamic compression screw

DMC dorsal microcirculatory chamber

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LDH Lactate Dehydrogenase

LISS Less Invasive Stabilization System

l-m latero-medial

MERF Medical Engineering Research Facility

micro CT micro computer tomography

MIO minimally invasive osteosynthesis

MIPO minimally invasive plate osteosynthesis

MIS minimally invasive surgery

mmHg millimetre Mercury

MRI magnetic resonance imaging (tomography)

ORIF open reduction and internal fixation

PC-Fix point-contact fixator

PDGF platelet-derived growth factor

STT soft tissue trauma

TGF-β transforming growth factor beta

TNF-α tumour necrosis factor alpha

VEGF vascular endothelial growth factor

X-ray conventional radiography

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SSttaatteemmeenntt ooff OOrriiggiinnaall AAuutthhoorrsshhiipp

“The work contained in this thesis has not been previously submitted to meet

requirements for an award at this or any other higher education institution. To the best

of my knowledge and belief, the thesis contains no material previously published or

written by another person except where due reference is made.”

Martin Wullschleger

Date:

xxi

AAcckknnoowwlleeddggmmeennttss First of all, I would like to thank my family, my wife Deborah, our boys Curdin,

Raffael, Jonas and Gian-Luca as well as our parents in Switzerland, for their big

support throughout the whole time, here in Brisbane.

Many thanks to my supervisors, Prof Michael Schuetz, Prof Mark Pearcy and Dr

Roland Steck, for giving me the opportunity to join the team of the Orthopaedic

Trauma Research group at Queensland University of Technology and supporting me

from the very beginning to the end of my PhD candidature.

Thanks to all collaborators and contributors of any part and at any time of my PhD:

International collaborators of the AO Research and Development Institute, Davos,

Switzerland: Prof Keita Ito, Romano Matthys and Peter Toggwiler.

The team of the Biological Research Facility of The Prince Charles Hospital

(nowadays: Medical Engineering Research Facility): Dr Kathleen Wilson, Bill

Sommers, Michael Lindeberg and Peter Lindeberg.

The team of the Orthopaedic Trauma Research group at the Institute of Health and

Biomedical Innovation (IHBI): Dr Beat Schmutz, Dr Devakar Epari, Prof Stephan

Perren, Dr Cameron Wilson, Dr Gongfa Chen, Rebecca Bibby and Caryll Clifford.

The technician team of IHBI: Greg Tevelen, Kimble Dunster and Melissa Johnston.

Medical and Medical Engineering students: Dr John Webster, Dr Adam Freeman,

Michael Uhr, Matthew Roberts and Daniel Broszczak.

Department of Radiology at The Prince Charles Hospital: Stan Redmond and his team.

Central Sterilisation Department of Princess Alexandra Hospital: Kayelene Wigg

The Medical Device Domain at IHBI: Kim Waddington, Prof Dietmar Hutmacher, Dr

Sadahiro Sugiyama, Dr Johannes Reichert, Dr Siamak Saifzadeh, Dr Ben Goss, Dr

Cameron Lutton and Dr Sarah Whitehouse.

The team of the Trauma Service at Princess Alexandra Hospital

AO Foundation for the AO Research Grant (05-W17)

AO Switzerland and AO International for the AO scholarships

University of Technology for the QUT fee waiver scholarship

Synthes Switzerland and Synthes Australia provided the instruments and implants

towards the project; especially Martin Altmann, Susan Watt and Adam Beesley.

1

CCHHAAPPTTEERR 11:: IINNTTRROODDUUCCTTIIOONN 1.1 BACKGROUND

In the last century, at the same time as numerous improvements and developments

across all medical specialties took place, advances in the field of fracture fixation also

occurred, improving the outcome for function and quality of life of patients with limb

fractures. While the healing progress of certain bone fractures clearly benefits by the

method of using a plate and screw fixation construct, in 1995 a new application of plate

fixation started. After a small surgical approach remote to the fracture zone, the fracture

fixation plate was applied by sliding it in a percutaneous fashion over the bone, passing

the fracture area and, finally, fixing the plate to the bone with screws. Prior to the

definitive fixation, the crucial realignment of the main fragments was carried out, if

possible, with closed, indirect reduction manoeuvres. This technically demanding

procedure was used instead of the standard method with a large surgical open approach

(open reduction and internal fixation: ORIF) including soft tissue dissection and

denudation of the periosteum to visualise the fracture fragments prior to the plate

fixation. Nowadays, this technique is widely accepted and more and more commonly

used.

This so called ‘minimally invasive plate osteosynthesis’ (MIPO) approach, was

developed to minimise the additional damage to the surrounding soft tissues of the bone

during the surgical procedure in order to optimise the biological conditions of the

fracture healing process. Clinically, this effect gains even more importance in critical

situations with severe soft tissue trauma and might be crucial in some anatomical areas

with thin soft tissue coverage of the bone, e.g. medial shin bone region.

However, the MIPO technique faces some significant drawbacks. Because of its

closed fashion without direct sight and overview of the fracture and the implant

manipulations, it requires training, manual skills and surgical experience to adequately

perform the procedure. In order to overcome the lack of visualisation, intra-operative

fluoroscopy with an image intensifier is used, but that inflicts the patients, surgeons and

nurses to a significantly higher exposure of radiation. If the operation including the

fracture reduction is not appropriately managed, complications may result, such as mal-

2

alignment and mal-rotation problems, as well as infections and delayed or non-unions.

Consequently, further secondary operations may be required to correct those conditions

and ensure the appropriate healing progress.

Clinically, many case collection studies indicate that the minimally invasive plate

osteosynthesis is a reasonable but serious treatment option; however, no randomised,

prospective study has proved this so far. Other evidence comes from the only

experimental animal study, performed by Schuetz et al. (1999) 1

Therefore, without clear evidence and in light of the increasing popularity of the

minimally invasive procedures in clinical practice, this study aims to answer the clinical

and scientific question: Does the surgical approach influence the fracture healing

process? Or, in other words: Is it worth undertaking a more challenging, complex

minimally invasive technique to perform plate fixation of bone fractures or should we

still rely on the simpler, open surgical method?

, which directly

compared the two different surgical approaches, minimally invasive versus open

technique. It did not show any significant differences in the mechanical integrity of the

healing fractures.

1.2 AIMS AND OBJECTIVES

In order to answer this question and to assess the effect of the surgical approach on

soft tissue recovery and the progress of fracture healing, the goal of this present study is

to develop and validate a comprehensive and suitable animal trauma model. Following

the development of this model the second step is to use it in an in-vivo animal

experiment to investigate, whether the healing progress of a defined, severe injury (soft

tissue damage and fracture) is better using a minimally invasive technique for the plate

stabilisation of the fracture in comparison with the standard, conventional open

approach for plate fixation.

• Development of severe trauma model to the distal femur and thigh in sheep:

Part I of the project:

o Development of an apparatus to inflict a severe soft tissue injury

(Tscherne III) 2

3

o Development of a mechanism to create a multi-fragmentary shaft

fracture (AO C-type 3

• Validation of the newly developed devices:

)

o In-vitro validation of the soft tissue injury apparatus

o In-vivo validation of the degree and extent of the soft tissue damage

o In-vitro validation of fracture creation on sheep bones

o In-vivo validation of fracture pattern

o In-vivo validation of combined trauma model

• Experimental study: (MIPO versus ORIF)

Part II of the project:

o Planning and performance of pilot experiments with 2 sheep (one each)

o Pilot study evaluation and final adjustments for main study

o Planning and performance of main series with 24 sheep:

• In-vivo monitoring (early soft tissue and fracture healing)

• Post-mortem testing (fracture healing progress)

• CT scanning

• Mechanical testing

• Histology

o Evaluation and analysis:

• Final Trauma model evaluation

• Soft tissue recovery evaluation

• Fracture healing evaluation

CHAPTER 2: LITERATURE REVIEW

2.1 HIGH ENERGY INJURIES

Data from the Australian Institute of Health and Welfare (AIHW) describes the

epidemiology of traumatic fractures in Australia for 2001/02 4 with forearm fractures

(21,931 patients per annum), fractures of the femur including neck of femur (21,353)

and fracture of the lower leg including ankle fractures (19,109), as the three most

common injuries for hospital admissions. Statistics shows an increasing trend for these

4

injuries, which leads to a substantial economic burden to the health system. Fifty four

percent of all traumatic fractures occur by falls, about 8% of them by high falls (> 5 m)

and 19% result from road traffic accidents. High-energy or high-velocity mechanisms

are mainly caused by those road traffic accidents and high falls.

Open or compound long bone fractures occur with a frequency of about 11.5 per

100,000 persons per year; the most common area is the tibial shaft (Figure 1), but

femoral shaft, distal femur and proximal tibial fractures tend to show up in the more

seriously injured patients and the soft tissue envelope is moderately to severely

damaged (Gustilo type II and III 5) in 68.6% of all those fractures 6.

Figure 1: Clinical picture of a severe lower leg injury sustained by a motorbike crash: large wound with a substantial, soft tissue damage (Gustilo type IIIB) and multiple bone fragments of the complex tibial shaft fracture.

Combined, severe bone and soft-tissue injuries challenge orthopaedic surgeons and

need to be treated as surgical emergencies. The damage to the morphological structures

caused at the time of trauma is set and further damage has to be prevented or

diminished through strict treatment modalities. The body itself responds to the injury in

a fundamental reaction to protect the tissue integrity, and the response aims to replace

lost cells and restore compound tissues by the physiological and biochemical processes

of wound healing 7. This wound healing sequence of overlapping reactions starts with

the initial coagulation phase, which takes some minutes and consists of two

mechanisms: vasoconstriction and haemostasis. The inflammatory phase (exudative

period) follows on with an active vasodilatation and increased blood flow to the injury

5

site. The first cells to move into the damaged tissues are neutrophilic granulocytes

(unspecific infection-control) and macrophages (removal of necrotic tissue and micro-

organisms as well as production and secretion of cytokines). The next stage is the

granulation phase with fibroplasia, angiogenesis, and epithelialisation and wound

contraction and takes a few days. After the establishment of a newly formed connective

tissue with rapid capillary proliferation and the contraction of the wound edges, the last

stage of the tissue repair, the scar formation phase occurs. This stage is of greatest

importance as it requires a balance of synthesis and degradation of collagen to re-

establish the tissue integrity and strength.

Severe injuries require a comprehensive understanding of those pathophysiological

mechanisms, clinical judgement skills as well as experience to adequately manage the

fracture and soft-tissue treatment in a timely and procedural manner. As part of this,

significant complications such as wound infections, soft-tissue loss, compartment

syndrome, non-union, chronic pain and stiffness, joint contracture, chronic

osteomyelitis, reflex dystrophy and even amputations have to be prevented 7

.

2.2 FRACTURE HEALING

Fracture healing is a complex, however, well-orchestrated, regenerative process that

is initiated in response to injury. During the repair process, the pathway of normal

embryonic development is repeated with coordinated participation from several cell

types. There are four components to the injury site: the cortex of the bone, the

periosteum, the bone marrow and the external soft tissues, all of which contribute to the

healing process. The extent to which each component is involved depends on the

conditions present at the injured tissue, such as the level of growth factors, hormones,

nutrients, pH-level, oxygen tension, the electrical environment and the mechanical

stability of the fracture 8

.

2.2.1 Types and phases of bone healing

Histologically, fracture healing has been divided into direct (primary) and indirect

(secondary) healing. Much of the current knowledge regarding cellular events and their

6

temporal and spatial characteristics has been elucidated from investigating fracture

healing in rats 8; 9

- Direct / primary cortical fracture healing:

:

Direct cortical fracture healing involves a direct attempt by the cortex to re-

establish the integrity of the bone by forming discrete remodelling units known as

‘cutting cones’, a process aimed at restoring mechanical continuity. Vascular

endothelial cells and perivascular mesenchymal cells produce the osteoprogenitor cells

that differentiate into osteoblasts. During this process little or no periosteal response is

noted (no callus formation) 10. Perren (2008) 11 even describes direct fracture healing as

an internal remodelling process and, therefore, not healing in a strict sense of the word,

but a side effect of internal removal of necrotic bone in the ‘fracture gap’. However,

this direct ‘remodelling’ of the broken cortex requires anatomical reduction of the

fracture fragments and almost complete stability (‘absolute stability’12) as well as

minimisation of the inter-fragmentary strains 13

- Indirect / secondary fracture healing:

. This is rarely seen in clinical practice.

Secondary bone healing occurs in the vast majority of bony injuries. It involves a

combination of intramembraneous and endochondral ossification with the subsequent

formation of callus. This type of healing benefits from motion 14 and is inhibited by

rigid fixation 13

Intramembraneous ossification involves the formation of bone directly, without

first forming cartilage. Committed osteoprogenitor and undifferentiated mesenchymal

cells that reside in the periosteum produce histological ‘hard callus’ within a few

millimetres from the fracture site

.

9. In this type of healing, the bone marrow with a high

cellular density contributes to the formation of bone during the early phase of healing,

when those endothelial cells transform into polymorphic cells, which subsequently

express an osteoblastic phenotype 15

Endochondral ossification consists of the recruitment, proliferation and

differentiation of undifferentiated mesenchymal cells into cartilage, which becomes

calcified and eventually is replaced by bone.

.

7

- Four stages of endochondral fracture healing:

- haematoma formation, inflammation and angiogenesis

This stage is characterised by formation of the fracture haematoma, which

resolves into granulation tissue with the typical inflammatory cascade. This

cascade is driven by signalling molecules: pro-inflammatory cytokines

(Interleukin-1, Interleukin-6 and tumour necrosis factor alpha (TNF-α); growth

factors such as transforming growth factor beta (TGF-β), bone morphogenetic

proteins (BMPs) and growth differentiation factors (GDFs); and the third group

of angiogenic factors (platelet-derived growth factor (PDGF), fibroblast growth

factors (FGFs), insulin-like growth factors (IGFs), vascular endothelial growth

factors (VEGFs) and Angiopoietin. The dynamic cascade of those molecules

interacts with platelets and mesenchymal stem cells and produces an

inflammatory response, including vasodilatation and hyperaemia, migration,

activation and proliferation, angiogenesis, chemotaxis of acute inflammatory

cells and further aggregation of platelets

: (~1-7 days post trauma)

10

-

.

formation of cartilage

This stage is characterised by the growth of the callus (soft callus). The

progenitor cells in the cambial layer of the periosteum and endosteum are

stimulated to become osteoblasts. Intramembraneous, appositional bone growth

starts on these surfaces away from the fracture gap, forming a cuff of woven

bone periosteally, and filling the intramedullary canal. It follows ingrowth of

capillaries into the callus and increased vascularity. In the fracture gap,

mesenchymal progenitor cells differentiate into fibroblasts or chondrocytes

(chondrogenesis).

: (~2-3 weeks post trauma)

- cartilage removal and calcification into bone formation

When the fracture ends are linked together by soft callus, the ‘hard callus’ stage

starts and lasts until the fragments are firmly united by new bone (~3-4 months).

As intramembraneous bone formation continues, the soft tissue within the gap

undergoes endochondral ossification and the callus is converted into rigid

calcified tissue (woven bone). It starts in areas with the lowest strain

:

8

(peripherally) and that reduces the strain more centrally, which then forms bony

callus.

- bone remodelling

The remodelling stage begins once the fracture gap has solidly united with

woven bone. The woven bone is then slowly replaced by lamellar bone through

surface erosion and osteonal remodelling.

:

The contemporary consensus and understanding of fracture healing implicates a

complex physiological process of an orchestrated series of biological events, which

involves multiple factors (on molecular and cellular levels) as well as biomechanical

principles.

2.2.2 Role of the soft tissues in the process of fracture healing

An ideal healing progress for bone fractures requires harmony between optimal

biology and optimal fixation 8. However, fractures are mostly associated with a certain

degree of soft tissue injury, which influences the treatment strategies of fractures and

consequently their outcome. Furthermore, terms like “biologic osteosynthesis” or “less

invasive surgery” emphasize the importance of adequate perfusion at the fracture site

and thus the integrity of the surrounding soft tissues. With severe soft tissue trauma and

apparent prominent oedema, conventional open approaches to the fracture site with a

wide dissection of soft tissues, including division of perforating vessels and exposure of

the fracture zone, may lead to major complications like infection, prolonged fracture

healing, non-union or a higher incidence of bone grafting 16-18. Some clinical studies

also give evidence for early soft tissue coverage of such denuded fracture areas,

especially in predisposed anatomical regions such as the tibial shaft, which benefits

from plastic reconstructive procedures 19; 20

Between 1960 and 1980, several groups published and emphasised the importance

and key role of the soft tissues on fracture healing: the periosteum as well as the

surrounding soft tissues

.

13. They were mainly focused on the preservation of the blood

supply 21, the development of an early extraosseus blood supply 22 and the cellular

activity within the processes of osteogenic induction 13; 23. With further research and the

detection of the molecular interactions and pathways during the healing process, it

9

became even more evident that it is necessary to protect and support the ‘biological’

environment of fractures. This inflammatory cascade is controlled by major signalling

molecules, such as interleukin 1 (IL-1), interleukin 6 (IL-6), transforming growth factor

β (TGF β), insulin like growth factor (IGF), fibroblast growth factor (FGF), platelet-

derived growth factor (PDGF) and the bone morphogenetic proteins (BMPs) 14

In recent years, experimental studies on rats have supported clinical experience with

these studies showing that the healing of bone fractures is influenced by the state of the

surrounding tissues

.

24; 25. This is especially the case in the early stage of recovery, as

well as in situations with very severe soft tissue damage (resection of large muscle

segment). However, Melnyk et al. (2008) 26

In summary, the role of the surrounding soft tissues on fracture healing is

significant in the early stage as it is critical to the supply of cells and molecules to

support the inflammatory stage. From clinical experience as well as experimental

studies, severely damaged soft tissue conditions adjacent to the fracture site challenges

and complicates fracture management and outcome. Therefore, in treating and studying

bone fractures the soft tissue condition has to be carefully considered and appropriately

treated to achieve an optimal fracture management outcome.

concluded from their rat model study, that

soft tissue damage without destruction of the bone-soft tissue interface is likely to have

only a limited effect on fracture healing.

2.3 FIXATION STRATEGIES

Simple bone fractures with minor soft tissue damage, normally caused by low

energy accidents, are managed by fixation principles according to biomechanical

characteristics of the injury, fracture pattern, availability of fixation methods and

implants, surgeons’ preferences and experience, as well as the patient’s requirements

and co-morbidities. Many different options of conservative and operative treatment

pathways include partial or full immobilisation with bandages, splints, casts or special

orthesis (e.g. adaptable range of motion braces), external fixation devices as external

fixators, pins and Kirschner’ (K-)wires, internal fixation devices such as wires (K-

wires, cerclage, tension band), screws, plates (compression, bridging, locking systems),

intra-medullary ‘nails’ as pins, un-reamed or reamed metallic rods, and several

10

combinations across those implants. Certain injuries may even be managed with a

combination of those implants.

High energy injuries with compound, contaminated, severely damaged soft tissue

and fracture conditions with defect wounds or loss of soft tissues or bony fragments, are

differently managed in a sequential manner: a step by step management is crucial for

the successful limb recovery. Initially, as soon as possible, a thorough washout and

debridement for decontamination, a temporary fixation (mostly with an external fixator)

and, if required, an urgent re-vascularisation with vascular reconstruction of the

damaged limb has to be carried out. This is called ‘damage control orthopaedics’. Then,

on a regular basis for the following days, or sometimes even a couple of weeks,

repetitive washouts and debridements are performed to achieve clean and early healing

soft tissue conditions. At a third stage, the definitive fracture fixation is defined and

performed, as well as the soft tissue management finalised by coverage of the bone and

the implant, if necessary by a plastic procedure with local or free soft tissue flaps. The

definitive fracture treatment includes joint reconstructions and in complex bone defect

situations, using special options such as limb shortening (temporary) or bone grafts

(autograft, allograft, bone substitutes), either primary or in a delayed timely fashion.

2.3.1 Plate osteosynthesis

The plate osteosynthesis was established at the end of the 19th century by the

surgeon C. Hansmann from Hamburg, Germany. He started to treat tibial shaft fractures

with a fixation method like a plate. He used a metal strip and fixed it with steel screws

to the bone fragments. In his publication 27 ‘A new possibility to fix bone fragments of

complicated fractures’ he presented the result of 21 cases treated with this method and

that was the foundation for all later techniques of plate osteosynthesis.

11

Figure 2: C. Hansmann worked in the middle of the 19th century in Hamburg (Germany) as a surgeon and is nowadays considered as the pioneer of plate osteosynthesis. In his publication 27

he presented his developed method for fracture fixation by plate.

An important step for surgical procedures in general and for the operative treatment

of fractures, in particular, was the development of asepsis which led to further

developments of fixation methods. Albin Lambotte was substantially involved in this

process 28. This Belgian surgeon created the term ‘osteosynthesis’ and brought forward

the concept of internal and external ‘splinting’. Today, these principles are still used in

almost all modern stabilisation methods. Apart from the external fixator, he developed

many different plate and screw designs, which made anatomical reconstruction and

early mobilisation of the limb and the patient possible.

Figure 3: Albin Lambotte (1866-1955) 28 was a Belgian surgeon, who defined the term: osteosynthesis. To the right side: A drawing of the treatment of a non reduced tibial shaft fracture by using a plate.

12

In the same period of time William A. Lane (1856-1943) performed surgery in

London and operated on fractures, first of all with cerclage wires, then after 1883 also

with screws, and finally at the end of the 19th century using plates. He published

extensively his experiences about operative fracture treatment in 1914.29 Another

Belgian surgeon, who is considered as the ‘Father of the modern osteosynthesis’, was

Robert Danis. In 1947, he published the development of a special compression plate

which permitted immediate mobilisation after fracture stabilisation 30

. With this kind of

fracture fixation with axial compression and rigid fixation the fractures healed without

radiological signs of callus formation. He described this finding as ‘primary fracture

healing’, in contrast to the ‘secondary fracture healing’ with callus formation by

conservative treatment. This conclusion led to the opinion that callus healing was

connected to instability with the associated tendency to develop delayed or non-unions

or risk of implant failure.

2.3.1.1 Open reduction and internal fixation (ORIF)

In the 1950s, the founders of the Swiss Association for the Study of Internal

Fixation (AO/ASIF) 31 standardised the use of plate systems. They described the main

goals of fracture treatment in the first edition of the ‘AO Manual of Internal Fixation’ in

1965 21 as the restoration of the function of the injured limb. Through performing a

stable osteosynthesis, the bone should get the primary strength to recover by early

functional aftercare. This could be achieved by a conventional, open surgical approach

for visualisation of the fracture site, open reduction of the fragments and stabilisation of

the reduced fracture with a plate, so called: open reduction and internal fixation

(ORIF). Complications, such as wound and bone infections (by large approaches and

wide dissections of the bone), mal-alignments and fracture disease caused by long-term

immobilisation of the limb and the patient, should be avoided. To reach this goal, four

fundamentals were set in the ‘AO Manual’ 21

1. anatomical reduction

:

2. absolute stability with inter-fragmentary compression

3. preserving blood supply throughout atraumatic operation technique

4. avoiding additional damage by immobilisation.

13

The original AO technique was carried out using lag screws or with a special

tension device. Later on the so called ‘dynamic compression plate (DCP)’ (Synthes,

Switzerland) made axial compression possible through eccentric screw insertion in the

special designed plate holes. Another concept to protect the lag screws was developed

using ‘neutralisation or protection plates’. All these plate techniques were implemented

to get primary fracture healing without callus formation. But these operation techniques

with sophisticated anatomical reduction required large open approaches and wide

dissections with additional damage and oppression of the blood supply to the fractured

bone fragments. As a consequence of such traumatic operation techniques, delayed

healings, pseudarthrosis and increased infection rates could be seen.

Parallel to this progress in the field of plate osteosynthesis, the intra-medullary

nailing procedure was initiated by Küntscher in the 1940.32 One important stage of

these further developments was the invention of locking nails in the 1970s. The

findings of this closed reduction and fixation technique with secondary callus healing,

even of large comminuted fractures, led to a change in the concept of fracture fixation

from the anatomical reduction and fixation with absolute stability to a fracture

reduction with correct axes, length and rotation of the bone and then a fixation

technique with relative stability. Consequently, bridging plates were performed without

touching or fixing of the multi-fragmentary fracture area. These plates were only

stabilised from one main fragment to the other main fragment (proximal and distal).

The advantage of this technique was to avoid further devascularisation of the small

fragments by the surgical procedure. Long plates were used to obtain optimal stress

distribution of the plates and implant failures could be reduced. The callus healing was

not an unwanted side effect anymore, but the aim of fracture treatment was a safe and

secure fracture healing with callus formation. Ganz suggested the term of a biological

plate osteosynthesis 33, which was characterised by an atraumatic surgical performance

without dissection of the fracture zone, indirect reduction techniques and stabilisation

with any bridging fixation device 34

The development of the plate design went parallel to the above mentioned stages.

The properties and shapes of the plates changed from simple, straight plate designs to

many different anatomically pre-shaped plates with different sizes adapted to the

.

14

different skeletal regions. These plates were all fixed and pressed to the bones to

overcome the friction forces, which were produced by loading of the limbs.

Experimentally, Perren and his group (1988) 35 found areas of osteonecrosis and

osteoporosis underneath the compression plates. If those cortical changes were purely

caused by biological reasons with reduced periosteal perfusion, or whether the

mechanical conditions changed to bone remodelling by stress shielding, is still

unproven. Clinically, the use of these plates was associated with higher risk for

infection and delayed or non unions in fracture healing 36-38

A first step to reduce the contact area between the plate and the bone was via plate

adaptation with undercutting of its under-surface with lower contact or point contact

design (LC-DCP and PC-Fix, Synthes, Switzerland). These constructs caused less

periosteal damage

.

39. Finally, with the development of the so called ‘internal fixator’,

the plates are positioned without any contact to the bone. To achieve this periosteal

perfusion preserving procedure, the screw-hole interface has been changed. Instead of

normal screws, which were inserted in the plate holes and worked only with

compression, the screw heads lock into the plate hole and function as an angular stable

device. This locking mechanism is achieved by a threaded screw head, which fits and

locks into the thread of the hole in the plate 40; 41. This improvement in terms of the

biological environment also changed the plate mechanics. In conventional compression

plates, the loads were transmitted by the friction between the implant under-surface and

the bone surface, and were therefore dependent upon the screw anchorage and the bone

quality. With this new angular stable screw-plate system, the forces are totally held

from the screws in one main fragment, transmitted by the screw-plate construct, over

the other main fragment. Because this involves the same concept as for an external

fixator, but its application is inside the body with coverage of soft tissues, it is called

‘internal fixator’. This technique of the internal fixator was realised with the so-called

PC-Fix 42, which was shown in animal studies to cause better cortical perfusion and less

cortical necrosis 41; 43; 44, advanced fracture healing in terms of mechanical conditions 41;

45 and a reduction in the incidence of infection 46. A further development of the internal

fixator technique was a new plate design including special instrumentation tools (such

as an aiming arm), named as Less Invasive Stabilisation System (LISS). This plate-

15

screw construct was developed to fix complex distal femur and proximal tibial fractures 47; 48. Clinical prospective collection studies 49-53

The last evolutionary development of plate design is the combination of both screw-

plate interfaces in one system. The LCP (Locking Compression Plate, Figure 4)

using the LISS method for distal femur

fractures showed high union rates of the fractures, relatively low complication rates

even in cases of severe injury and lower rates of bone grafts.

54 is

designed with special plate-holes: the configuration of the holes correspond to a figure

‘eight’-shape: on one side the conventional compression hole and, on the other side, the

threaded hole for angular stable screw insertion. Thus, the LCP can be used as a

conventional compression plate, as an internal fixator or for certain applications as a

combination of both techniques 55. The LCP system is regarded as technically mature

and offers numerous fixation possibilities and has proven its worth in complex fracture

and revision situations 56

.

Figure 4: The design of the Locking Compression Plate (LCP) 54

with the ‘eight-shaped combi’ hole, the left part for conventional, compression screw insertion and the right part for head locking screw insertion.

2.3.1.2 Minimally invasive plate osteosynthesis (MIPO)

In the era of minimally invasive surgery (MIS), procedures such as endoscopic,

laparoscopic or video-assisted thoracoscopic operations became more and more

important and nowadays these techniques are well established. The intention of those

minimally invasive methods was to reduce and minimise the additional, iatrogenic

damage caused by open surgical approaches. For several indications and procedures

16

morbidity, complication and, even, mortality rates could be decreased. In the field of

orthopaedic trauma surgery, percutaneous insertion of pins, K-wires, screws or external

fixators has been performed since the early stages of operative fracture fixation. This so

called minimally invasive osteosynthesis (MIO) technique involves a closed and mostly

indirect reduction of the fractures and afterwards fracture fixation by implants using

small incisions and approaches. The aim behind this technique is to avoid or minimise

additional operation damage and therefore optimise the biological conditions and

environment for fracture healing.

As mentioned in the chapter 2.3.1.1 above, plate osteosynthesis is still quite often

performed with an open surgical approach, dissecting the soft tissues to visualise the

fracture zone and stabilising it with a plate device after performing fracture reduction

(ORIF). In contrast to the AO principle of ‘preserving of blood supply throughout

atraumatic operation technique’, the conventional plate osteosynthesis technique often

led to a wide open dissection with too much additional trauma and consequently to an

increased rate of complications such as wound healing problems including infections,

delayed fracture healing or pseudarthrosis (non-unions), implant failures as well as the

incidence of bone grafting 17; 18

In order to minimise such additional operation trauma and optimise the soft tissue

and fracture healing, Gerber et al. (1990)

.

57 and Claudi et al. (1991) 58 described the

term ‘biological internal fixation’. Baumgaertel et al.(1994) 59 published a case series

of 19 patients, in which they performed this kind of biological plate fixation on

comminuted femur fractures with an open approach. This led in all patients to a good or

excellent clinical outcome with fracture healing on schedule. These findings stimulated

surgeons to develop further technical improvements in the operation technique in a

sense of minimising the surgical damage. Wenda et al. (1995) 60 described the

application of a ‘slide-in plate technique’ on femur shaft fractures. After sufficient

indirect fracture reduction under extension and this new type of slide-in plate fixation,

fast developing callus formation and homogenous remodelling could be achieved.

Krettek (1997) 61, who called this technique ‘minimally invasive plate osteosynthesis

(MIPO)’, using the ‘Dynamic Compression Screw (DCS)’ plates for femur shaft

fracture fixation, stated that this procedure can be safely and successfully performed,

17

but it is technically demanding and the limb alignment must be carefully handled.

Helfet (1997) 62 performed this method for the first time at the distal tibia region and

described it as a feasible and worthwhile method of stabilisation, while avoiding severe

complications. In addition, the technically advanced LISS plates supported the

minimally or less invasive technique for the treatment of complex distal femur and

proximal tibia fractures 47

The introduction of the LCP system led not only to a general improvement of

internal plate fixation, including complex fractures and difficult bone conditions (e.g. in

osteoporosis), but also to further clinical applications in terms of MIPO. Finally, from

the clinical perspective Sommer et al. (2005)

.

63

stated in a review article: “This

technique has received widespread acceptance under the term of MIPO during the last

five years, especially with the new angular stable screw-plate systems (LISS, LCP).”

The main problem of the MIPO technique is and remains the reduction (no direct

manipulation possible) and their intra-operative assessment (no direct visualisation).

The balance between the degree of invasivity and the achieved quality of reduction and

stability is often difficult to define and must be related to several factors (anatomical

region and type of the fracture, local soft tissue conditions, quality of the bone, age and

requirements of the patients, available implants, experience and preference of the

surgeons, etc.). New technologies such as improved imaging, intra-operative navigation

and percutaneous reduction tools will help to further reduce the invasivity of fracture

surgery in the future.

2.3.1.3 Clinical evidence of MIPO

In the last decade, many clinical non-randomised case collection studies from

different anatomical regions have been published or presented and illustrate that the

MIPO technique displays high fracture healing rates, a small number of complications

as well as a low amount of mal-reductions and mal-alignments:

- proximal humerus shaft: Sommer et al. (2005) 63

- middle to distal third of humerus shaft: Zhiquan et al. (2007)

64

- distal radius: Imatani et al. (2005)

65

- distal femur: Schuetz et al. (2003)

66, Ricci et al. (2006) 67, Kolb et al. (2008) 50

18

- femur shaft: Apivatthakakul et al. (2009) 68

- proximal tibia: Cole et al. (2004)

69, Oh et al. (2005) 70

- distal tibia: Helfet and Suk (2004)

71, Krackhardt et al. (2005) 72, Wullschleger et al.

(2006) 73, Collinge et al. (2007) 74, Hasenboehler et al. (2007) 75

The only study published so far comparing the two approaches (MIPO and ORIF)

for plate osteosynthesis, is a retrospective case collection series with 33 patients

included

.

76

As a conclusion of all those studies, the MIPO technique has been proven to be a

reliable and reasonable treatment option to the ORIF method, and satisfactory results

are achieved in terms of soft tissue healing and fracture union rates, as well as

functional outcome.

. They could not show any significant difference in their evaluation, but

some advantages in terms of reduced incidence of iatrogenic radial nerve palsies and

accelerated fracture union. The functional outcome with respect to shoulder and elbow

function was similar.

2.3.1.4 Experimental evidence of MIPO

In an experimental study investigating biological plate fixation, but still performed

via an open surgical approach, Baumgaertel et al. (1998) 77

In a human cadaver injection study, Farouk et al. (1999)

showed in a comminuted

proximal femur fracture model in sheep, that the fracture healing progress was faster

and more efficient in terms of the mechanical properties using the indirect reduction

and bridging plate technique compared to the anatomical reduction and standard plate

fixation method. 18

The first animal study analysing the influence of the operative approach onto soft

tissue and fracture healing in the field of internal plate fixation was presented by

Schuetz et al. (1999)

presented that the

minimally invasive approach to the lateral aspect of the femur compromised the bone

perfusion significantly less than the conventional open approach.

1. This group used an ovine trauma model of a simple, mid-shaft

tibia fracture with a concomitant moderate soft tissue injury to the lateral muscle part of

the lower leg (at the same level as the tibia fracture). In that experimental design, they

compared the open (ORIF) versus the minimally invasive plate osteosynthesis (MIPO)

19

in terms of the bone perfusion and the rate of fracture healing, but no statistical

differences could be found. This might be explained by the low severity of the trauma

model with simple tibial shaft fracture and very localised, moderate soft tissue damage

on the lateral side (opposite to the surgical approaches). The moderate soft tissue

damage recovers quickly and does not significantly compromises the fracture healing

outcome 26

However, we believe that the minimally invasive technique (MIPO) from our

clinical experiences, supported by the clinical studies, is advantageous for the healing

process of the soft tissues and the fractured bone in comparison to the open surgical

method (ORIF). From the above outlined experimental study, we also hypothesise, that

potential differences in the healing outcome after those two surgical approaches and

plate fixation could be demonstrated in an animal model with a more serious injury

pattern, such as severe soft tissue damage and complex fracture configuration, as well

as in an anatomical area with more and circumferential soft tissue and muscle coverage

of the fractured bone, like the femoral region.

. Additionally, healing of simple, diaphyseal fractures in sheep is known as

robust, and those fractures treated with an appropriate fixation device heal fast and

without problems. On the other hand, the surgical approaches done on the medial side

of the tibia in an anatomical area without any muscle coverage does not differ largely

between the two techniques, nor does it significantly impact the fracture healing

process because the blood supply and the muscle coverage of the tibia is located

laterally and posteriorly.

2.4 ANIMAL TRAUMA MODELS

Numerous reports have been published which describe experimental trauma models

in laboratory animals over the last decades. Several different methods and devices have

been developed to create either soft tissue damages or bone fractures or a combination

of both together. In the following chapters (2.4.1 to 2.4.3) those experimental trauma

models are presented, first the soft tissue trauma models, thereafter the fracture models

and thirdly, the combined trauma models.

20

2.4.1 Soft tissue trauma models

Several groups developed and applied different soft tissue injury models in small

laboratory animals to study the influence and alterations of the traumatic damage on the

local pathophysiological reactions, including the microcirculation, and the infection

resistance of the soft tissues, mainly the muscles. The basis of all those techniques was

an impacting device, which hit the defined anatomical area in a ‘controlled and

reproducible’ manner to induce a mild to severe soft tissue damage. The mechanisms to

impact the soft tissues showed a variety of setups and included the use of: a spring-base

hammer device 78; 79 with a standardised trauma impact of 50% of the energy needed for

fracturing the adjacent bone; a drop weight mechanism with a round-tipped brass

cylinder with 23g weight sliding down a tube of 117 mm length 80; the use of a

computer-assisted and controlled-impact apparatus 81; 82 with a compressed nitrogen gas

source for the impact with a velocity of 7 m/s; or a pneumatically driven and computer-

controlled impact device (cylinder) 83, simulating a closed, high velocity injury of the

lower hind limbs of rats. Some of those above mentioned soft tissue trauma devices 81-83

were modified from originally developed models to produce standardised closed brain

injuries in rats. Dixon (1988) 84 presented a fluid percussion injury model with a direct

brain deformation. Lighthall (1989) 85 and Goodman (1994) 86 used a different type of

percussion model with a controlled cortical impact by a compressed air-driven metallic

piston. Marmarou et al. (1994) 87 implemented a special weight drop model, which was

one of the most frequently used constrained rodent models of impact acceleration head

injury. It consisted of a column of brass weights falling freely by gravity from a

designated height through a Plexiglas tube to hit a stainless steel disc positioned on the

brain region to be damaged. All those soft tissue trauma models were successfully used

and proven to reproduce the pathophysiological changes and morphological responses

of the damaged soft tissue regions. The characteristics of those changes included the

reduction in functional capillary density values of soft tissues, increased leakage of

macromolecules and activation of leukocyte adherence leading to activation of the local

inflammatory process. One of those studies 83 showed that selective cyclooxygenase-2

inhibition was highly effective in restoring the disturbed microcirculation, as well as in

reducing the inflammatory sequelae. The soft tissue trauma study of Kaelicke (2003) 81

21

with rats presented a significantly higher infection rate in the animals with soft tissue

injury compared to the non-injured animals. Those results highlight the relevance of the

soft tissues in the healing process and the importance of having suitable animal models.

For clinical reliability reasons, as well as for comparison to the human pathophysiology

of soft tissue injuries, large laboratory animal models would help, but none has been

presented or published so far.

2.4.2 Fracture models

In the clinical practise of the orthopaedic trauma surgeon, the management of bony

fractures belongs to the daily activities. The fracture treatment modalities evolved over

the last century, from purely conservative management with all sorts of traction and

splinting methods to many more methods with conservative and operative fixation

options. Those alterations resulted in earlier fracture re-union with significantly lower

complication rates. This development process involved clinical knowledge and

experience gained from clinical studies, as well as experimental evidence from animal

research. To study the biological and mechanical properties and conditions of the entire

process of fracture healing, suitable and standardised fracture models were required.

The creation of such a realistic and ‘natural’ fracture is difficult. Many different models

have been developed thus far; the challenges and their disadvantages are outlined in the

following section.

In small laboratory animals, experimental fractures were first produced by manual

loading of the bones, but the fractures created varied too much in respect to extent and

configuration. It was challenging to precisely control the forces to create reproducible

fractures 88-90. Jackson’s group (1970) 90 reported the first standardised femur fracture

model in rats. The ‘pre-pinned’ femur with an intra-medullary wire was subsequently

fractured with a blunt guillotine, driven by a pneumatic punch press powered by a

cylinder of compressed air. This standard closed fracture was thought to be superior to

an open osteotomy through elimination of the added variable of local wound healing

and the associated soft tissue trauma was considered to be minimal.

22

Because of the additional soft tissue damage around the created fracture site, further

modifications of this blunt fracture model will be discussed in details in Chapter 2.4.3

with the combined trauma models.

Several reports used ‘open transverse osteotomy’ models; Oni et al. (1988) 91 and

Herzog et al. (2002) 92 for the tibia in rabbits, Bak et al. (1992) 93 and Utvag et al.

(2003) 25 for the tibia in rats, Oszwald et al. (2008) 94 on the femur in rats and Harry et

al. (2008) 95

The first three-point bending fracture model in rabbits was reported by Ashhurst et

al. (1982)

on mice tibia. Despite the defined and very consistent fractures, the

criticism of those osteotomy models was that the surgical approach interacted and

influenced the pathophysiological environment of the healing bones.

96 and resulted in a reproducible fracture pattern. Park et al. (1994) 97

In recent years, new stabilisation devices have been established in rats and mice to

fix the fractures created to study fracture healing and bone regeneration. These internal

devices are either intra-medullary locking nails

published a further modification by loading in three-point bending in an arbour press.

Using this new fracture model either transverse or oblique fracture at any desired angle

are produced by variations of the position of the cortical drill hole relative to the central

loading point. This method resulted in a consistent location of the fracture, with 88%

reproducibility of the fracture angle and only minor damage to the adjacent soft tissues.

98; 99 or locking plates with rigid or

flexible mechanical properties 100

As a summary, fracture models in small laboratory animals developed towards a

very standardised fracture creation (either by simple open osteotomy, by three-point

bending mechanism (closed) or by weight-drop technique (see also Chapter 2.4.3). The

fractures created are highly reproducible and suitable for experimental investigations,

especially with mechanically defined stabilisation techniques, to gain a more

comprehensive understanding of the biomechanical environment and the dynamics and

interactions of the biological and mechanical processes throughout the different stages

of fracture healing.

. Intra-medullary nails are used in closed fracture

models, while plate fixation procedures are performed in open fracture models.

The creation of a ‘natural’ and reproducible fracture type in large laboratory

animals is challenging, due to the high forces that have to be overcome to break the

23

bone (especially in the shaft region with strong cortical properties), and the enormous

tendency of those bones to split, which causes anisotropic shapes and unpredictable

configurations. Therefore, in order to manage these challenges, simple osteotomies with

a high reproducibility have been widely used. Schenk and Willenegger (1963) 101

presented the first standardised fracture model by carrying out an open osteotomy of the

tibia in dogs. Several other groups used this kind of model 102; 103, while further

developments of the osteotomy model were implemented, producing either a

comminuted fracture with a triple-wedge osteotomy 104 or a spiral fracture with a

consistent pattern 44. However, osteotomies are not fractures; while highly reproducible

fracture patterns might be achieved, there are also certain drawbacks associated with

this method. Primarily, heat necrosis may develop due to high temperatures occurring

during the sawing 43 with insufficient irrigation. This heat necrosis subsequently

influences the vascular density and therefore the remodelling of the bony fragments 105.

Furthermore, the separation of the bone into regular bony fragments with smooth edges

and surfaces does not replicate the fracture patterns observed clinically and provides

less intrinsic stability due to reduced friction between the bone fragments, when

compared to the jagged bone surfaces of a “natural” fracture. In light of these

drawbacks of the osteotomy models, several different three-point bending devices were

developed that should fracture the bones in a reproducible, but more irregular and

realistic fracture pattern. Examples of such fracture models have been applied to large

laboratory animals. With a three-point hydraulic bending device, simple transverse

fractures of the ulna and radius in dogs could be shown 106. A similar three-point

bending technique was used to create a transverse tibia fracture in dogs, reproducibly

tested on a series of 14 animals 107

As a further next step towards achieving a more reproducible, as well as ‘natural’

fracture pattern, was the development of combined procedures with the use of

osteotomies and different bending force techniques. Kregor et al. (1995)

.

43 presented

the creation of a short mid-shaft spiral tibial fracture in sheep by using a micro-

oscillating saw to perform a 2 mm deep partial osteotomy at the medial side. Following

this, the three-point bending apparatus was placed and an external rotation force applied

to the tibia. The final step was to initiate the fracture with a blow of a slap hammer

24

against the three-point bending fracture apparatus. Tepic et al. (1997) 41 published a

method to generate an oblique fracture of the sheep tibia: a stress raiser was produced

by a 1 mm deep saw cut in the mid-shaft area (on the medial aspect) using an

oscillating saw. Then the hook of the fracture jig was carefully passed around the

periosteum and the hook tightened against the block of the jig, which led to a preload of

a three-point bending moment. Following a torsion load to the tibia by hand, the final

impact was added to the hook of the jig with a slide hammer. In their series of the in-

vivo experiment, all fractures (56 animals) but one obtained by this method were

oblique and could be fixed in the planned manner with DCP or PC-Fix plates. A further

modification was the application of a four-point bending impact device which produced

transverse or short oblique tibia fractures in a sheep model with even higher

reproducibility of the fracture characteristics 108

A third fracture model combining different techniques was introduced by

Baumgaertel et al. (1994)

.

39. This group created comminuted proximal femur fractures

in sheep with a three step procedure: firstly, a longitudinal bicortical osteotomy limited

by drill holes as breakpoints and to avoid further bone splitting, secondly, applying a

static lateral bending load of 0.2 kN with an external fixator and, finally, a sudden

three-point antero-posterior compression force was created with a blow from a hammer

against the bending device to initiate the fracture. This method was successfully used in

36 sheep. The latest combined fracture model was presented by Dumont et al. (2008) 105

Most of the above described fracture models produce simple fractures. Those

fractures are easier and to be created consistently, hence they provide a very reliable

and comparable approach to study the different stages and characteristics of the process

of fracture healing and bone regeneration. From the clinical perspective, however,

fracture patterns are mostly irregular and more than half of all fractures, depending on

the anatomical region, are multi-fragmentary or comminuted (three or more fragments).

Especially in high-velocity injuries, those more irregular and complex fracture patterns

producing a short oblique (30°) tibia fracture in sheep: a partial saw corticotomy to

one-third of the cortical circumference in the transverse plane was carried out, and the

fracture was completed by applying a manual force with a roller device functioning as a

hypomochlion. This fracture model was successfully used on eleven sheep.

25

are common. So far, in the experimental setup, only two fracture models with multi-

fragmentary fractures have been described. Heitemeyer et al. (1990) 104 studied on the

significance of postoperative stability for bony reparation of comminuted fractures and

used a triple-wedge osteotomy model in ovine tibiae. Baumgaertel et al. (1994) 39

presented a different multi-fragmentary fracture model in an ovine experiment

assessing the biological plate osteosynthesis in the proximal femur region. They used a

combined technique involving a longitudinal osteotomy, the application of a static

lateral bending load via an external fixator and, finally, a sudden three-point antero-

posterior compression impact on the bending device by a hammer to initiate the

fracture. Both groups were struggling in the trial stage to create and fix those more

complex fractures, but the results of their models were impressive, especially as they

showed to simulate a more clinical relevant, complex fracture pattern and their

outcomes of the different healing conditions.

2.4.3 Combined animal trauma models

Most of the animal models were developed and used to study either soft tissue

recovery or fracture healing. In real life, a soft tissue injury might be sustained without

a bony breakage, but if a bone fractures, in almost every case the soft tissues are

compromised or damaged, too. Therefore, to investigate fracture healing in as natural as

possible conditions, a certain defined fracture pattern should be accompanied with

matching soft tissue damage. From the clinical experience, fractures with severe soft

tissue injury are much more challenging and sometimes even critical to manage. The

bone healing process is dependent on the soft tissue conditions and impacts on

complication rates, particularly with severe, compound injury sustained from a high-

energy impact. A first model, mainly focussed on the fracture model, but also creating a

soft tissue damage is the weight-drop “guillotine” method, which was developed by

Jackson et al. (1970) 90 and modified by Bonnarens and Einhorn (1984) 109. This blunt

trauma model consists of four component parts: the frame, which holds the other

components in place, the animal support system to secure the correct position of the

animal and the guillotine ramming system with the impact disc, which is driven

downward by the dropping weight. Before the fracture was created, they performed an

26

intra-medullary pinning of the femur. This method produced 38 transverse femur shaft

fractures and two short oblique fracture types out of the 40 cases. The authors

summarised their model as simple in its design, creating reproducible fracture

configurations and ‘minimal degree’ of soft tissue trauma; it is an excellent technique

for the production of a standard closed fracture in laboratory animal bone. This

‘standard’ model was slightly modified and then used by An et al. (1994) 110 to create

closed tibia fractures in rats, which was also successfully performed with reproducible

fracture types. Tatari et al. (2007) 111

In small laboratory animals, only three reports published so far have assessed the

influence of the soft tissue injury on fracture healing, focussing on a better basic

understanding of pathophysiological interactions and processes of bone and soft tissue

healing. Utvag et al. (2003)

introduced a new pendulum device with the

weight drop from different angles to produce tibia shaft fractures post pinning. They

concluded their technique was a simple apparatus to create easy and reproducible tibia

fractures in a standard fashion without fracture displacement, ideal for bone healing

studies. The best position of the weight was identified as a 60 degrees angle, which

produced only minimal soft tissue trauma.

25 studied the healing of intra-medullary pin-fixed tibial

osteotomies in combination with different degrees of soft tissue injuries inflicted by

either crushing the adjacent muscle group with a surgical forceps or by removing parts

of the adjacent muscle group by surgical dissection. Claes et al. (2006) 24 and Melnyk et

al. (2008) 26 used a newly developed trauma model with a two step procedure: firstly,

with a blunt impactor as a dropping weight (170 g, impact velocity of 6 m/s) the closed,

severe soft tissue damage (grade II according to the Tscherne classification 2) was

applied and secondly, with a three point bending configuration the dropping weight

(650 g, 1.6 m/s speed) created the tibia fracture. All three trauma models were

successfully performed and evaluated. The results showed that only in the very early

stage of the healing process (within the first three days) the blood flow at the fracture

gap is reduced (not significantly) in the animals with additional moderate (grade II) soft

tissue trauma. The fracture healing in the cases with soft tissue damage was slightly

delayed in the early phase (until day 14), and did not differ significantly in histological

and biomechanical properties after four weeks. With a complete muscle group excision

27

at the fracture site, the fracture healing outcome at four weeks was significantly

reduced compared to the group without soft tissue compromise. The conclusion of

those three studies in rats suggests that the soft tissue envelope influences fracture

healing, but if the damage does not completely destroy the bone-soft tissue interface,

the effect is likely to be only limited on fracture healing.

In large laboratory animals, the only trauma model combining a soft tissue injury

and a bone fracture was reported by Schmeling et al. (2000) 108. This ovine model

included a simple mid-shaft tibial fracture (AO classification Type A) and a

concomitant defined soft tissue injury (Tscherne II) to the lateral tibia region of the

sheep. In a first step, the moderate soft tissue contusion was created with a pneumatic

cylinder device, which hit the defined area with an impact speed of 6 m/s and impact

duration of 200 ms. Secondly, after repositioning the animal on the operating theatre

table, the short oblique, mid-shaft tibial fracture was performed. Through a small

medial approach, a partial transverse osteotomy (1/3 of the circumference) was carried

out and a custom designed four-point bending device subcutaneously inserted and fixed

with two mono-cortical Schanz’ screws. A pre-bending moment was applied by pulling

on the screws through a turnbuckle. Finally, the fracture was created by the impact load

of a slap hammer. The results showed a high consistency of tibial fractures (96% short

oblique fractures according to AO types A2 and A3) and a reproducible, moderate soft

tissue injury (Tscherne II). This trauma model, which reflects a rather low-velocity

injury, is limited by its moderate severity in terms of studying the influence of soft

tissue damage on fracture healing, as shown by Melnyk et al. (2008) 26

. Consequently,

the low severity of the injuries achieved with this model limits the conclusions, as such

low grade injuries usually recover quickly and, from the clinical perspective, those

injuries are not very challenging. In order to develop and study methods, implants and

surgical procedures that improve and optimise the treatment of complex bone fractures

with concurrent severe, soft tissue injury, a more realistic and severe trauma model for

large animals is required.

28

2.5 METHODS TO MONITORING SOFT TISSUE DAMAGE

In the previous chapter several animal models have been discussed on the creation

and the impact of soft tissue injuries. Those studies investigated structural, biochemical

and functional changes of the soft tissues, mainly the muscles, through the recovery

process. In-vivo monitoring methods focus on functional and metabolic measures, such

as the blood flow or the oxygen supply, pressures or the temperature within the

analysed tissues. Post-mortem analyses assess morphological characteristics at the

molecular, cellular and structural levels.

In small laboratory animals, the following methods are used to measure the

functional and micro-structural environment of intact and damaged soft tissues as well

as their longitudinal changes during the recovery period (in-vivo):

In-vivo monitoring

Laser Doppler Flowmetry (LDF) is a generally accepted and commonly used

method to determine tissue blood flow by measuring the movement of the erythrocytes.

Several groups 24; 26; 80; 92 investigated the tissue perfusion in rats and rabbits, and

presented good results with a sufficient reproducibility of the measurements as a

validation of blood flow. A second method to assess microvascular parameters of soft

tissues was implemented by Smith et al. (1993) 80, implanting a dorsal microcirculatory

chamber (DMC) in rats. With the use of video-monitoring, they measured

microvascular perfusion and venular and arteriolar diameters. In addition,

photomicrographs were taken to assess skeletal muscle microcirculation. This

observation chamber (DMC) was further developed to perform intravital fluorescent

microscopy in mice, rats or rabbits 79; 82; 83; 92. Those groups assessed different

monitoring protocols to measure the functional capillary density (FCD) of the blood

vessels, diameter of arterial, venous and lymphatic vessels, the velocity of the blood

flow and the extravasation index for determining the macromolecular leakage

(microvascular permeability). Sticking and slowly rolling leukocytes were also

measured 79; 82; 83. The third technique to measure the muscle blood flow was published

by Utvag et al. (2003) 25, injecting radioactive marked microspheres and countering the

flow rate through the tissues with a Packard Auto Gamma spectrometer. Of those three

described methods, the Laser Doppler Flowmetry is preferred, because of its non

29

invasive assessment, instead of the microsphere method or intravital microscopy, since

these are affected by flow changes due to the injected tracers and requirement for

surgical intervention, respectively. Local temperature within the soft tissues 79 and

intramuscular pressures 82

Post-mortem assessment

were monitored, too, with special measuring probes.

Histology is the method of choice for the post-mortem assessments of damaged soft

tissues. To determine the extent of the muscle injury in their trauma model, Bonnarens

et al. (1984) 109 carried out a descriptive, micro-pathological muscle histological

analysis in the very early stage (24 hours post-traumatic). Other groups, such as Hurme

et al. (1991) 78 performed muscle histology for ultrastructural and

immunohistochemical analyses, and Gierer et al. (2005) 83

In large laboratory animals, adequate monitoring and measuring of soft tissue injury

is still an outstanding question and remains difficult. In the clinic, soft tissue injuries

are assessed and regularly followed-up by clinical examinations to determine the extent

of the soft tissue damage, as well as to rule out the existence of a compartment

syndrome. A useful tool, especially in situations with high suspicion of the

development of a compartment syndrome, is the measurement of the compartment

pressure, which, in relation to the diastolic blood pressure, can give hints to the severity

of a soft tissue injury

performed histology and

immunohistochemistry for cellular assessments and immunoreactivity measurements.

108. Another method to evaluate the presence of a soft tissue injury

is the measurement of intramuscular oxygen partial pressure in the contused area 112; 113

Epari et al.(2008)

.

The partial oxygen pressure is expected to increase in low to moderate soft tissue

damage through the hyper-perfusion of the inflammatory reaction, while in severely

damaged regions, the partial oxygen pressure tends to drop because of the reduced

blood flow in ischemic tissue areas. Unfortunately, the results obtained with these

studies were relatively unspecific and the variability of the values was often high,

attributed to technical difficulties and problems of a steady position of the probes

during the period of measurements. 114 used a special, multi-parameter catheter (Neurovent PTO,

Raumedic AG, Germany) for the measurement of tissue pressure, oxygen tension and

temperature at the osteotomy gap of the tibia in a sheep study. They recorded the values

30

for the first ten days of the fracture healing process. Those probes are highly sensitive,

but the fibre-optic cable for transmitting the signal of oxygen tension and the

temperature tends to fail (break) in such a setup with live sheep.

Alternatively, for the purpose to detect and monitor the extent and the course of soft

tissue injury on a systemic basis, certain blood serum markers are used in clinical

practise, such as Creatine Kinase (CK) and Lactate Dehydrogenase (LDH). The main

markers as investigated by Strecker et al. (1999) 115 with significant changes after a soft

tissue injury were Serum Interleukin-6 and Serum Interleukin-8, CK and LDH. Also,

Oni et al. (1989) 116 and Tepic et al. (1997) 41

showed that CK and LDH serum

concentration levels correlate with the severity of soft tissue injury. Creatine Kinase is

an enzyme expressed by various tissues and cell types and catalyses the conversion of

creatine and consumes adenosine triphosphate (ATP) to create phosphocreatine and

adenosine diphosphate (ADP). Any damage to the tissue releases CK into the blood

stream, and therefore it is used as a serum marker of myocardial infarction (heart

attack), rhabdomyolysis as severe muscle breakdown, muscular dystrophy, and is also

elevated in acute renal failure. Lactate Dehydrogenase is an enzyme present in many

different tissues of the body and exists in five isoforms in the heart, the reticulo-

endothelial system, the lungs, the kidneys, in the liver and the striated muscle. It

catalyses the interconversion of pyruvate and lactate and converts pyruvate to lactic

acid if oxygen is absent or in short supply, and it performs the reverse reaction during

the cori cycle in the liver. Because of its general origin, released in the blood stream,

LDH is a serum marker of general tissue breakdown, and is not as specific as CK for

muscle breakdown. Elevated LDH levels indicate for example, haemolysis, tissue

turnover (general tissue breakdown), and pathologic conditions as cancer, meningitis,

encephalitis, acute pancreatitis and HIV can cause increased LDH levels.

2.6 METHODS TO ASSESS FRACTURE HEALING

Bone fracture healing is a highly complex process, which is still by far not

completely clear and scientifically understood. In general, a broken bone, with at least

two pieces, progresses through different stages of the recovering process and finally

ends with the remodelling into one fully functional integrity (see also Chapter 2.2.1).

31

This multi-factorial healing process can be separated in two entities: the biological and

the mechanical environment, which also depend on and interact with each other. To

capture the bone regeneration process, different methods have been developed to study

one or more of those aspects. In the early stage of the fracture healing process, the

monitoring starts with the characterisation of the fracture haematoma by molecular and

cellular activities, as well as the development of the blood supply. The callus formation,

especially the growth of the calcified areas, is determined with imaging tools, such as

conventional radiography or computer tomography to measure the morphology

parameters. On the other hand, mechanical tests are performed either in-vivo to

investigate functional properties, such as fracture gap movements or strains and load

changes related to implant characteristics, or post-mortem to determine the mechanical

competence of the bone at defined time points. Finally, different bone histology

techniques are available to further characterise the morphological properties of bone

and callus formation in details.

Small laboratory animals: With the conventional radiographic technique, for a long

time it was difficult to achieve reasonable results in examining small animal bones.

More recently, micro-radiographic investigations by Utvag et al. (2003) 25 and by

Holstein et al. (2009) 117, presented good results, measuring the cross-sectional area of

callus or the size and the density of the callus formation in rats and mice. The second

radiographic method, the micro-CT technique, reveals more detailed and more specific

information of the process of fracture healing and bone regeneration; parameters such

as the tissue mineral density, total callus volume, bone volume fraction of the callus

and even vasculature of the callus region, visualised by post-mortem injection of a

chromate-based contrast agent, were measured 117

Mechanical testing of long bones from small laboratory animals are challenging

because of the size of those bones, especially in mice. Nevertheless, several studies

described their results in assessing the mechanical properties of fractured bones in rats.

Most commonly three-point bending tests were used and analysed for maximal

structural stiffness (rigidity), maximal load at failure, fracture energy absorption and the

area moment of inertia

.

24-26; 93. A four-point bending apparatus was developed for the

testing of bones of mice and evaluated for bending stiffness, load at failure and energy

32

absorbed to failure 95. Bonnarens et al. (1984) 109 also tested rat bones for their torsional

strength and stiffness, angular deformation and energy absorption at failure. A third

method, involving axial loading, was also implemented with good results by Holstein et

al. (2009) 118

Bone histology was one of the routine methods to assess callus formation and bony

regeneration morphologically. The descriptive histology involved several staining

procedures to point out certain areas of interest, and immunohistochemical methods

were used to perform cellular and molecular analyses

.

117. On the other hand,

quantitative evaluation of the callus formation was widely recognised to measure

defined regions and their properties. The so called histomorphometry assessed in a

standardised manner: height of newly formed bone, height of fibrous cartilage, height

of fibrous tissue, length of newly formed bone, length of fibrous cartilage, length of

fibrous connective tissue and the periosteal diameter close to the fracture line 24.

Further densitometric properties were measured, such as total cortical bone mineral

content, total cortical bone volume, cortical bone mineral density and the area moment

of inertia 95

In large laboratory animals, the focus of the fracture healing monitoring has mostly

been on the development and progress of callus formation and the remodelling

processes. Similar to what has been reported in small laboratory animals, the three

options of radiography, mechanical testing and histology are commonly undertaken.

Conventional radiographs, nowadays processed in a digital manner, are widely used to

investigate many different parameters: periosteal callus area

.

106, cortical bone density 43, callus area at three regions of interest41, callus density measurements (expressed as a

percentage of intact collateral bones) 39; 105, area of periosteal callus and the average

area of callus 119, area of the projected callus formation at two defined regions of

interest 120, bridged cortices counted in two perpendicular planes and four bridged

cortices considered as healed 121; 122. Tepic et al. (1997) 41, Hente et al. (1999) 123 and

Dumont et al. (2008) 105 reported the use of microradiographic methods to characterise

the fracture healing progress in more details: quantitative and qualitative assessments of

callus formation at certain regions of interest in terms of fracture consolidation,

determining areas of implant loosening and possible spots of osteolysis.

33

Two different techniques to determine the mechanical integrity of the healing bone

have been performed by applying either torsional or bending forces. The protocols of

testing in torsional direction included torsional stiffness, ultimate torque at failure,

energies-to-rupture and the moment of inertia 106; 121; 122. The bending test protocols

were separated according to their differently applied forces: three-point or four-point

bending tests. A three-point bending apparatus was used by Claes et al.119 (1997), while

other groups 41; 43; 105; 123

In the field of histology, many different procedures and techniques have been

developed to gain more structural information about specific areas of the healing bones

at defined time points. The descriptive approach assessing histological slices ranges

from routine bone histology to special staining methods, as well as to investigations

with special light microscopes including fluorescence microscopy and electron

microscopy. Histomorphology can be used to assess the degree of vascularisation,

callus extension, callus resorption, fragment resorption

applied a four-point bending test concept to determine

mechanical properties, such as stiffness and strength, as well as the location of the re-

fracture.

41; 105, as well as screw

loosening regions, cortical microcracks, granulation tissue and resorption cavities

within the fracture area or adjacent to the implants 105. Cytological studies can indicate

cellular activities within specific regions of interest by electron microscopy 106.

Furthermore, with the use of a special light microscopy technique the degree of cortical

bone perfusion can be analysed 43. A more ‘dynamic picture’ about the process of callus

growth and the bone remodelling phases can also be achieved by serial in-vivo injection

of different substances, such as tetracyclines, Xylenol orange and Calcein green. With

the technique of fluorescence microscopy, labelled regions can be ‘highlighted’ and are

therefore accessible for evaluation 105; 106. For qualitative assessment of the callus

morphology, histomorphometry can establish total callus area, mineralised bone,

fibrous tissue and cartilage via measurement using standardised protocols 105; 121; 122

Further options of measuring the perfusion of the bone and the fracture region have

been reported using either intra-vital Laser Doppler Flowmetry

.

43 or vessel infiltration

(Hypaque and Prussian blue pigment) at the time of sacrifice to micro-angiographically

34

assess the peripheral blood supply and obtain an impression of the bone vascularisation 39; 106

.

CCHHAAPPTTEERR 33:: RREESSEEAARRCCHH DDEESSIIGGNN

3.1 GENERAL RESEARCH PLAN

The present study aims to develop and validate a new animal trauma model with

severe soft tissue damage and a complex fracture pattern, and then, in a second step,

use this model to investigate the effect of different surgical approaches on soft tissue

recovery and fracture healing outcome.

To address this clinically relevant research topic, a ‘realistic’ trauma model is

required, which should reflect a typical trauma case scenario, such as a pedestrian hit

by a car bumper. To meet these severe trauma criteria and convert them into a

reproducible, animal trauma model, the following trauma regime and treatment design

is proposed:

• Anatomical region:

The distal part of the femur (supra-condylar) and the thigh are ideal to study. This

region contains circumferential muscle coverage of the bone (femur) with perforating

vessels for the required blood supply. The distal metaphysis of the femur (supra-

condylar region) is a clinically recognised region for potentially complicated fracture

healing.

• Soft tissue injury:

A severe, soft tissue injury is aimed with a circumferential damage to the entire soft

tissues, critical for the development of a compartment syndrome (Tscherne III

according to the closed, soft tissue classification 2

• Fracture model:

).

A high-energy impact to a long bone often results in a complex fracture pattern. A

comminuted, multi-fragmentary fracture matches these requirements and is categorised

as an AO C-type fracture 3. This fracture configuration is defined by the proximal main

fragment, one or more intermediate cortical fragments and the distal main fragment;

most importantly, the main fragments completely lose direct contact with each other,

35

because of the intermediate fragments. This sort of fracture pattern causes a highly

unstable, skeletal condition.

• Therapeutic management plan:

According to the standard, clinical practice, such a severe injury to the distal region

of the femur/thigh is treated with a two-stage procedure: The initial management

involves a temporary stabilisation with an external fixator frame, and secondly, after

distinct recovery of the soft tissue injury (about five to ten days post injury), the

definitive fracture management is performed by stabilizing the fracture with an internal

fixator.

The following sub-chapters report the chronological steps of developments of the

trauma model and its detailed validation.

3.2 DEVELOPMENT OF TRAUMA MODEL

An ‘all-in-one’ trauma model with one single impact to create the soft tissue

damage and the fracture at the same instance would be preferable. The literature

review, along with the experiences of the author in developing a novel trauma model in

sheep resulted in the conclusion that such a single ‘perfect’ trauma model was not

achievable, because of technical and reproducibility reasons. The high impact required

to create a severe injury pattern would cause a complex fracture shape with multiple

smaller and bigger fragments as was co-incidentally shown during the first in-vivo

experiment testing the soft tissue trauma device (see also chapter 3.2.1, Figure 17).

These inconsistent fracture configurations would not meet the requirements of

reproducibility. Moreover, the different fracture patterns could not be stabilised with

one sole, mechanically consistent fixation construct. Therefore, the development of the

trauma model was performed in two separate interventions, and is presented in two

separated sections: firstly, the development and validation of the soft tissue trauma

model and secondly, the development and validation of the fracture creation model.

36

3.2.1 Development and validation of soft tissue trauma model

3.2.1.1 Requirements on the degree and extent of the soft tissue damage:

The aim was to produce a closed and severe soft tissue injury grade III according to

the Tscherne classification2

This defined high-grade injury translates into the ovine soft tissue trauma model as

outlined in the following description:

. The definition of a grade III soft tissue damage includes

extensive skin contusion, distinct damage of muscles and subcutaneous tissues

including degloving (décollement), high risk for the development of compartment

syndrome and possible vascular lesion (not mandatory). The impact which causes such

severe soft tissue damage is comparable to a direct hit to a pedestrian leg by a car

bumper with a speed of more than 30 km/h (high-energy injury pattern).

The trauma impact has to be created in a closed manner (without skin laceration or

open wound), affecting the entire circumference of the soft tissues around the distal part

of thigh and extending over a length of approximately 5 cm. The damage has to be

reproducible, at any time, and technically requiring a simple mechanism apparatus, so

that the impact is easily achieved.

3.2.1.2 Development of the device:

Following discussion with the international collaborators from the AO Research

and Development Institute, Davos, Switzerland, several possibilities of how to achieve

this defined soft tissue damage were proposed (Appendix E). Three general impact

mechanisms were outlined:

• guillotine apparatus: simple mechanism with vertically dropping weight

o advantages: simple construction (design), no base (trestle) required, possible

to be placed on top of operating theatre table, height and weights of the free

falling impactor easy adjustable to change impact energy, no space to the

sides required (as pendulum), reproducible mechanism, reasonable ‘low’

material and production costs

37

o disadvantages: vertical space required (high ceiling), free fall to be

controlled (friction during ‘sliding’ mechanism)

• pendulum device: simple mechanism with turning pendulum impactor

o advantages: simple mechanism with adjustable height, pendulum length

(lever arm) and impact weights, side impact on very defined spot, only

friction spot at turning axis, reproducible mechanism, reasonable ‘low’

material and production costs

o drawbacks: apparatus requires strong base (trestle) to hold against impact

energy, side space necessary (pendulum)

• pneumatic cylinder:

o advantages: ‘small’ technical device, team experience from another study

used such a pneumatic driven cylinder108

o disadvantages: technically complex mechanism/device with risk for

malfunction or failure, complicated design and production with high

expenditure

(ISO 6431, Sempress,

Mississauga, Canada)

It was decided that the two more simple constructs (guillotine and pendulum

devices) were most feasible, and due to technical construction reasons, the pendulum

device was favoured. Peter Toggwiler, supervised by Romano Matthys, drew the

principle outfit of the pendulum device (Figure 5). After final refinements of the design,

the soft tissue trauma device was produced and assembled (Figure 6) in Switzerland for

preliminary tests, and then delivered to the animal laboratory of the Biological

Research Facility on The Prince Charles Hospital campus.

38

Figure 5: First sketch (drawing) of the soft tissue trauma device.

Figure 6: Picture of the newly developed soft tissue trauma device.

Trestle base

Support frame

Head support

Table

Impactor

Legs (6)

Beams (3)

Pendulum

Release mechanism

39

3.2.1.3 Description of the soft tissue trauma (STT) device (Figure 6)

The soft tissue trauma device consists of two parts: the base for positioning and

holding of the sheep, and the support frame with the attached pendulum to hit the right-

sided thigh. The sheep is laid on the flat table on top of the base in prone position,

while the legs hang downwards on each side of the table. To maintain a strong stand,

six legs are built onto the table and at the bottom of these legs, three flat beams are

attached in perpendicular direction to the table, to secure the trestle from falling over

during the blow. A head support is also fixed on the front end of the table and enables a

safe positioning of the head during the general anaesthesia. At the rear end of the

trestle, the support frame with the pendulum is fixed to the right-sided middle and rear

legs. The two curved beams of the support frame are connected on the top with two

metal rods, which sets the turning point vertically above the table. The pendulum is

suspended on the upper metal rod and the release mechanism of the pendulum is

constructed with the lower metal rod. The pendulum consists of the pendulum arm and

the impact device, which is adjustable in height and the number of weights it can take

(Figure 8). The impactor has got a groove on the inner side (Figure 7), which protects

the bone (femur) from being crushed during the impact. The impact counterpart (Figure

7) is fixed between the curved beams at the right side of the back of the base.

After the sheep is safely and properly positioned on the trestle, the right hind limb

(5 cm above knee joint line) is placed between the two grooves of the impactor and its

counterpart. Then, the pendulum is raised to the top and locked in. When the setup is

complete, the pendulum is released and it hits the sheep’s leg at the pre-defined position

and location.

Technical data of the soft tissue trauma device

Dimensions of rod (lever arm): 2 x 2 x 122 cm

:

Distance of centre of impactor from axis: 104 cm

Weight of impactor adapter minus impactor: 1.95 kg

Dimensions of impactor 4 x 20 x 2.5cm

Impactor weight 0.75 kg

Material: Aluminium

40

Figure 7: Top view from the impactor Figure 8: Impactor from the outside. and its counterpart.

3.2.1.4 Soft tissue trauma device: tests and validation

After the first trials of the newly developed soft tissue trauma device in the

workshop of the AO Development Institute in Davos (Switzerland) with foam material,

the STT device was moved to Brisbane and re-built. Further in vitro tests to measure

and validate the impact speed with a high-speed camera were carried out. Then,

cadaver legs were used to check and adjust the position of the impactor and its

counterpart. Finally, in vivo tests were performed with five anaesthetised sheep.

Following these experiments, further evaluation and validation of the degree and

extension of the soft tissue damage was done.

In clinical practice, usually a soft tissue injury is extensively characterised using

imaging methods such as MRI and CT scan. Unfortunately, those techniques MRI and

CT scan were not available for in vivo testing. Instead, the extent of the damage was

quantified by macroscopic dissection performed by experienced surgeons and post-

mortem CT scan.

High-speed camera trials and results

To validate the reproducibility of the STT device in terms, the exact speed of the

impactor at the time of the hit to the leg was determined with a high-speed camera (see

Figure 9). Dr Roland Steck installed the high-speed camera device and we tested the

41

device once with a foam noodle and also by placing the cadaver leg between the

impactor and its counterpart.

Methodology

: A ruler was fixed to the trestle to determine the position of the

impactor at each time point (movie frame) (Figure 9). To dampen the impact a foam

cushion (swim noodle) or a cadaver leg was attached to the impactor counterpart

(Figure 10). With the high-speed camera (MotionXPro X3, Redlake MASD LLT,

Tucson, AZ, USA) we filmed the final course of the impactor and its deceleration at

1000 frames per second. The movie was recorded and saved on a laptop with the x-

vision software (IDT, Tallahassee, FL, USA; Figure 11). The frame-by-frame analysis

(Dr Roland Steck) was performed to determine the impactor movement (distance/time)

and the final speed defined with plotting of displacement versus time curves (Microsoft

Excel, Redmond, WA, USA, Figure 12 and 13).

Figure 9: Picturial overview of Figure 10: Ruler fixed perpendicular to the high-speed camera setup. the impactor and counterpart. The foam noodle is positioned to dampen the hit.

42

Figure 11: Illustration of frame-by-frame evaluation with x-vision software.

260

265

270

275

280

285

290

295

300

305

0.115 0.116 0.117 0.118 0.119 0.12 0.121 0.122 0.123 0.124 0.125

time [s]

dist

ance

[mm

y = -7400x + 1157.2R2 = 0.9978

282

284

286

288

290

292

294

296

298

300

0.1155 0.116 0.1165 0.117 0.1175 0.118 0.1185

time [s]

dist

ance

[mm

Figure 12: Displacement vs time curve 1: Figure 13: Displacement vs time curve 2: Linear portion of the curve Determination of the velocity representing the velocity of the impactor. Slope of the before the impact. linear portion = Impact velocity

Results: Two trials with the high-speed films were evaluated. The first impact

velocity was 7.40 m/s, the second impact velocity was 7.40 m/s. Therefore, the average

impact velocity was consistent at 7.4 m/s (26.6 km/h).

Conclusion: The validation of the pendulum device in terms of the reliability of the

impact speed shows that both trials with high-speed camera measurements present

exactly the same values of 7.4 m/s impact speed. Hence, with the given impactor

weight and the constant impact speed, the energy of the impact is consistent and very

reliable.

43

Report of in vivo tests

Following the foam noodle and cadaver leg tests, the new pendulum device was

tested and validated in vivo.

Four sheep (Merino ewes, approximately seven years old, slightly underweight

(<30 kg), but in good general health condition) were anaesthetised and later sacrificed

for other scientific purposes. At the end of the general anaesthesia and with an adequate

analgesic dose, the sheep were positioned on the pendulum trestle with the distal thigh

(5 cm proximally from the knee joint) within the groove of the impact counterpart and

the soft tissue trauma is induced. The subsequent hit was carried out with the sheep

turned around on the device and re-positioned to fit the contra-lateral thigh in the

counterpart groove. In order to create and detect several degrees of the severity of the

soft tissue injury, different impactor weights were chosen and monitored. The following

are the denominations of how the experiments were performed with the additional

weights: Sheep 1 - No additional weight;

Sheep 2 – 2 kg additional weight (refer to Figure 14),

Sheep 3 - 0.5 kg additional weight

Sheep 4 - No additional weight.

The second traumatic blow to the leg was timed to be 15 minutes before sacrifice,

so that the there was sufficient interval to determine the ‘full’ extension of the soft

tissue damage, as well as the borders between the damaged and the undamaged tissues.

Immediately after the sacrifice, the regions of interest (distal part of the thigh on both

sides) were dissected by scalpel to evaluate the macroscopic extend and depth of the

emerged damage. Sheep 3 alone was managed differently: After euthanasia, both hind

limbs were amputated (hip exarticulation) and a native post-mortem CT scan (Philips,

Brilliance 64, Netherlands) was performed in the Department of Radiology at The

Prince Charles Hospital. Then, the legs were transported back to the animal laboratory

and macroscopically dissected, in the same manner as the other six legs.

The above-mentioned test series helped us to validate the exact position of the lever

arm distance as well as the best angle of the impactor and its counterpart (see Technical

data in Chapter 3.2.2 and Figure 15).

44

Figure 14: Outer side of impactor Figure 15: Final position of the counter- showing 2 kg additional weight. part block for blow against the right thigh.

All eight trial blows were successfully performed (Figure 16). During the trial

blows, the sheep did not show any sign of pain or discomfort. The first hit included an

‘accidental’ femur fracture (Figure 17), which occurred, because of the mal-positioning

of the sheep’s leg between the grooves of the impact blocks.

45

Figure 16: (Left) In vivo trial of soft tissue trauma device: The pendulum is moving towards the point of impact on the distal thigh. The ‚marked cross’ on the right picture shows the point of contact of the pendulum with the sheep.

Figure 17: Surgical dissection to visualise the damage incurred. This figure also shows incidental femur shaft fracture caused due to the mal-positioning of the sheep’s leg.

46

Figure 18: The team involved in the first in vivo trial of the soft tissue trauma device in the animal operating theatre of The Prince Charles Hospital, Brisbane.

Dissection Results

The soft tissue trauma was in vivo tested on five sheep, under general anaesthesia

and 15 minutes later the sheep were sacrificed. Straight after their sacrifice a careful

dissection in layers, from skin to bone, was carried out. The macroscopic assessment

included the detailed description of the skin, subcutaneous and muscle conditions such

as bruises, contusion marks, signs of ruptures and haematomas as well as the conditions

of the nerve and vascular structures at the postero-medial localisation of the distal thigh

(refer to Appendix F). This assessment was performed by the candidate and confirmed

by Dr Kathleen Wilson and Dr Roland Steck.

47

Summary of the assessment of macroscopic dissections:

• The soft tissue trauma device was highly reproducible in terms of its impact speed

and impact energy.

• The soft tissue trauma model was able to produce moderate (Figure 19) to very

severe soft tissue damage (Figure 20). In one particular scenario, even a ‘crush’

fracture was produced (Figure 17).

• The impact weight and the severity of the soft tissue damage correlated with each

other; 2 kg additional impact weight caused significantly more destructive damage

(Figure 20) compared to the trials without any additional weight to the pendulum

(Figure 19).

• The slight modification to the counterpart block to the pendulum impactor during

the last trial protected the postero-medially located vital structures, such as the

sciatic nerve and the femoral vessels.

• The leg preparation with circumferential shearing and shaving at the impact

location, as well as a relaxed muscle tone by the chosen leg position was important

considering the reproducibility of the soft tissue injury.

Figure 19: This macroscopic dissection of the left picture shows a moderate muscle contusion to the lateral vastus muscle and the right picture shows an intact sciatic nerve with superficial perineural haematomas. Both the pictures represent the damage resulted from a pendulum hit without any additional weights.

48

Figure 20: These pictures represent the extensive damage after the pendulum hit with 2 kg additional weights. In the left picture, the lateral vastus muscle is severely damaged, partially disruptured, the subcutanous layers degloved with small bruises and haematomas. The right picture shows the sciatic nerve substantially contused with epineural haematomas, but longitudinally intact. The deep soft tissues are bruised with underlining haematomas and edematous.

Evaluation of computer tomography:

For further evaluation and validation of the soft tissue trauma device and the

damage it inflicts, a post-mortem CT scan was performed 15 minutes after the soft

tissue injury. For this purpose, one of the sheep (no 336) was euthanized and its right

leg was amputated at the level of the hip joint. Then, the amputation site was wrapped

in sterile, moist gauzes and the entire leg was transported to the Radiology Department

for imaging with the high-resolution CT scanner (Philips, Brilliance 64, Netherlands:

slice thickness 0.67 mm, pixel size 0.625 mm, 120 kV, 250 mAs, rotation time 1 s).

After the scan, the leg was brought back to the animal laboratory for macroscopic

dissection analysis.

Results: The native scan without intravenous contrast of the entire right leg is

reconstructed in all axial, sagittal and frontal planes. The native scan in sagittal plane is

shown in Figure 21. The areas of damaged muscles are visible as slightly darker tissue

spots within the homogenous normal muscle tissues. The muscles are all in anatomical

place and there is no evident disruption in their positioning. The anterior part of the

rectus femoris is severely bruised and some areas contain intramuscular haematomas.

49

The exact quantification of the extent of the damage is difficult because of the limited

resolution of this non-contrast scan.

Figure 21: High-resolution CT scan of a right sheep leg after soft tissue trauma trial showing sagittal plane of the entire leg (Sheep 336).

Figure 22: CT scan with mid-sagittal reconstruction of Figure 23: Axial view of the right thigh. Yellow spots show: intramuscular bruises the distal thigh region. and haematomas.

Although the images could not quantitatively describe the damage inflicted, the

images acquired by post-mortem CT scan without intravenous contrast qualitatively

50

indicates significant muscle damage. This finding corresponds to the macroscopic

dissection findings that shows severe amount of bruising in all muscles around the

femur including some intramuscular haematomas. Further analysis of the exact extent

of the damage is difficult because of the non-contrast scan that does not clearly

distinguish between the normal and damaged tissues. Fifteen minutes time interval

between the soft tissue trauma and euthanasia of the sheep was sufficient to show the

muscle damage, but for a more precise evaluation of the extent of the soft tissue injury,

a longer period of time would be necessary.

Conclusions of the soft tissue trauma trials:

This newly developed soft tissue trauma device has demonstrated its ability to

create moderate to very severe soft tissue injury to a well-defined area of the sheep’s

leg. The severity of the damage depends on the amount of additional weights loaded on

the pendulum impactor. The clinically comparable Tscherne III soft tissue damage 2

This successful setup of the soft tissue trauma device will be applied in the pilot

study on two animals as part of the combined trauma model. This model will be

employed to study the effect of the surgical approaches on soft tissue and fracture

healing. The soft tissue damage will be monitored and re-validated prior to the main

series of the animal experiments.

was obtained by the impactor weight alone. The results show a significant soft tissue

bruise of all layers including subcutaneous and intramuscular haematomas as well as

disruption of the shear or degloving injury mechanisms. The vital anatomical structures

including the sciatic nerve and femoral vessels are still preserved.

3.2.2 Development and validation of fracture creation model

3.2.2.1 Requirements of the fracture model

In real clinical circumstances, the previously described soft tissue injury model will

ideally inflict a comminuted fracture as a result of the high-energy trauma that is

involved. Therefore, a fracture model is required to create a multi-fragmentary fracture.

This fracture pattern will include two main fragments, proximal and distal, and at least

51

one intermediate fragment. The intermediate fragment splits the two main fragments

without any direct contact with either of the main fragments. The fracture created will

correspond to an AO C-type fracture as classified by the Association for the Study of

Internal Fixation (AO/ASIF). The fracture will be around 3 cm in length and will be

located at the distal end of the femur shaft, in the transitional area of the metaphyseal to

the diaphyseal region and 5 cm proximal to the condylar line (Figure 24). All these

fracture properties fulfil the requirements of a defined, complex fracture and ensure an

accurate and consistent fixation construct. The following are the requirements to satisfy

such a fracture type:

• a reproducible fracture pattern in terms of fracture length and numbers of

fragments created

• a less invasive and constant approach to create the fracture, and minimize

iatrogenic soft tissue as well as bone damage

• simple tools applied in a well-defined sequence to cause breakage of the

bone

Figure 24: Multi-fragmentary distal femur fracture – a butterfly type, AO C-type fracture 3

of a cadaver sheep bone.

3.2.2.2 Development of the fracture model

The required fracture pattern emulating an AO C-type fracture was constructed in

collaboration with Romano Matthys (AO Development and Research Institute, Davos,

Switzerland) (See Appendix E).

52

The fracture pattern constituted:

• two transverse fracture lines (distal and proximal)

• one bicortical, longitudinal fracture line to separate the middle fragment in two

small intermediate fragments (Figure 24)

The resulting “H” shaped fracture pattern evidently meets the criteria of an AO C-

type fracture 3

The fracture pattern was created via the following steps:

.

• Step I

•

: Weakening of the cortical bone with total or partial osteotomies and by

drilling holes into the bone.

Step II

o Mediating direct impact to fracture area by either

: Initiating the final fracture by

A blunt blow with a heavy weight – hammer, pendulum, etc.,

A sharp hit with a chisel or

With an expanding device inserted in drill holes or osteotomy

gaps

o Mediating indirect impact to leg or bone by:

Applying external force either manually or with a torque

machine

The analysis of the merits and demerits of the fracture model enabled us to infer the

following:

1) The use of osteotomies was not preferred, because of its potential to cause

‘unnatural’ fracture gaps with smooth fragment ends and heat necrosis.

2) Creating the fracture with a ‘blunt blow’ method was discarded due to its inability to

create a reproducible fracture pattern (Figure 17).

3) Any technique requiring a larger surgical approach or a technically complex

apparatus was also abandoned.

Out of the remaining possibilities the challenge was to find the optimal combination

and sequence of the following tools (instruments): oscillating saw, drill, blade bar and

chisel. Three different blade bars with two, three and four blades (‘wings’, Figure 25),

53

were designed and produced in the AO Development Institute, Davos (Switzerland) and

sent to Brisbane. The other instruments were delivered from Synthes, Australia and

Switzerland.

Figure 25: Custom made blade bars with two, three and four blades.

To start with and measure of the forces to break a sheep femur in the shaft region,

three femur bones (supplied from the local butcher, Westfield Mall, Chermside) were

tested with the Instron machine, applying a three-point bending moment. The bones

fractured at an average force of 3493±629 N (SD) as ultimate bending failure. Figures

26, 27 and 28 show the bending test, the degenerated bending to failure curve and the

resulted fracture respectively.

Figure 26: Right femur bone tested with the Instron machine for three-point bending properties.

54

Three point bending test of sheep femur

0

500

1000

1500

2000

2500

3000

3500

0 1 2 3 4 5 6 7distance (mm)

Forc

e (N

)

Figure 27: Test curve showing breakage of an ovine femur on application of three-point bending forces by the Instron machine (Figure 26).

Figure 28: Fractured femur resulted after the application of three-point bending forces. The image shows a random fracture configuration with a posterior intermediate split fragment.

All the in vitro fracture model tests were carried out with cadaver femurs of sheep

supplied from a local butcher (Westfield Mall, Chermside) and tested on the vice of the

biological bench in the Biomechanical Engineering laboratory, Gardens Point,

Queensland University of Technology.

As a part of the validation process, an ‘H’ fracture was created on several femur

bones using different numbers, sizes and distribution of drill holes. The femur was

fractured by turning the proximal and distal external fixator frames towards each other

(Figure 29 left). Examination of the fractured femur showed that the fracture lines were

randomly distributed, sometimes not even through the weakening drill holes (Figure 29

right).

55

Figure 29 (left): Cadaver femur bone with drill holes in H shape. Two external fixator frames, proximal and distal to the fracture, are inserted for fracture initiation. Figure 29 (right): Fractured femur with random fracture lines. There is no evident fracture seen through some of the drill holes.

A subsequent optimisation included the creation of two transverse fracture planes

aimed to crack the thick and strong anterior cortex without any longitudinal splits. In

order to achieve that, it was decided to perform two partial, anterior osteotomies, at the

expense of a small surgical approach. Further tests on many cadaver bones to refine the

model were carried out, including implementation of chisel hits to initiate the fracture

planes more precisely. Hence, the fracture creation model was further optimised and the

following sequence of fracture creation pattern was tested on 16 femur bones (Figure

30 and Appendix G):

• Partial anterior osteotomy was performed (1/5 of circumference) 50 mm

proximal from knee joint line with a sagittal saw. The sagittal saw blade used

was narrow and thin (Stryker, 2108-150, USA) (Figure 30).

• Partial anterior osteotomy was performed (1/5 of circumference) 80 mm

proximal from knee joint line with a sagittal saw. The same blade was used as

before.

• Two holes of 2.5 mm were drilled through each of the anterior osteotomies.

Four other holes of 2.5 mm were drilled in the remote cortex in posterior and

postero-lateral directions. The drill was purchased from Synthes, 310.240,

Switzerland (Figure 30).

56

• One hole of 3.5 mm was bicortically drilled in antero-posterior direction 65 mm

proximal of the knee joint line with the 3.5 mm drill bit from Synthes, 310.360,

Switzerland (Figure 30).

• Two, unicortical holes of 3.5 mm were drilled in the lateral cortex 50 and 80

mm proximal to the knee joint line.

• A 2.5 mm drill bit was inserted in each of the lateral 3.5 mm drill holes. Then,

the remote, medial cortex was perforated with 2.5 mm drill holes in two

different directions (medial and postero-medial).

• Blade bars with two sharpened wings were inserted bicortically into the 3.5 mm

hole in antero-posterior direction with the support of a slotted hammer. Slotted

hammer was purchased from Synthes, 332.200, Switzerland (Figure 30).

• Chisels of 8mm width were inserted into the two anterior osteotomies with the

help of the slotted hammer used previously. This initiated a transverse fracture

that manifested as a crack in the remote cortex (transverse fracture initiation).

Chisels were flat and straight and were purchased from Synthes, 397.970,

Switzerland.

• The longitudinal fracture line was created with the rotation and removal of the

blade bar.

Figure 30: All the instruments used on the fracture model. From left, saw blade, drill bit 3.5 mm, drill bit 2.5 mm, blade bar with two wings and chisel respectively.

Results: As mentioned before, the fracture creation model was tested on the sixteen

distal femur bones of sheep. The average bone length was 18.2 cm. The proportion of

the partial osteotomies to the entire circumference was 19% for the proximal osteotomy

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and 20% for the distal osteotomy. The longitudinal fracture initiation with the blade bar

and transverse fracture initiation with the two chisels was incomplete in three cases. All

fractures were C-type fractures according to the AO classification. Except for one case

of three-fragment fracture, in all the other fifteen cases, the fractures consisted of four

main fragments (Figure 31). The measured maximal fracture length was 4.3 cm in

average. In four cases (25%) the fractures were more than 5.0 cm long due to a

longitudinal split at the proximal lateral side (Figure 31).

Figure 31: C-type fracture with long lateral fragment of 66 mm.

Conclusion

: This newly applied fracture model with partial anterior osteotomies

and several drill holes creates reproducible C-type fractures. The longitudinal splitting

that produced longer intermediate fracture fragments in 25% of all cases is not ideal,

and would have caused inconsistency for fracture fixation (with the same plate

construct), for mechanical testing and histological analysis. Therefore, two steps of the

fracture sequence had to be adjusted: Firstly, the partial osteotomies will be slightly

deeper, especially on the lateral proximal aspect to avoid the longitudinal fracture

splitting. Secondly, to initiate the longitudinal fracture line with the blade bar, the

insertion will be performed only unicortical. Both the above-mentioned changes should

ensure that the fracture length will not be longer than 4.0 cm in further experiments.

Second Test Series:

These minor changes of the previous fracture series were implemented and the

second in vitro series were performed with 20 bones.

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Results

: The average bone length of 20 femurs was 17.9 cm. The osteotomies were

slightly deeper with proximally 22.6% and distally 22.9% of the whole circumference

(Figures 32 and 33). The fracture initiation was constantly done by unicortical blade bar

insertion and chisel hits. In all 20 cases, the fracture configuration resulted in an AO C-

type butterfly fracture with four main fragments (Figures 34 and 35). In one case, a

small fifth fragment was produced. The maximal fracture length averaged at 3.5 cm. In

two cases, a short proximal lateral split towards the middle shaft region was seen. In

five cases, the longitudinal fracture line was created by bicortical blade bar hits without

any complications.

Figure 32: Proportion of partial proximal Figure 33: Proportion of partial distal osteotomy to entire femur circumference. osteotomy to entire femur circumference.

Figure 34: Fracture configuration with intact Figure 35: Fracture pattern (periosteum periosteum and fragments in place. removed and fragments laid to sides).

Proximal Osteotomy Femur

22.6%

77.4%

Distal Osteotomy Femur

22.9%

77.1%

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Conclusion

: The outcome of this second series clearly shows that the developed

fracture model creates highly reproducible fractures of consistent type and length. The

technique employed is reliable and easy to perform without complications. Therefore,

this combination of fracture model with two partial anterior osteotomies, antero-

posterior and latero-medial drill holes, unicortical blade bar insertion and chisel hit

initiation, will be continued to be employed for further validation.

Cadaver leg tests

To further validate the fracture model, in vitro tests were carried out with the entire

sheep’s leg instead of just the sheep’s femur bone. This validation process using a

standard lateral approach would test the fracture model’s performance and its reliability

through the soft tissues. The entire fracture model was performed on six cadaver hind

limbs of Merino ewe’s sheep (Figure 36).

Figure 36: Ovine cadaver leg fixed in the vice of the bench: After the fracture model was performed, the outcome was checked by a standard lateral approach to the femur shaft.

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In all of those six cadaver leg experiments, the fracture model was successfully

carried out through an antero-lateral, trans-muscular approach with a skin incision of 3

cm (Figure 37 left). No complications occurred and the fracture configuration was

consistent (AO type C3) with two intermediate fragments (Figure 37 right) and fracture

length shorter than 4 cm. To minimise tension forces during the procedure, an antero-

lateral external fixator frame was inserted prior to the testing of the fracture model

(Figure 38).

Figure 37 (left): 2.5 cm skin incision required to perform the fracture model. Figure 37 (right): Standard lateral surgical approach to visualise and evaluate the fracture outcome.

Figure 38: Picture with external fixator frame in situ and the fracture model performed by blade bar and chisel insertion through 3 cm skin incision.

Conclusion: The newly developed fracture model was successfully validated to

create a multi-fragmentary fracture of the distal femur in sheep. This model generated

fracture patterns of consistent and reproducible configurations and lengths. The final

sequence of the fracture model (Figure 39) consists of two partial anterior osteotomies,

antero-posterior and latero-medial drill holes. Also, the longitudinal split was initiated

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between the two osteotomies using a sharpened blade bar inserted unicortically into the

anterior middle drill hole. To generate the ultimate butterfly fracture, slightly oblique

fracture lines were created from transverse osteotomies using two chisel hits. This

sequence is easy and reproducible, even through a small (< 3 cm) skin incision and a

trans-muscular approach to the antero-lateral distal aspect of the femur shaft.

In all the in vitro trials, ovine femur bones were used to test the fracture model and

the outcome was very successful. Further experiments required the testing of the

combination of the fracture model and the two step fixation method (see Chapter 3.3.1).

Female sheep bones would not be ideal for these experiments due to their thin cortical

bone quality and hence, the probability of further bone splitting it presents. Therefore,

male sheep (Merino wethers) with stronger and slightly larger bone properties were

chosen for future experiments.

Figure 39: Fracture creation sequence: 1) Two partial transverse anterior osteotomies performed at 30 mm apart from each other. 2) In between the osteotomies, an antero-posterior (a-p), bicortical, 3.5 mm drill hole was placed. 3) Through the anterior osteotomies, four bicortical, 2.5 mm, oblique holes were drilled at 30 degrees to each other. 4) In the latero-medial direction: four bicortical, 2.5mm diameter holes were

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drilled at 30 degrees to each other. 5) To split the middle segment longitudinally into two fragments, a sharpened blade bar was inserted into the a-p drill hole by soft hits with a hammer. 6) Finally, two chisels were used to initiate the two transverse fractures by a strong hit with the hammer.

3.2.3 Validation of combined trauma model

The soft tissue trauma model as well as the fracture creation model were tested and

validated as separate applications so far. To perform both models as an ‘all-in-one

impact’ procedure would reflect the real-life circumstance better. Such an experimental

scenario is unrealistic in terms of reproducibility and reliability, mainly because of the

fracture configuration (as discussed in previous chapters). Therefore, the combined

trauma model was decided to be setup as a two step concept: firstly, the production of

the soft tissue damage on the pendulum trestle, and secondly, the creation of the desired

fracture on the operating theatre table. To assess and validate the entire course of the

two steps p, an in vivo setup was required. A female Merino sheep, 8 years of age and

weighing 30Kgs was sacrificed for other research purposes. Before euthanasia and

under general anaesthesia, the combined trauma model with two steps was tried out.

The soft tissue trauma was produced at the distal thigh of the right hind limb without

any additional impactor weight. This caused moderate to severe soft tissue damage as

assessed by macroscopic dissection. Immediately after, the fracture model was

performed at the ‘pre-injured’ right leg. The fracture model sequence resulted in an AO

C-type fracture of 3.8 cm with four main fragments and one smaller intermediate

cortical piece. After the combined trauma model was successfully completed, the

fracture fixation model with an external fixator frame was tested (see chapter 3.3.1 and

Figure 44). Although a longitudinal split occurred in the femur shaft during the

insertion of the proximal Schanz’ screws, the implant was positioned successfully. In

the end, a stable fixation construct was created over the distal femur fracture. To

prevent the occurrence of longitudinal splits in future experiments, the external fixator

pins have to be inserted prior to the fracture creation and the Schanz’ screw should be

positioned as far away from the fracture region as possible. In addition, stronger bones

with thicker cortical properties such as those of male sheep (Merino wethers) will be

employed.

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Conclusion of the combined trauma model:

This newly developed trauma model represents the first combined severe trauma

model in large animals. Because of its safe and reliable performance with consistent

soft tissue injury and fracture configuration, this trauma model is considered as a

valuable tool for experimental, in vivo studies.

3.2.4 Development and validation of ‘Supporting Trolley System’

Outlining the problem

During the early stage of recovery from anaesthesia and injury, the animals require

clinical observation and support for their impaired leg. Research shows, that other

animal laboratories in Australia (Adelaide and Sydney), Europe (Davos, Switzerland

and Berlin, Germany) and the United States (Cleveland) use supporting sling systems

or similar adapted devices to aid in recovery. These devices have been used for several

years yielding optimal recovery outcomes, low rates of complications and reduced loss

of animals. The main goal of these devices is to protect the sheep and the operated leg

from strain that is caused during lying down and getting up. These movements usually

lead to high shear forces at the fracture site and potentially could cause implant failure.

In our experimental setup, these devices would also stabilise the external fixator during

the first five days and also aid to regularly monitor the soft tissue conditions. If not for

the devices, the sheep would have to be caught at least twice a day for regular

monitoring. This poses a high risk of additional injuries, dislocation or breakage of the

soft tissue measurement probes.

Dr Roland Steck designed a new Supporting Trolley System after collating various

possibilities with the currently available animal supporting systems (see Figure 40).

Four supporting trolley system was constructed at the mechanical workshop in O block,

Gardens Point, Queensland University of Technology.

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Figure 40: Supporting Trolley System with the first sheep in trial based at the animal housing on The Prince Charles Hospital campus.

Description of the Supporting Trolley System

The Supporting Trolley System consists of a stainless steel trolley frame supported

by four wheels capable of 360 degrees of rotation. The trolley frame is provided with

two hinges at the front and rear end. These hinges allow the trolley frame to be folded

inwards or outwards and hence, help in the stability as well as transport of the trolley.

At the top of the metal frame six ring screws are fixed for hanging up six chains. At the

bottom end, a shade cloth is suspended through these six chains (Figure 41). The shade

cloth were designed by the candidate and produced by Advanced Shade Systems,

Capalaba, Australia. The shade cloth bears five holes, four for the legs and the middle

one for excretion purposes. The length of the chains can be adjusted to position the

animal depending on its size and shape. The sheep in the trolley would be able to move

and walk around, if the wheels are unlocked.

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Figure 41: Shade cloth with five holes.

Sheep trials

The trolleys were transported to the animal facility of The Prince Charles Hospital.

The competency of the complete trolley frame was tested by placing a healthy sheep

overnight under the supervision of Dr Kathy Wilson, Michael Lindeberg and William

Sommers. The behaviour of the sheep was recorded to be normal with no evident

adverse event. For further validation, a second trial with another healthy sheep was

carried out for five days.

Results: The sheep showed normal behaviour with regular food and water

consumption during the entire experimental duration. The daily clinical assessments of

the general condition as well as the state of the skin and wool around the legs

(especially shoulders and thighs) showed a completely healthy animal without any

signs of pressure or abrasions. A weight loss of nearly 600 g was observed, which was

within the expected range, due to the change of environment with a certain amount of

minor stress.

Conclusion

For future experiments, the two pilot sheep will stay for one night in the trolleys

prior to the experiment. This will allow the sheep to get used to the trolleys and their

environment. After the pilot test experiments, the trolley trial will be re-assessed for its

use and efficiency over the main series of experiments. Any necessary refinements or

changes will be applied accordingly.

: This newly designed ‘Supporting Trolley System’ is well tolerated and

efficient to keep the sheep for at least five to seven days regularly monitored, supported

and protected.

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In general, sheep are shy animals and they normally live in groups. Therefore, we

will keep at least two sheep together in one cage, but only one of them will be

supported in the trolley (Figure 42).

Figure 42: Trolley trial with another ‘free’ sheep in the same cage.

3.3 STUDY DESIGN

The newly developed and validated combined trauma model with the established

soft tissue injury and the multi-fragmentary fracture will now be applied to study the

soft tissue recovery and fracture healing process within a specific area of clinical and

experimental interest. This study will focus on the differences in surgical approach

techniques, and whether the operative procedures influence the healing outcome. To

assess and compare the different surgical approaches such as the minimally invasive

technique and standard open approach, a realistic fracture fixation model is required.

3.3.1 Establishment of fracture fixation model

This trauma model that simulates a high-velocity injury is capable of causing a

significant damage distal femur of the animal leg. In a clinical situation, the treatment

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regime for equivalent injury to the leg is commonly a two stage procedure: an external

fixator is temporarily applied at the initial acute phase. After a certain period of time,

the definitive fixation procedure is performed by replacing the external fixator with an

internal fixation device to aid the soft tissue recovery. In the case of distal femur

damage, often a plate-screw construct is chosen as definitive fixation method. This

treatment strategy with proven successful clinical outcome will be modified and applied

to the sheep model.

To start with, the right pin and frame configuration was developed and established

to secure a successful early fracture stabilisation. The anatomy of the ovine femur was

studied and landmarks such as the condylar block, femoro-patellar notch and the

concave ‘groove’ between the lateral condyle and the anterior part of the femoro-

patellar notch were defined. From the mechanical point of view, two 4.5mm Schanz’

screws were fixed in each of the two main fragments, mid-proximal shaft and condylar

block. The pins were inserted in latero-medial direction and a unilateral external fixator

construct was created such that all the pins were parallel to each other along with two

longitudinal bars attached with clamps. With lamb femurs and sheep cadaver legs,

several trials were carried out to test the practicability of the external fixator

configuration in relation to the fracture model and the secondary plate fixation

procedures. The following pictures (Figures 43, 44, 45 and 46) illustrate the four steps

in the optimisation process for configuration of the external fixator in a cadaver leg test:

Figure 43: First step

: The four Schanz’ screws were placed in relation to the approach and application of the fracture model. In this case, the figure illustrates a small anterior approach marked in bold blue in longitudinal direction and fracture zone is marked with transverse blue lines. The proximal two pins were positioned parallel and in longitudinal direction and the distal two pins were positioned in a perpendicular plane, slightly convergent to each other.

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Figure 44: Second step

: After attaching the ‘unilateral external fixator’ to the pins, the fracture creation model was performed via the small anterior approach. The figure also shows the chisel and blade bar being inserted.

Figure 45: Third step

: The percutaneous plate insertion was tested with the support of the locked drill guide at the end of the plate. This external fixator configuration clearly shows, that without removal of the frame, the plate can not be fully inserted and that the longitudinal rod crosses the percutaneous screw insertion. Therefore, the distal condylar block pins have to be re-arranged and the rod must be moved to the anterior region of the pins.

Figure 46: Fourth step

: The last part of the trial concludes with the performance of the open, lateral surgical approach, but as stated in the previous step, the rods and clamps of the external fixator have to be re-positioned or even removed to proceed with the open approach. Distally, this pin configuration is too close to each other and must also be altered, otherwise the plate can not be fixed properly.

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The final configuration of the external fixator (Figure 47):

• The two distal pins in the condylar block had to be changed into the

perpendicular plane of the shaft axis, because the proximal end of the two pins

was placed too close to the fracture area. This increased the risk for a fracture

extension, and would have compromised the lateral plate fixation of the

secondary, definitive fixation procedure. Therefore, the new position of the two

distal pins was defined as, the anterior one positioned dorsally and parallel to

the anterior femoro-patellar notch in antero-lateral to postero-medial direction,

and the second pin from the lateral condyle into the medial condyle in ‘true’

latero-medial direction.

• The proximal two pins had to be inserted from antero-lateral to postero-medial

direction so that they do not interfere with the plate fixation of the second stage

procedure.

• Because of the new positions of the four pins the configuration of the bars had

to be adjusted. One of the bars was longitudinally fixed with clamps to the three

antero-lateral pins. The bars were placed at the anterior side of the pins.

Through this plating procedure, it is ensured that the minimally invasive

technique is not compromised, while the external fixator frame remains in situ,

until the plate fixation is finalised. The second rod is fixed in oblique direction

to connect the two proximal pins and the posterior lateral condylar pin. This

external fixator frame is constructed in a V-shape and provides sufficient

mechanical stability.

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Figure 47: Lateral view (left) and axial distal view (right) of the external fixator frame (’V-shape’ type) and the locking compression plate (LCP) fixed onto the antero-lateral distal femur of a plastic ovine bone model.

The second part of the fixation model includes the definitive fracture fixation. This

is executed by the application of the ‘internal fixator’. This type of fixation device

consists of a metal plate with at least two screws on each side of the fracture. The

specialty of such an internal fixator is the locking mechanism between the screw heads

and the plate, which leads to a different construct mechanics with transferring the loads

from one bone fragment, onto the screws, then through the screw-head – plate-hole

interface on the plate. The load crosses the fracture area and gap within the plate and

again, is transferred via the interface between the plate hole and the screw head onto the

screws and finally to the opposite bone fragment. This kind of fixation construct, which

does not directly fix the intermediate fracture zone, is called ‘bridging plate’

configuration.

The requirements of the internal fixator are:

• Appropriate mechanical properties: The construct has to resist all loads of a

sheep hind limb, without risking an implant failure. The chosen implant is the

4.5mm (narrow) stainless steel locking compression plate (LCP) used with

5.0mm head locking screws (HLS).

• Correct length of the plate: The created fracture is approximately 3.5 to 4cm

long, and the screw insertion points have to be in a safe distance from the

fracture site, not to risk further fracture extension with bone splitting. Therefore,

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the shortest possible plate is a 7-hole 4.5mm LCP. Additionally, this plate is not

too long and will not potentially irritate the lateral soft tissues along the femur.

• Optimal plate-screw configuration: The 7-hole LCP is fitted with two, bicortical

head locking screws (HLS, 5.0mm) in the proximal and the distal main

fragment and three plate holes in the middle are empty (Figure 48). This

configuration satisfies all the above mentioned requirements and provides a safe

and stable implant position.

Figure 48: The internal fixator configuration with a 7-hole narrow 4.5mm, stainless steel LCP with four 5.0mm head locking screws.

3.3.2 Experimental design and description of operative techniques

The research question is to compare the minimally invasive plate osteosynthesis

versus open reduction and internal plate fixation on the soft tissue recovery and fracture

healing. We aimed to address and study this question in an experimental animal model

with sheep. The previously developed and validated trauma model with a multi-

fragmentary distal femur fracture with concomitant severe soft tissue damage is applied

for this purpose

Initially, the external fixator as discussed in the previous chapter is fixed. Within

the following five days, the animals were clinically observed and their soft tissue

conditions were monitored. Following which the sheep undergo the definitive fracture

fixation with an internal fixator (Figure 48). This plate fixation procedure is performed

either with the standard, open surgical approach (ORIF) or by minimally invasive

technique (MIPO). Following this, the external fixator was removed and the sheep were

clinically observed and monitored for nine days. The fracture healing progress was

regularly assessed with biweekly conventional radiographs. Two groups with twelve

sheep each were sacrificed at four and eight weeks, respectively. Post-mortem analysis

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was carried out by CT scan to assess the callus morphology followed by mechanical

testing to investigate the mechanical integrity of the healing femur bone.

In the following paragraphs, each of the above mentioned procedures is described in

detail, and the following chapters will detail the entire course of the animal experiments

(pilot study and main series), including the aspects of animal care and welfare, as well

as post-mortem investigations and evaluations with all specific aspects and features.

Description of operative techniques:

1) Application of external fixator

The application of external fixator was performed during the fracture model

procedure (see also Chapter 3.2.2). While the sheep was still under induced general

anaesthesia, the sheep was positioned in left lateral recumbent position with the right

leg on top of the operating theatre at the animal laboratory. The leg has been entirely

shorn and shaved before the soft tissue trauma. After cleaning with 0.5% red

Chlorhexidine in 70% Ethanol supplied by the pharmacy of The Prince Charles

Hospital and draping the right leg accessible from the greater trochanter to the tibia

head, the four external fixator pins were inserted by mini-incisions: the two proximal

pins in antero-lateral position parallel to each other, the two distal pins in antero-lateral

position just behind the femoro-patellar notch and in postero-lateral direction into the

lateral condyle of the femur (Figure 47 and 49). Then, the fracture model was applied

over the antero-lateral small approach to the distal, metaphyseal part of the femur.

Before fracture initiation, the longitudinal external fixator rod was fixed to the three

antero-lateral pins and only the distal clamp was loosened. Afterwards, the fracture was

initiated by blade bar insertion and two chisel hits, and the leg was rotated in the

fracture plane at 90° ante flexed and 90° retro flexed positions to complete the

disruption of the periosteum. After this final manoeuvre of the fracture model, the

external fixator frame is applied. Initially, the distally loosened, longitudinal rod was

clicked in the distal clamp again at the same position. After preliminary fracture

reduction by manual adjustment of the two intermediate fragments through the small

antero-lateral approach, the second oblique rod is fixed to the pins (Figure 51). Finally,

:

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the wounds were washed out and the fracture model approach was closed with sutures

in layers and the pin sites were dressed.

Figure 49: Figure shows the insertion of the four external fixator pins. The two proximal pins at the left are positioned longitudinal to the femur shaft and the two distal pins at the right are placed perpendicular to the proximal once.

Figure 50: Figure showing the small antero-lateral approach employed for the application of the fracture creation model.The oscillating saw was used to carried out the partial anterior osteotomies .

Figure 51: External fixator frame (two carbon rods) on the upper part of the right hind limb of a sheep. The three stitches of the fracture model approach are seen in front of the longitudinal rod. The fixation tool of the probe to monitor the muscle condition such as compartment pressure and partial oxygen tension is placed on top of the proximal clamps.

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Five days after the trauma model and the external fixator was applied, the sheep

was brought back to the animal laboratory for applying the internal fixator. Internal

fixator is going to be applied by either the open surgical approach or the minimally

invasive method.

2) Operation of the open reduction and internal plate fixation (ORIF)

The sheep was anaesthetised and positioned in left lateral recumbent position on the

operating theatre table. After cleaning with 0.5% red Chlorhexidine and draping the

right upper part of the hind limb, including the external fixator, the rods and clamps of

the external fixator were removed. Then, a standard lateral surgical approach of 15 cm

length to the right femur was performed. After the skin incision, the subcutaneous

layers (Figure 52) and the fascia of the ileo-tibial tract were longitudinally split. The

lateral vastus muscle was mobilised into anterior direction and the perforating blood

vessels were dissected between ligatures. Behind the lateral vastus muscle, the lateral

aspect of the femoral shaft was prepared from the subtrochanteric to condylar region

including visualisation of the fracture area (Figure 53). Initially, the fracture

haematoma was removed and the gaps and cavities were washed out with sterile saline.

Subsequently, the fracture lines -two transverse planes and one longitudinal split were

identified and the fracture margins were cleaned including removal of 5 mm adjacent

periosteum. Under slight axial traction on the pins of the external fixator, the fracture

fragments were anatomically reduced and temporarily held in place with pointed

reduction forceps and small K-wires of 1.25 mm. If the accurate fracture position was

achieved, the internal fixator (7-hole 4.5mm narrow stainless steel Locking

Compression Plate (LCP, Synthes, Switzerland) was then placed on the lateral side of

the femur and the distal end of the plate was centred in the groove anteriorly to the

lateral condyle. Four threaded drill guides were turned into each of the screw holes

before drilling two distal holes followed with two bicortical head locking screws (HLS)

with the correct length inserted (Figure 54). If necessary, any final adjustments to the

fracture position were made. Following which two proximal screw holes were drilled

and two bicortical HLS inserted. All screw heads were locked in the plate with the

torque limited screw driver of 4 Nm. All temporary fixation devices and the external

:

75

fixator pins were removed (Figure 55). The entire wound was thoroughly washed out.

The wound closure was performed in layers, with a continuous suture of the fascia

(Monocryl 2-0), with interrupted sutures of the subcutaneous layers (Vicryl 2-0) and

with interrupted ‘Donati’ stitches of the skin (Novafil 2-0) (Figure 56). After cleaning

thoroughly, the wound was disinfected with a moisture vapour permeable spray

dressing. The spray dressing was bought from Opsite, Smith & Nephew Pty. Ltd.,

Australia.

Figure 52: Conventional lateral open surgical approach: The dissection of the subcutaneous layers is being performed with the ultrasound knife. The pins of the external fixator are still in situ and might be supportive during the fracture reduction process.

Figure 53: The lateral aspect of the femur shaft is being prepared for visualisation by retracting the lateral vastus muscle anteriorly using a metal retractor ‘Langenbeck hook’.

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Figure 54: LCP is being placed on the lateral aspect of the femur and fixed with screws. The two proximally applied drill guides act as a guide to drill the holes in exact, perpendicular direction to the plate.

Figure 55: The LCP was fixed with four screws (HLS), two proximal and two distal. The two intermediate fracture fragments are shown to be anatomically reduced, but not fixed as supposed to with the bridging plate technique.

Figure 56: The final result of the open surgical approach with many skin sutures in place. The soft tissue monitoring probe shown as the orange cable fixed with the blue caped device is still in situ for post-operative measurements.

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3) Operation of the minimally invasive plate osteosynthesis

The sheep was anaesthetised and positioned in left lateral recumbent position on the

operating theatre table. After prepping with red Chlorhexidine 0.5% and draping the

right upper part of the hind limb, including the external fixator, a short (2 to 3 cm)

longitudinal skin incision was made between the distal pins of the external fixator at the

lateral femur condyle. The subcutaneous layers and the fascia were longitudinally split

and the periosteum in the ‘lateral groove’ anteriorly on the lateral condyle prepared.

The image intensifier (Philips, BV 25, Netherlands), with a sterile plastic bag covering

the top of the C-arm, was used in the operation field to check the fracture reduction and

control the implant position during the procedure (Figure 60). With a blunt bony

elevator, the epi-periosteal tunnel was prepared without damaging the periosteum. The

fracture region was not visualised during the entire procedure. Further, the 7-hole

4.5mm narrow stainless steel LCP with one threaded drill guide locked in the most

distal plate hole was inserted in a percutaneous fashion (Figure 57 - 61). A second

threaded drill guide was percutaneously inserted through a stab incision of the skin in

lateral projection to the planned plate hole. This drill guide was fixed in the most

proximal plate hole. With the support of those two drill guides and under image

intensifier control, the plate was placed on the femur bone in the correct position in

both radiographic planes (Figure 60). Before fixing the plate to the bone, the fracture

reduction was checked. If the fracture position had to be adjusted, the external fixator

frame was slightly loosened and changed into a better position. Once the fracture and

implant position was correctly established, four bicortical head locking screws were

percutaneously inserted. The screw configuration is the same as with the open

technique. After the final control with the image intensifier, the external fixator was

removed and the distal wound was closed in layers, with a suture of the fascia

(Monocryl 2-0) and a couple of skin stitches (Novafil 2-0). The three stab incisions

were closed with only a skin suture (Novafil 2-0) (Figure 61). The operated area was

cleaned and the wound finally disinfected with a moisture vapour permeable spray

dressing (Opsite, Smith & Nephew Pty. Ltd., Australia).

:

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Figure 57: The LCP with a threaded drill guide fixed at the most distal plate hole is being slid into the small incision.

Figure 58: LCP is percutaneously advanced passing the fracture region.

Figure 59: LCP is positioned with the distal plate end in the ‘condylar groove’ between the two distal pins of the external fixator.

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Figure 60: The image intensifier (Philips, BV 25, Netherlands) is used to check and control the fracture reduction as well as the implant position during the whole procedure.

Figure 61: The postoperative clinical picture showing few skin stitches and the soft tissue monitoring probe in situ.

3.3.3 Instruments and implants

The following instruments and implants were used for this study:

• Instruments for the fracture creation model:

o general tools and instruments: scalpel, surgical forceps, scissors and

wound retractors including small ‘Hohmann’ hooks, rulers

o sagittal oscillating saw (Stryker, Stryker 5 system, USA) with oscillating

blade (Stryker, 2108-150, USA) (Figure 30)

o drill (Stryker, Stryker 5 system, USA) with drill bits (2.5 mm and 3.5

mm) and drill guides (Figure 30 and 62)

o blade bar (designed and made by AO Development Institute, Davos,

Switzerland) (Figure 25 and 30) and sliding hammer

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o surgical chisel (Synthes, 397.970, Switzerland) (Figure 30)

o Kirschner wires (Synthes, 1.25 mm, 292.120, Switzerland)

• Surgical instruments for the operations (Figure 62):

o general surgical tools (scalpel, forceps, scissors, towel clamps, arteries,

retractors, needle holders, suction device)

o drill (Stryker, Stryker 5 system, USA) with 4.3 mm drill bits and

threaded drill guides (4.3 mm)

o screw drivers (large fragment and 4.0 Nm torque limiting device,

Synthes, 324.052, Switzerland)

o pointed reduction forceps

o bony elevator

o Kirschner wires (Synthes, 1.25 mm, 292.120, Switzerland)

• Implants (Figure 47 and 48):

o external fixator (Synthes, large AO external fixator, Switzerland)

carbon fibre rods (Synthes, 11.0 mm diameter, 150 mm length,

394.820, Switzerland)

clamps (Synthes, 393.978, Switzerland)

self-drilling Schanz’ screws (Synthes, 4.0 mm x 100 mm,

294.776 (Titanium), Switzerland)

o internal fixator

7-hole 4.5/5.0 mm narrow, stainless steel LCP (Synthes,

224.571, Switzerland)

5.0 mm self-tapping HLS (Synthes, 213.328-213.346,

Switzerland).

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Figure 62: General surgical instruments used for the fracture creation model and external fixator application except the implants, the sagittal saw and the drill.

3.4 ANIMAL CARE AND WELFARE

This chapter outlines the exact description of the animals used, their handling and

care as well as the details of the peri-operative and post-operative management.

3.4.1 Sheep data

In this experimental study, male sheep (ovis aries, Merino species) were used.

These sheep were supplied from a local breeder at Lyle Nation, Gympie. These adult

wethers were fully grown with a strong and mature skeleton, weighed around 40 kg and

were about four to six years. Male sheep were chosen because of their bigger size,

heavier weight and stronger bones than the ewes. The sheep were housed in a separate

yard at the Biological Research Facility of The Prince Charles Hospital, Chermside

(Figure 63). The sheep were tested for Q-fever, vaccinated with Glanvac 6 (100 ml im),

treated for worms every six weeks and ear tagged for identification. These protocols

followed the regulations of the Queensland Health and the Department of Primary

Industries (DPI).

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Figure 63: Sheep flock in the yard at the Biological Research Facility of the Prince Charles Hospital; nowadays, Medical Engineering Research Facility (MERF), Chermside.

3.4.2 Perioperative management

The day before the experiments were conducted, the animals were moved to a

separate area within the animal house including daylight access, for preparation and

adaptation to this new environment. The next morning, the sheep were weighed on the

animal scale for baseline weight measurement and later, transported to the animal

laboratory of Biological Research Facility at The Prince Charles Hospital with a custom

made, large animal trailer. The animals were kept in small in-house cages until the

procedure started.

The animals were caught in the cage by two animal nurses, put into a special trolley and

then, the entire right hind limb from the heel up to greater trochanter and the iliac crest

as well as the neck from the jugular notch to the jaw angle was appropriately shorn and

shaved. After the sheep were moved to the operating theatre room, Dr Kathleen Wilson

or Dr Sadahiro Sugiyama managed the peri-operative care. Under sterile conditions,

one tri-luminal central venous line was inserted into right external jugular vein by

Seldinger technique. Before inducing the general anaesthesia, 10 ml blood was

aspirated for examination of serum concentrations of Creatine Kinase and Lactate

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Hydrogenase as pre-operative baseline parameters. The general anaesthesia was

induced with propofol (10 mg/ml) 0.5 mg/kg body weight as bolus intravenous

injection. Then, the sheep were endotracheally intubated and ventilated with 100%

oxygen on the ULCO Campbell ventilator (MK.5PC, ULCO Engineering Pty. Ltd.,

Australia). The anaesthesia was maintained with propofol at 4 mg/kg body weight/hour

and buprenorphine (0.01 mg/kg body weight) was also administered intravenously to

provide sufficient analgesia. Before the experimental procedures began, two

intravenous antibiotics (gentamycin 80 mg and cephalothin 1 g) and intra-muscular iron

supplementation (Ferrosig 2 ml) were injected, too. The animals were then carefully

positioned on the pendulum trestle for application of soft tissue trauma to the right

femur after the supra-patellar thigh circumference was measured. Afterwards, the sheep

were re-positioned onto an operating table in left lateral recumbent position and the

multi-fragmentary fracture created and the external fixator applied as described

previously.

Post-operatively, conventional radiographs were taken (see chapter 3.6.1) and then

the anaesthesia discontinued. The animals were extubated and were kept under close

clinical observation until they were directly transported in a special trailer) to the

animal facility. While still in the post-operative recovery stage, the sheep were safely

put into the supporting trolleys and the height of the shade cloths adjusted according to

the size of the animals.

3.4.3 Post-operative management and care

The sheep were looked after and fed by the animal nurses according to the protocol

and standards of the animal facility. Twice a day, the investigators supervised by the

veterinary surgeon carried out clinical assessments of general medical condition as well

as local wound care for the sheep. The pain management included a second dose of

buprenorphine (0.01 mg/kg body weight), and further doses were given if required.

The sheep spent four days closely monitored in the supporting trolleys and on day

5, the animals were again transferred to the animal laboratory for the second procedure.

The induction and maintenance of the general anaesthesia was performed in the same

manner as for the first operation. After the x-rays were taken at the end of the definitive

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plate fixation procedure, the general anaesthesia was terminated and the sheep were

transported back to the animal facility and put into the supporting trolleys for another

night to fully recover from the operation and anaesthesia. Following which the sheep

were safely released into a separated outside paddock and closely observed by the

animal nurses. Within the following week, further blood samples were taken from the

central venous line, and after following the complete blood sample collection

procedure, the lines were removed on day 9 after the second procedure. The skin

stitches were taken out 14 days post-operatively.

As a precaution, the sheep spent at least two weeks post-operatively in this

separated paddock in a small group of only four to six sheep. After this period of time,

the animals were released back to the flock into the main yard of the animal facility.

3.4.4 Euthanasia

After the defined recovery time (six weeks for the two pilot sheep, and four to eight

weeks for the main series animals, respectively), the sheep were finally weighed and

transported to the animal laboratory. The animals were laid on the special laboratory

trolley and euthanized by an intravenous injection (external jugular vein) with

pentobarbitone sodium (Lethabarb, 325 mg/ml) 0.5 ml/kg body weight.

3.5 METHODS TO MONITOR THE SOFT TISSUE DAMAGE

In this chapter, the methods of measuring the extent of the soft tissue damage and

its recovery are described. The soft tissue damage is monitored in two different ways:

locally via an in situ probe to measure compartment pressure and partial oxygen

pressure and systemically by collecting blood to assess blood serum markers for cell

and soft tissue damage.

3.5.1 Local soft tissue assessments

Compartment pressure, partial oxygen pressure and temperature

To monitor the soft tissue damage and recovery over time, special catheters were

used to measure the compartment pressure and the oxygen tension (partial tissue

oxygen pressure) within a defined area of the soft tissues. The probe (Neurovent PTO,

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No. 095 008, Raumedic AG, Germany) was inserted in the rectus femoris muscle of the

right hind limb (Figure 64) at the end of the first operation, after the fracture was

created and the temporary external fixation. The probe was percutaneously inserted

from the proximal anterior aspect of the thigh and tunnelled into the central part of the

rectus femoris muscle. The location of the probe was manually controlled from the

small antero-lateral fracture creation approach. The cannulated needle with a

longitudinal split on one side is removed and the catheter at the skin entry point fixed

with two sutures (Figure 65).

Local soft tissue monitoring:

antero-lateral trans-muscular fracture creation approach

compartment measurement probe in rectus femoris muscle

lateral surgical approach for open fracture fixation method (ORIF)

Figure 64: This graph shows the localisation and direction of the surgical approaches and the inserted probe in a cross-section through the middle part of the ovine thigh. The red lines indicate the planes of the two surgical approaches and the bold black line presents the direction of the inserted probe.

86

Figure 65: Clinical post-operative site of the proximal right hind limb with the external fixator frame. The figure also shows the sutures, the inserted soft tissue monitoring probe as well as the cables secured in a circle.

The cables of this soft tissue measurement probe are connected to the monitor

(datalogger MPR 2 logO, No. 095 254, Raumedic AG, Germany). Three parameters are

collected twice a day at 7am and 5pm) for the first 14 days. The parameters recorded

are compartment pressure and partial oxygen pressure. To ensure accurate

measurements especially of the compartment pressures, the animals were either

monitored in a calm standing position in the trolleys or in a laying, lateral position on

the special animal trolley under serene circumstances.

Data were collected on paper and later transferred to a Microsoft Excel

database/worksheet.

3.5.2 Systemic investigation:

Blood serum tests

Within the first two weeks after the soft tissue trauma, blood samples were taken to

assess the serum concentration (Units per liter: U/l) of specific cell damage markers,

such as Creatine Kinase (CK) and Lactate Dehydrogenase (LDH). Five ml of blood was

collected through the central venous catheter and directly transported to the Queensland

Health Pathology Laboratory at The Prince Charles Hospital for analysis. The blood

samples were collected before induction of the general anaesthesia as well as post-

operatively, before discontinuing the general anaesthesia. On day 1, 2, 3 and 4 samples

were taken at 7 am. On the day of the second procedure (day 5), pre-operative and post-

87

operative samples were collected just as before. The same procedure for blood sample

collection was repeated on day 6, 7, 8, 9, 11 and 14. All values were copied from the

Pathology Laboratory sheets into a Microsoft Excel table and analysed thereafter.

3.6 METHODS TO ASSESS FRACTURE HEALING

3.6.1 Conventional radiographs

To monitor the progress of the fracture healing over time, conventional radiographs

(x-rays) were taken every fortnight. The first two x-rays were taken post-operatively

after the first and the second operation in the operating theatre (Shimadzu MU 125,

Japan). The latero-medial x-ray was taken with 60 kV and 6 mAs, the antero-posterior

picture with 80 kV and 10 mAs. The x-rays after two, four and six weeks were acquired

in the animal facility with an image intensifier (fluoroscopy; Philips, BV 25,

Netherlands). The latero-medial x-ray (Figure 66) was shot with 40 kV and 3.2 mAs

and the antero-posterior (Figure 67) with 40 kV and 4.0 mAs. The final x-rays after

four and eight weeks, respectively, were captured directly post sacrifice in the animal

laboratory with the Shimadzu device in the same manner as described above. All the

radiographic plates were delivered to the Department of Radiology of The Prince

Charles Hospital and digitally developed and saved as bitmap files (Digitizer, ADC

Compact plus, AGFA, Type 5146, Belgium).

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Figure 66: Latero-medial x-ray taken using the image intensifier in the animal facility. The sheep positioned in lateral recumbent position on the special large animal trolley. The ‘black’ radiographic plate is placed on the medial, inner side of the femur and the x-ray is taken from the top.

Figure 67: Antero-posterior x-ray taken with the BV 25. The radiographic plate is placed on the back side (posterior) of the leg and the x-ray shot taken from the front.

The first post-operative, digitized radiographs were evaluated qualitatively, with

respect to fracture pattern according to the AO classification of long bone fractures 3

and quantitatively with the computer software AMIRA (Visage Imaging GmbH, Berlin,

Germany). This software was used to determine the mean length of the fracture zone in

the anterior, lateral (middle) and posterior aspect of the lateral view radiographs (Figure

69) and the medial, anterior (middle) and lateral in the a-p view (Figure 68).

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Figure 68: This screenshot of the AMIRA programme that illustrates the measurements of the fracture length at three areas (left to right: lateral, middle, medial) from the antero-posterior view of the post-operative x-ray after the fracture creation and external fixation procedure.

Figure 69: This cropped AMIRA screenshot from the lateral view of a post-operative x-ray shows posterior, middle and anterior (left to right) fracture length measurements.

To monitor the fracture healing course and determine potential problems, such as

change of fracture, implant failure or re-fractures biweekly x-rays of all sheep were

compared and evaluated. The fractures and implant positions were measured and the

callus morphology was assessed by determining the number of bridging cortices (in

quarters) (Figure 70 and 71).

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Figure 70: X-rays taken at 4 weeks of Figure 71: X-rays taken at 4 weeks of MIPO group with medially and posteriorly ORIF group with medial and posterior complete callus bridges (2 bridges). callus, but not completely bridged (0). 3.6.2 Computer tomography (CT)

Computer tomograms of all femur bones were taken post-mortem. After euthanasia

of the sheep in the animal laboratory, the entire right femur bone was carefully

explanted (Figure 72) and the implants (LCP plus four screws) removed (Figure 73).

The femur bones were wrapped in saline soaked gauzes and put in two plastic bags.

Afterwards, these bones were transported to the Department of Radiology at The Prince

Charles Hospital and a high-resolution computer tomography scan was performed

(Philips, Brilliance 64, Netherlands: slice thickness 0.67 mm, pixel size 0.625 mm, 120

kV, 250 mAs, rotation time 1 s). After scanning the femora were stored frozen at -20

°C.

91

Figure 72: Right explanted femur without soft tissues, but implant still attached.

Figure 73: Right femur after the implant removal.

The CT data sets of all femur bones were evaluated with the software AMIRA

(Visage Imaging GmbH, Berlin, Germany) and 3D reconstructions generated (Figure

74 left). After determining the density threshold between the cortical bone and the

newly developed, mineralised callus, the callus was visualised and evaluated for its

total callus volume in mm3 and callus length in mm (Figure 74 right). Further

measurements from this dataset included the cross-sectional surfaces in mm2 of the

callus formation in the proximal and the distal transverse fracture planes (Figure 75).

To analyse the fracture reduction and fracture configuration, the anterior fracture

length, the maximal fracture length as well as the maximal distance between the most

displaced fragments were assessed (Figure 76). All the three measurements were

performed in 3D.

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Figure 74: With the AMIRA software, (left) a three-dimensional histogram is generated and the proximal and distal callus planes are defined. After cropping this middle callus field, (right) the total mineralised callus volume is visualised and measured (mm3

). The longitudinal length of that callus volume defines the total callus length.

Figure 75: This picture, gained with the AMIRA software, shows the areas of the callus formation in the proximal and the distal, transverse fracture planes. Example from the eight week ORIF group.

93

Figure 76: This three-dimensional reconstruction of the cortical bone fragments shows the two intermediate fragments (lateral (left) aligned, medial (right) distally displaced). The anterior fracture length (23.27 mm), the maximal fracture length (25.77 mm) and the maximal distance of fragment displacement (6.15 mm) is measured and displayed.

To more specifically investigate the callus morphology of this relatively complex

fracture pattern, a novel approach was developed to semi-quantitatively analyse the

callus morphology. With a scoring system, the callus morphology, especially the callus

bridges were assessed. This score consists of two different measurements: firstly, we

determined the number of callus bridges that were linking the fracture gaps, and

secondly, the thickness of the callus bridges was measured. A score was given for each

bone according to the following criteria:

Number of callus bridges

• Proximal transverse fracture plane divided into four areas or quarters: anterior,

lateral, posterior and medial areas. Each quarter gets 1 point (maximal 4 points)

:

• Distal transverse fracture plane divided into four areas or quarters: anterior,

lateral, posterior and medial areas. Each quarter gets 1 point (maximal 4 points)

• The proximal and distal fracture quarters are added together

• The score for the number of callus bridges will range from 0 – 8

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Size of callus bridges

• Proximal and distal callus bridges are counted separately

:

Scoring pattern for callus bridges:

• No callus bridge at all = 0

• All callus bridges with callus thickness < cortical thickness = 1

• Some callus bridges (partially) with callus thickness > cortical thickness = 2

• All callus bridges (complete) with callus thickness > cortical thickness = 3

• Both fracture planes are added together: maximal 6 points

• Callus bridge reaches over proximal and distal fracture plane + 1

• The score for the size of the callus will range from 0 – 7

Total score of callus morphology

• maximal number of bridges x maximal size of callus bridges

:

• The total score will range from 0 – 56

This scoring system was applied into a two- and three-dimensional analysis pattern:

For the two-dimensional method, two longitudinal planes were defined in antero-

posterior and latero-medial direction (Figure 77) and those two slices were generated

and extracted from the 3D-CT dataset (similar to virtual histology slices). Then, the

callus morphology was assessed according to the callus bridging score (Figure 78).

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Figure 77: With the AMIRA software, two perpendicular planes cut the 3D CT model (red - callus, blue - cortical bone).

Figure 78: (left) Cross-sectional surface in medio-lateral direction and (right) cross-sectional cut in antero-posterior direction. Numbers and quality of callus bridges are assessed according to the morphologic scoring system. This example belongs to one of the four weeks ORIF group with medial and posterior callus formation bridging the fracture gap distally and proximally, respectively.

The three-dimensional method was performed on the entire 3D-CT dataset. In the

AMIRA programme evaluation of numbers and sizes of the callus bridges was carried

out dynamically by rotating and moving the dataset throughout the assessment. The

entire circumference of the callus dataset was divided into four parts: anterior, lateral,

posterior and medial (Figure 79).

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Figure 79: Clockwise from left top: lateral, anterior, medial and posterior view of a 3D CT-reconstruction. A representative picture of the four weeks MIPO group.

Blinded test approach was used to evaluate all CT scans and the results were

collected and analysed with Microsoft Excel software.

3.6.3 Mechanical testing

The frozen femora were slowly thawed over a period of two days at room

temperature in the biomechanical laboratory at the Institute of Health and Biomedical

Innovation. The proximal and distal ends of the bones were thoroughly dissected to the

cortical surfaces and embedded with polyurethane in two specially designed stainless

steel cups (Figure 80). The left and right femurs were then appropriately positioned and

tested mechanically in the Instron machine (Instron 8874). The testing algorithm

included a first trial run with minimal torsional load. This was followed by the two

defined runs to assess the torsional stiffness and finally, the maximum torque at failure

of the healed femurs (Figure 81). The results were reported as absolute values (Nm) and

as a percentage of the values from the intact contra-lateral side, as determined

according to a procedure previously described 124.

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Figure 80: Dissected femur proximally and distally embedded and fixed into metallic cups. It was attached to the Instron testing machine to perform the torsional test of stiffness and strength.

Figure 81: This picture shows the spiral fracture in the distal part of the femur shaft (right femur of sheep 418) after performing the mechanical test run to assess the ultimate torque.

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3.7 STATISTICAL ANALYSIS

All experimental data was saved electronically and analysed using Microsoft Excel

software, and the statistical analysis done using the SPSS (Statistical Package for Social

Sciences) software with the assistance from a bio-statistician (Dr Sarah Whitehouse).

Inter-approach variances in mechanical testing which is normalised to the intact contra-

lateral femur, computer tomography and general sheep data were compared using

independent t-test or Mann-Whitney test. Serum marker levels which were normalised

to pre-operative were compared between approaches using a repeated regression model

analysis with time as repeat parameter. Because of normal distribution of those

parameters, ANOVA test was performed with a statistical significance of p<0.05.

3.7.1 Estimation of sample size and power analysis

In a previous study 1, the tibia treated with the ORIF approach had bending stiffness

of approximately 11 Nm/deg with a standard deviation of 1.0 Nm/deg. Based on these

values, a sample size of 6 in each group (MIPO/ORIF for each survival duration) will

have 80% power to detect a difference in means of 14% (MIPO-ORIF / ORIF) using an

independent t-test with a 0.05 one-sided significance level. In the study of Tepic et al.41

,

the mechanical parameters were normalised to the contra-lateral intact tibia similar to

the present animal model. The tibial fractures treated with an internal fixator in ORIF

manner obtained 64.5% (standard deviation 5.1%) of the intact tibial strength after 12

weeks. Based on these normalised values, a sample size of 6 in each group will have

80% power to detect a difference in means of 12% (MIPO-ORIF / ORIF) using an

independent t-test with a 0.05 one-sided significance level. Although a larger sample

size would have a similar 80% power to detect even smaller differences, the clinical

relevance of such small differences between approaches may be questionable.

3.8 ANIMAL ETHICS DOCUMENTATION

To perform animal studies at Queensland University of Technology, animal ethics

application was submitted to the QUT ethics committee prior to the start of

experiments. On the 13th of September 2005, the animal ethics application was

conditionally granted by the University Animal Ethics Committee (UAEC). Further

99

information and more detailed descriptions of the project were submitted and on the

26th

• Species: Sheep (Ovis aries)

of October 2005, the UAEC decided to grant the project: "Effect of surgical

approach on bone vascularisation, fracture and soft tissue healing by internal fixation:

comparison of less invasive to open approach" the approval to perform animal studies.

Reference number for this application was: QUT Ref No 4222A .The following species

and number of animals were approved:

• Numbers: 2 pilot animals + 36 sheep (main series)

The researchers complied with the policies and guidelines issued by the NHMRC

and AVCC (including the Australian Code of Practice for the care and use of animals

for scientific purposes (7th

Through the stage of further developments within the project, three minor changes

to the approved version of the animal ethics protocol were proposed and therefore, two

additional animal ethics applications were submitted and approved:

Edition 2004).

• intramuscular measurements for monitoring the soft tissue injury and changes in

the postoperative care with the use of special designed trolleys (28th

• soft tissue monitoring using the method of microdialysis (7

February

2006) th

Annual animal ethics progress reports (2005, 2006, 2007, and 2008) were submitted

to the UAEC as part of the standard regulations after which the project was allowed to

be further continued.

July 2006)

3.9 PROCEDURES AND TIMELINE

This chapter summarised the compilation of previously described developments and

methods into one pilot project. The detailed construction and course taken is outlined

with the results presented and discussed. Conclusions were drawn to optimise the main

series of the experimental study and its outcome. Furthermore, both parts of the project

include comprehensive project plans and timelines.

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3.9.1 Pilot study

The purpose and the aim of the pilot study were to try and assess:

• The newly developed animal trauma model in vivo and throughout the entire

time of soft tissue recovery, which had to be evaluated and defined.

• The application of the animal trauma model to study the effects of surgical

approaches on the soft tissue and fracture healing processes.

• The performance of the experimental study and evaluate the course and

outcomes towards further refinements and adjustments for the main study.

• The overall performance of the animal experiments to report back to the animal

ethics committee.

3.9.1.1 Methodology and timeline

The pilot tests were carried out with two adult, male sheep (Merino species) and the

entire experimental setup was run and tested. As described in the experimental design

of this study, the initial procedures, namely, combined trauma model (soft tissue injury

and fracture creation) and external fixator application were the same for both pilot

sheep. The subsequently operations were split into two experimental methods: the first

pilot sheep underwent the internal plate fixation via the standard open surgical

approach, while the second pilot sheep was fixed via the minimally invasive plate

osteosynthesis technique. After thorough planning and preparation of every step and

part of the experimental setup, the pilot experiments for the first and second sheep were

started on the 13th and 20th

September 2006 respectively. The detailed pilot study

timeline is attached in Appendix H.

3.9.1.2 Results

General data:

Both pilot sheep (Merino wethers) had highly similar characteristics: 7-8 years of

age, and 44.9 and 45.0 kg of body weight. The four general anaesthetics went smoothly

with the postoperative clinical observations performed by the candidate, the veterinary

surgeon and the animal nurses demonstrated no signs of any adverse effects. The day

after the second surgery, the animals were released from the trolleys and kept in the

101

facility for further clinically assessments such as blood sample collections and soft

tissue monitoring. Slippery concrete floor on which both sheep slipped and fell over a

couple of times posed to be the only problem during this protocol. After 14 days of

close observation and monitoring, the sheep were released outside into the paddock of

the animal facility.

Times of the procedures:

The times of the four general anaesthesia and the operations are listed in Table 1.

first procedure second procedure anaesthesia operation anaesthesia operation Sheep 1 (ORIF) 155 min 95 min 130 min 80 min Sheep 2 (MIPO) 135 min 60 min 130 min 50 min

Table 1: General anaesthesia and operation times

Soft tissue trauma evaluation:

The condition of the muscle damage in the anterior compartment of the distal thigh

(rectus femoris muscle) was locally measured and evaluated:

For the first sheep, the compartment pressure measurement (Figure 82) after the

second surgery (ORIF) demonstrated normal values of 0 to 6 mmHg with a peak of 12

mmHg. In the second sheep, the compartment pressures started with higher values of

12-14 mmHg, which decreased to normal values (around 6 mmHg) after the second

intervention.

Compartment pressure measurement

02468

10121416

post 1 2 3 4post 6 7 8 9 10 11 12 13 14

time (days)

mmHg pilot sheep 1pilot sheep 2

Figure 82: This graph presents the compartment pressure values of the two pilot sheep from the rectus femoris muscle compartment measured twice a day over a period of 14 days post trauma.

The second parameter measured in the anterior thigh compartment was the partial

oxygen pressure (Figure 83). In the first pilot sheep, the partial oxygen pressure

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increased to 80 mmHg within the first 24 hours post-operation and steadily diminished

to about 45 mmHg before the second operation. After the second intervention, the

pressure peaked to 160 mmHg at day 7 and fell back to a constant level of around 70 to

80 mmHg over time. In the second pilot sheep, the starting curve was similar to the

curve for the first sheep, with an increase of up to 70 mmHg and then a steady decrease

again. The values of the second sheep showed only a small second increase (60 mmHg)

after the second operation and distinct fall to about 10 mmHg from day 9 onwards.

Partial oxygen pressure (pO2) measurement

020406080

100120140160180

post 1 2 3 4post 6 7 8 9 10 11 12 13 14

time (days)

mmHg pilot sheep 1pilot sheep 2

Figure 83: This graph shows the measurements of the partial oxygen pressures of two pilot sheep in the anterior muscle compartment of the thigh.

On the other hand, to monitor the soft tissue damage systemically, daily blood

samples were collected and assessed for Creatine Kinase (CK) and Lactate

Dehydrogenase (LDH) serum levels:

The CK levels of pilot sheep 1 (Figure 84) showed a distinct peak (~1550 U/l) on

the first postoperative day and a recovery to normal levels within the following two

days. After the second operation, there was another increase (~450 U/l) on day 6 with a

full recovery over the next day. Pilot sheep 2 presented a similar pattern, but both peaks

on day 1 and day 6 were lower than those of pilot sheep 1. An additional short peak

occurred on day 4, with the level reaching normal the day after.

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Creatine Kinase (CK) serum levels

0200400600800

1000120014001600

pre post 1 2 3 4pre post 6 7 8 9 11 14

time (days)

U/L pilot sheep 1pilot sheep 2

Figure 84: This graph shows the serum Creatine Kinase levels of both pilot sheep over the first two weeks of the experiments.

The pattern of the LDH serum concentration curve was similar to those of CK

curves (Figures 84 and 85). The postoperative LDH concentration (day 1) of pilot sheep

1 showed a higher peak than pilot sheep 2. Over the following days, the LDH levels

recovered and presented a second smaller peak (day 6) after the second procedures (day

5) with a quicker recovery of the LDH levels by pilot sheep 2.

Lactate Dehydrogenase (LDH) serum levels

300

400

500

600

700

800

900

pre post 1 2 3 4pre post 6 7 8 9 11 14

days

U/L pilot sheep 1pilot sheep 2

Figure 85: This graph presents the serum Lactate Dehydrogenase levels of both pilot sheep over the first 14 days of the experiments.

Fracture healing evaluation:

Conventional radiographs:

The post-operative x-rays for Pilot sheep 1 (Fig. 86/87) showed a good implant

position which was slightly anterior in the distal condylar fragment. However, the

implant was not perpendicular to the distal screws, which is a non-ideal screw position,

resulting in potentially non-locked screw heads in the two distal plate holes. The

fracture reduction was acceptable, with a slight varus (3°) and ante curved (2°) position.

104

Despite the successful open fracture reduction, the intermediate fragments were not

reduced. Within the first four weeks of recovery, the fracture position changed with an

increased varus (12°) and ante curve (20°) misalignment, due to the non-locked

application of the distal screws. However, between Week 2 and Week 4, the visible,

mineralised callus formation progressed distinctly and by Week 6, the medial and

posterior hard callus was bridging the fracture area.

Post-op 1 Post-op 2 2 weeks 4 weeks 6 weeks

Figure 86: Conventional radiograph series of recovering fracture in Pilot sheep 1 (antero-posterior view).

Postop 1 Postop 2 2 weeks 4 weeks 6 weeks

Figure 87: Conventional radiograph series of recovering fracture in Pilot sheep 1 (lateral view).

The postoperative x-rays following the first and second procedures in Pilot sheep 2

(Fig. 88 and 89) presented an almost anatomical reduction of the fracture. The internal

fixator consisting of a bridging plate was placed at a distance of approximately 1.5 cm

105

away from the femur. The most proximal screw was slightly angled, but otherwise the

positioning of the fixation device was appropriate. After two weeks of recovery,

beginning callus formation was already visible both medially and on the posterior. At

Week 2, the position of the plate and the fracture also remained in good alignment. On

the other hand, a new ‘cortical shadow’ was detected retrospectively, proximal to the

internal fixator at this time point. Secondly, a proximal, subtrochanteric fracture was

identified. Over the following four weeks, the callus formation progressed in size and

density, and after six weeks of recovery, the fracture area appeared to be bridged on the

medial, lateral and posterior side.

Post-op 1 Post-op 2 2 weeks 4 weeks 6 weeks

Figure 88: Conventional radiograph series of Pilot sheep 2 (antero-posterior view).

Postop 1 Postop 2 2 weeks 4 weeks 6 weeks

Figure 89: Conventional radiograph (x-ray) series of Pilot sheep 2 (lateral view).

106

Computer tomography

After six weeks of recovery, the two pilot animals were sacrificed following the

removal of the femur explants containing the plate and screw implants. These were

wrapped in soaked saline gauzes, and directly transported to the Department of

Radiology of The Prince Charles Hospital where they were scanned using a high-

resolution computer tomograph (Philips, Brilliance 64, Netherlands).

The Pilot sheep 1 (Fig. 90) which was operated with the standard open surgical

approach, presented a large callus mass and the integration of the two intermediate

cortical fragments together with what appears to be the beginning of a callus bridging.

The regions with the most callus formation were posterior, medial and lateral of the

femur. The least amount of callus formation was found on the antero-medial side.

Anterior Middle Posterior

Figure 90: Coronal reconstruction of CT scan of Pilot sheep 1.

Pilot sheep 2 (Fig. 91) which was fixed with the minimally invasive technique,

showed less callus formation and a reduced fracture position, especially in terms of the

two intermediate fragments as compared to Pilot sheep 1. In terms of the fracture

107

healing progress, some bridging callus was detected in Pilot sheep 2 as well. The

second proximal femur fracture which was initially suspected was confirmed, and

appeared clearly on the sagittal reconstruction of the CT scan (Fig. 91). Also around

this fracture site, callus formation was apparent on the scan indicating that this fracture

occurred at the earlier stage of the trial (prior to Week 2).

Figure 91: Sagittal reconstruction of CT scan of Pilot sheep 2. The accidental, second displaced fracture is clearly visible proximal to the most proximal screw hole.

Mechanical testing:

After six weeks of recovery, the relative torsional stiffness which is defined as the

percentage of the intact contra-lateral femur was smaller in Pilot sheep 1 (ORIF;

36.0%) than in Pilot sheep 2 (MIPO; 60.6%). The relative ultimate torque was also

smaller in Sheep 1 (22.6%) than in Sheep 2 (56.2%). The absolute values of both pilot

sheep are listed in Table 2.

Torsional stiffness Nm/Deg Ultimate torque Nm Femur side: Right (fractured) Left (intact) Right (fractured) Left (intact) Sheep 1 (ORIF) 0.18 0.51 14.2 62.7 Sheep 2 (MIPO) 0.39 0.65 35.4 63.1

Table 2: Mechanical test results of Pilot sheep 1 and 2.

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3.9.1.3 Discussion and Conclusion

The purpose of the pilot study which used two sheep as experimental models was to

examine the entire experimental setup and course of recovery of fractures created in the

femurs of sheep. In terms of animal recovery, the pilot tests were successful as both

sheep underwent the two-stage procedure and recovered successfully afterwards. The

animal behaviour appeared normal and any weight lost within the first week post-

procedure was subsequently regained. After the first two to three weeks, the gait of the

sheep returned to an almost normal pattern with the right hind limb able to support the

full weight bearing on it. At the site of the procedure, no adverse events occurred and

the wounds appeared to be healing well.

The use of sheep femur fractures as a model to study trauma and fixation

demonstrated an overall good outcome in terms of its ability to examine the process of

recovery. However, there was a mix of both constants and fluctuations among the

parameters measured. All anaesthetic and surgical operations were performed safely

and as planned. The soft tissue trauma showed elevated values of compartment

pressures but without any signs of compartment syndrome. High post-traumatic peaks

of the serum markers CK and LDH as an indication of severe soft tissue damage were

also not observed. The fracture model was very consistent with an AO C-type fracture

with two intermediate fragments in both animals. After the second operation,

monitoring of the soft tissue showed alterations from the first procedure. However, this

data was limited after the damage of one of the compartment probes. These events

demonstrate the importance of careful positioning and handling of these delicate and

fragile probes and cables. The external fixator application worked well as a temporary

fixation method. The position of the Schanz’ pins proved to be crucial for the second

procedure as the location of the pins may affect the positioning of the plate screws.

Another point of consideration in this minimally invasive procedure is to ensure the

complete insertion of the external fixator frame, and the positioning of the internal

fixator. Also, leaving the frame in place until the end of the plate fixation is vital. The

second operations which were either open or minimally invasive were successfully

performed without wound or fracture healing complications.

109

The position and the reduction of the fracture fragments were different, which is

due to the bridging osteosynthesis. From the clinical perspective, this difference is

acceptable. However, as part of the experimental model, the reduction of the axes and

rotation should be improved. Another important factor of the minimally invasive plate

fixation procedure is the plate position. It should be as closely as possible located to the

bone as when done using the open technique.

The recovery of the soft tissue damage was, as expected, occurred within the first

10 to 14 days of the experiments. This indicated that no changes to the original methods

used were necessary. The fortnightly conventional radiographs presented a useful visual

sequence of events occurring during the fracture healing process. These radiographs

indicated significant callus formation from week 4 onwards. The results of the

computer tomography and the mechanical testing confirmed these findings by showing

a similar progress in fracture healing. The computer tomography revealed a

morphologically clear callus formation while experimental data on the mechanical

integrity of the callus indicated the strength of the healed fracture.

These results were presented and discussed in a video-conference meeting with our

international collaborators, Prof Keita Ito, Romano Matthys, Peter Toggwiler and Dr

Simon Pearce from the AO Research and Development Institute, Davos, Switzerland.

From this meeting, it was concluded that the trauma model produces a reproducible

severe soft tissue damage and multi-fragmentary distal femur shaft fracture. To treat the

damage and examine the processes involved in healing, the two-stage fixation model

was deemed suitable. Due to the advanced fracture healing progress seen after six

weeks of recovery time and to investigate differences in the early stages of recovery

which may lead to different healing outcomes, the recovery times of the two

experimental groups was changed from six to four weeks and from twelve to eight

weeks.

3.9.2 Main test series

After thorough planning and preparation including alterations to the main study, the

test series was initiated in November, 2006. The entire timetable of the main series is

shown (Appendix I). The first experiments were conducted on groups of two or four

110

animals over a course of eight weeks and were completed in April, 2007. The next set

of experiments was carried out on the animals of in the four weeks recovery group and

data was collected by October, 2007.

CCHHAAPPTTEERR 44:: RREESSUULLTTSS

4.1 CHARACTERISATION OF TRAUMA

4.1.1 Soft tissue trauma recovery

Results of local, intra-compartmental soft tissue monitoring

In a group of 16 sheep, the intra-compartmental probe was routinely inserted at the

end of the first procedure after the external fixator application was completed. The

intra-compartment pressure measurements (Fig. 92) in 16 animals revealed peak values

immediately following the operation of 6.2 ± 6.0 mmHg (mean value ± SD) within the

rectus femoris muscle. These values remained below the threshold of a pressure reading

indicating the onset of a compartment syndrome. The elevated pressure decreased over

the next five days with the lowest value measured on day 5 of 1.2 ± 1.6 mmHg.

Compartment pressure (cp) measurements (n=16)

0

2

4

6

8

10

12

14

2 16 26 40 50 64 74 88 98 112

time (hours postop)

mmHg

Figure 92: Mean values of the intra-compartmental pressures of 16 animals over a time span of five days. Critical compartment pressures (> 20 mmHg) were not reached.

Partial oxygen pressures (Fig. 93) could only be measured in seven of the 16

animals, due to defects in the probe catheter or cables which were inserted in these

animals. The results showed slightly elevated partial oxygen pressure within the first

two days after trauma, with the highest value of 69.8 ± 24.3 mmHg (mean ± SD)

111

measured after 48 hours. After this peak, the partial oxygen pressure decreased

continuously to reach final values of 44.8 ± 27.9 mmHg five days after the procedure.

Partial oxygen pressure (pO2) measurements (n=7)

0102030405060708090

100

2 16 26 40 50 64 74 88 98 112time (hours postop)

mmHg

Figure 93: Partial oxygen pressure monitoring within the rectus femoris muscle of the right thigh following operations. Due to damage to measuring equipment, only seven animals remained for evaluation. The mean values (n=7) and the standard deviations are listed.

Results of systemic soft tissue monitoring by serum markers

In all the 24 animals included in this study, daily blood samples were taken from

the central venous line over five days. The evaluation of the serum levels of CK and

LDH, as markers of cellular break down, yielded peak values of both CK and LDH one

day after trauma and surgery, with 1140.2 ± 672.0 U/L and 627.9 ± 130.6 U/L,

respectively. These values decreased over the following two days back to

physiologically normal serum levels (Fig. 94).

112

Creatine Kinase (CK) and Lactate Hydrogenase (LDH) serum levels (n=24)

0

500

1000

1500

2000

pre-op post-op 1 2 3 4 5time (days)

U/L

LDH

CK

Figure 94: Mean values (n=24) and their standard deviations of the CK and LDH serum levels over a period of five days.

4.1.2 Fracture model

The fracture creation technique was successfully performed in vivo in 24 sheep

through a small (3 cm) antero-lateral, trans-muscular approach. The conventional post-

operative x-ray radiographs showing both the antero-posterior and latero-medial aspects

of the fracture as well as the evaluation of computer tomography data confirmed the

AO C3-type fracture pattern in all of the 24 fractures (Fig. 95/96). Further analysis

based on 3D-CT scan data showed 21 fractures (87.5%), where the middle segment was

divided into two fragments as intended (Fig. 95) and only three fractures (12.5%),

where the middle segment was divided into three fragments (Fig. 96). The average

length of the fracture zone, measured on the post-operative x-rays in both planes (Fig.

68/69) was 30.1 ± 2.7 mm. Fracture lengths (Appendix J) showed consistency across all

fractures. The measurement of the reduction angles of the bone axes in both planes

(Fig. 97) showed slightly varus position of 3.2 ± 5.5 degrees and a minor ante-curved

position of 2.6 ± 7.0 degrees. All 24 distal femur fractures were stabilised using the

same V-shaped external fixator construct (Fig. 51) without any complications observed

clinically during the five days.

113

Figure 95: These post-operative x-rays show the fracture configuration (AO C-type with two intermediate fragments), the aligned fracture reduction as well as the appropriate positioning of the external fixator.

Figure 96: The three-dimensional CT reconstructions of the cortical fragments illustrates the multi-fragmentary fracture pattern (AO C-type) with three intermediate fragments, the smallest fragment located in the proximal posterior region. The images are from the antero-lateral and medial perspectives respectively.

114

Figure 97: These two post-operative x-ray radiographs processed with AMIRA show the angles of alignment in both axes. On the left, a varus position of 7 degrees, and the x-ray on the right shows a slight retro-curved position of 3 degrees.

4.2 MINIMALLY INVASIVE VERSUS OPEN PLATE OSTEOSYNTHESIS

4.2.1 General results

The 24 sheep used in this experimental study had a mean age of 5.4 years, a mean

starting body weight of 39.1 kg and a mean final body weight of 37.9 kg. The thigh

circumference of the completely shorn and shaved right hind limbs was an average of

32.0 cm. The detailed physical database is listed in Appendix K.

All the procedures conducted had no adverse effects on the animals. Also, during

the soft tissue trauma and the postoperative aftercare, there were no clinical signs of

any distress or discomfort caused by pain. All surgical interventions were performed by

the same surgeon. The operation time for the minimally invasive technique of 51 ± 16

minutes was significantly (p<0.005) shorter compared to the open surgical method of

76 ± 16 minutes. For the first six days, the sheep remained in the supporting trolleys,

which caused some distress and sleeping problems in the animals. Despite an

appropriate food and water intake during the initial post-operative period, all the

animals lost approximately 10% of their body weight. Overall however, every sheep

stayed in general healthy condition. Regular observations of the animals’ behaviour in

115

the outside paddock showed that most of the animals started to use the operated hind

limb within the first two weeks with touch or even partial weight bearing. After

approximately four weeks, the sheep appeared to be fully weight bearing and the

injured leg fully functional.

From the experience of the pilot study with a fall and the occurrence of the

proximal femur fracture, precautions were undertaken to prevent the animals from

slipping and falling on the slippery concrete floor of the animal facility. Nevertheless,

four animals had significant falls and sustained fractures and implant failures, which

were confirmed radiologically (Fig. 98). These animals were sacrificed immediately

and replaced. One animal from the 4 weeks group, which had been operated with the

minimally invasive method, developed a wound infection at one of the proximal pin

sites after the second operation. After regular washing and disinfection of the wound,

the infection appeared to have healed. The post-mortem examination via dissection and

explanation of the femur revealed no clinical signs of any ongoing soft tissue

infections. However, osteolysis around the proximal holes of the external fixator as

well as of the internal fixator was seen. The decision was made to exclude this sheep

from further evaluations.

Figure 98: X-rays of a sheep which sustained a fall on the first post-operative day confirmed a suspected fracture extension with proximal implant failure.

116

4.2.2 Soft tissue recovery

For the first 14 days of the experiments, the effect of the soft tissue injury and its

recovery were monitored locally using the compartment measurement probes and

systemically via the analysis of serum markers. Blood for the serum marker analysis

was collected from all sheep for the entire duration of the experimentation period. All

16 compartment measurement sensors were operational after insertion during surgery.

Within the first seven days, two of the minimally invasive group and five of the open

group lost the partial oxygen pressure probes or cables due to technical failure. Due to

issues concerning reproducibility and small sample sizes, analysis of the partial oxygen

pressure measurement was therefore not performed.

The compartment pressures recovered to normal levels after peaking following the

first procedure and presented a rise after the second operation (Fig. 99). The second

peak of the compartment pressure tended to be higher in the ORIF group (9.8 ± 10.0

mmHg, p = 0.08) than in the MIPO group (3.3 ± 4.5 mmHg). Over the next one and

half days the compartment pressure fell to normal levels below 5 mmHg.

Intra-compartmental pressure measurement (n=8)

0

2

4

6

8

10

postop

11 /

am1 /

pm2 /

am2 /

pm3 /

am3 /

pm4 /

am4 /

pm

preop 2

postop

26 /

am6 /

pm7 /

am7 /

pm8 /

am8 /

pm9 /

am9 /

pm

10 / a

m

10 / p

m

11 / a

m

11 / p

m

12 / a

m

12 / p

m

13 / a

m

13 / p

m

14 / a

m

14 / p

m

time (postop days)

mmHg

MIPO

ORIFpost 1st procedure: trauma and external fixation

post 2nd operation: plate fixation

Figure 99: Intra-compartmental pressures over the entire soft tissue monitoring time of 14 days. After the second, randomised operation (MIPO and ORIF), the peak of the ORIF group is higher (p=0.08) than the MIPO group.

The evaluation of the systemic soft tissue monitoring by serum markers during the

first 14 days following the procedures showed two peaks, one following the initial

operation and creation of the fracture and soft tissue trauma, the second occurring

following the second operation to replace the external fixator with the internal fixation

device (Fig. 100/101).

117

The serum Creatine Kinase (CK) levels on the first post-operative day after the

second procedure were significantly higher (p = 0.01) in the sheep operated with ORIF

approach (1029 ± 827 U/L) compared to MIPO (286 ± 260 U/L). Serum levels of CK

returned to normal levels (<200 U/L) on the 9th

Creatine Kinase serum level

0

500

1000

1500

pre post 1 2 3 4 pre post 6 7 8 9 11 14

Time (days)

Cre

atin

e K

inas

e co

ncen

trat

ion

(U/L

)

ORIF

MIPO

*p < 0.05

post 2nd operation: plate fixation

post 1st procedure: trauma and external fixation

post-operative day in both groups.

Figure 100: The Creatine Kinase serum levels (n=12) show two peaks, after the trauma and external fixator application as well as after the second operation for definitive plate fixation. The second peak is significantly higher with the ORIF group compared to the MIPO group.

A similar trend was observed for the serum LDH levels. On the first (ORIF 617 ±

179 vs. MIPO 484 ± 91 U/L), second (ORIF 559 ± 197 vs. MIPO 408 ± 81 U/L) and

third (ORIF 483 ± 98 vs. MIPO 397 ± 78 U/L) postoperative days following the second

operation the LDH levels were significantly higher (p < 0.05) in the ORIF group.

118

Lactate Dehydrogenase (LDH) serum levels

200

300

400

500

600

700

800

900

preop postop 1 2 3 4 preop postop 6 7 8 9 11 14time (days)

LDH

ser

um c

once

ntra

tion

U/l ORIF

MIPO

*p < 0.05*p < 0.05

*p < 0.05

post 2nd operation: plate fixationpost 1st procedure: trauma and external fixation

Figure 101: Serum LDH concentrations (n=12) over a period of the first 14 days. The first peak indicates the soft tissue damage after the first procedure (trauma and external fixator application). The second peak follows the second operation of the randomised plate fixation: the first three days post-operatively (day 6, 7 and 8) the LDH levels are significantly higher in the ORIF group than in the MIPO group.

4.2.3 Fracture healing

Conventional radiography

The progress of the fracture healing was monitored with bi-weekly conventional

radiographs taken in two planes, perpendicular to each other (antero-posterior and

latero-medial). The postoperative x-rays after the second operation showed good

reduction and alignment of the two main fragments with appropriate implant positions.

The fracture reduction of the middle fragments was better aligned with the open method

(ORIF) as compared to the minimally invasive technique. The analysis of the

fortnightly x-rays over the eight week period of time showed the beginning of callus

formation at week 2. Further expansion of the calcified callus areas including the

formation of callus bridges was apparent at week 4. At week 6, further calcification

with more callus formation was evident and finally at week 8, callus formation had

progressed and showed clear signs of callus bridges. These observations are confirmed

by the assessment of the number of callus bridges on radiographs (Fig. 102). At all time

points there is the trend of more progressed callus formation across the fracture gaps for

the MIPO group at 4 weeks. The difference of callus formation in this group as

119

compared to the ORIF group is statistically significant (p = 0.006). Two representative

x-rays from each experimental group are attached in Appendix L.

Number of callus bridges from conventional radiographs

0

1

2

3

4

2 weeks 4 weeks 6 weeks 8 weeks

MIPO

ORIF

Figure 102: Callus bridges at each of the fortnightly time points from the conventional radiographs in both planes. At four weeks recovery time, MIPO group presents significantly higher callus bridges than the ORIF group (p = 0.006).

Computer tomography (CT scan)

The 3D-reconstructions of the post-mortem CT scans were analysed for differences

in the fracture configuration and the callus morphology. The analysis of the callus

morphology was divided into three parts: the callus volumes and lengths, the cross-

sectional callus areas through the proximal and distal fracture planes as well as the

morphological callus bridging score measured in two and three dimensions.

The evaluation of the fracture configuration, in terms of differences in the fracture

reduction, showed no significant differences in the fracture length, neither the maximal,

3D fracture length (ORIF 35.8 ± 9.1 mm (mean ± SD) and MIPO 31.1 ± 2.8 mm) nor

the anterior fracture length from the proximal to the distal anterior osteotomy (ORIF

27.6 ± 4.9 mm and MIPO 25.7 ± 2.7 mm). The measurements of the maximal 3D

fracture displacement, however, highlighted a significantly larger distance in the

minimally invasive group with 9.8 ± 2.2 mm (p=0.04) as compared to the open

reduction method with 7.5 ± 2.9 mm.

Callus morphology between both surgical approaches was measured using the total

mineralised callus volume and the total callus length (Table 3). At both time points, 4

p<0.05

120

and 8 weeks, similar mineralised callus volumes were determined in both groups.

Similarly, no significant differences in callus length were observed at either time point.

Callus volume (mm3 ORIF ) MIPO P Value

4 weeks 19100 ± 8500 17400 ± 4800 p = 0.70

8 weeks 24400 ± 8500 20700 ± 5700 p = 0.39

Callus length (mm)

4 weeks 89.7 ± 14.0 80.5 ± 15.5 p = 0.33

8 weeks 72.9 ± 10.1 76.7 ± 7.2 p = 0.47

Table 3: Total mineralised callus volumes and callus lengths determined by 3D-reconstructions of the images from the CT scans at 4 and 8 weeks post-operatively.

The measurements of the cross-sectional callus areas were made in the proximal as

well as in the distal fracture plane. At 4 weeks, the callus areas of the MIPO group were

higher than those at 8 weeks for the ORIF group (Table 4) but none of those results

were statistically significant (p>0.05).

Callus area proximal (mm2 ORIF ) MIPO P Value

4 weeks 256.5 ± 151.9 337.3 ± 142.9 p = 0.39

8 weeks 538.6 ± 92.8 490.4 ± 140.7 p = 0.50

Callus area distal (mm2 )

4 weeks 390.0 ± 139.7 437.6 ± 106.2 p = 0.54

8 weeks 559.3 ± 159.7 508.2 ± 77.2 p = 0.50

Table 4: Cross-sectional callus area in the proximal and distal fracture plane of both groups and at both time points.

The semi-quantitative callus bridging score indicated statistically significantly

higher callus bridging rates at the 4 weeks time for the MIPO group (38.2 ± 8.9 (SD);

p=0.007 with the 3D method and 27.6 ± 15.3; p=0.039 with the 2D method) compared

to the ORIF group (20.0 ± 8.6 with the 3D technique and 10.7 ± 7.4 with the 2D

technique). At 8 weeks of recovery, this difference of the callus bridging score was not

statistically significant (p>0.05).

121

Mechanical Testing (Figure 103)

Mechanical tests were conducted to obtain absolute and relative measures of the

mechanical properties including torsional rigidity and ultimate torque at failure. After 4

weeks, the absolute torsional stiffness was significantly higher in the MIPO group (0.21

± 0.06 Nm (mean ± SD) with p=0.006) as compared to the ORIF group (0.06 ± 0.07

Nm). The absolute ultimate torque also presented higher values for the MIPO group

(12.7 ± 8.1 Nm; p=0.15) than for the ORIF group (5.8 ± 6.3 Nm).

After 8 weeks, the absolute torsional stiffness showed similar values of both groups,

slightly higher for the minimally invasive approach (MIPO 0.62 ± 0.12 Nm/deg (mean

± SD; p=0.46) and ORIF 0.56 ± 0.15 Nm/deg). The absolute torsional strength was 37.6

± 10.0 Nm/deg for MIPO group and 37.2 ± 11.9 Nm/deg for the ORIF group.

After 4 weeks, the relative torsional rigidity of the fractured femur as a percentage

of the intact contra-lateral bone was significantly higher (p=0.018) in the MIPO group

(30.1 ± 10.6%) compared to the ORIF group (9.8 ± 12.4%). The mean torsional

moment at failure was higher (p=0.11) in the MIPO group (20.7 ± 11.0% versus 9.6 ±

9.8%).

After 8 weeks, the relative torsional rigidity tended to be higher (p=0.36) in the

MIPO group (130.4 ± 61.9%) compared to the ORIF group (100.4 ± 41.4%). The

torsional moment at failure after 8 weeks also tended to be higher (p=0.26) in the MIPO

group (79.0 ± 23.6 vs. 64.4 ± 18.3%).

122

Mechanical test results

64.4

9.6

100.4

9.830.1

20.7

129.4

79.0

4 weeks torsional rigidity

8 weeks torsional rigidity

4 weeks ultimate torque

8 weeks ultimate torque

% o

f int

act c

ontr

a-la

tera

l fem

ur ORIF

MIPO

Figure 103: The mechanical properties of torsional rigidity and ultimate torque in relation (percentages) to the intact, contra-lateral femur are higher in both timelines with MIPO than in ORIF technique. After four weeks, the MIPO group shows significantly stiffer mechanical properties than the ORIF group (p=0.018).

Correlation analysis

Any correlations between the mechanical test results and the morphological

findings of the callus formation from the CT scans were then analysed. Firstly, the

relationship between the results of the absolute torsional stiffness and the proximal

cross-sectional callus area at 4 weeks was found to be reasonable with R²=0.43 (Figure

104). The correlation between the distal cross-sectional callus area however was

considerably smaller with R²=0.27.

Correlation between mechanical property and proximal callus area at 4 weeks

y = 960.37x + 156.54R2 = 0.4254

0100

200300400

500600

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35

torsional stiffness (Nm/deg)

prox

imal

cal

lus

area

(m

m2 )

Figure 104: This graph shows the good correlation (R²=0.43) between the absolute torsional stiffness (Nm/deg) and the cross-sectional callus area of the proximal fracture zone of all eleven animals at four weeks.

p<0.05

123

Secondly, the relationship between the mechanical properties and the

morphological callus bridging score (3D data) showed a high correlation at 4 weeks

with R²=0.79 (Fig. 105) and a reasonable correlation at 8 weeks with R²=0.35 (Fig.

106).

Correlation between mechanical property and callus morphology score at 4 weeks

y = 0.7354x + 14.285R2 = 0.7939

0

10

20

30

40

50

60

0 5 10 15 20 25 30 35 40 45 50torsional stiffness (% of intact bone)

brid

ging

sco

re

Figure 105. Correlation between the relative torsional stiffness and the 3D callus bridging score of all eleven sheep at four weeks recovery time.

Correlation between mechanical property and callus morphology score at 8 weeks

y = 0.1747x + 35.3R2 = 0.3483

30

35

40

45

50

55

60

40 50 60 70 80 90 100 110 120ultimate torque (% of intact bone)

brid

ging

sco

re

Figure 106: Correlation between the relative ultimate torque at failure and the 3D callus bridging score of all twelve animals at eight weeks observation time.

124

CCHHAAPPTTEERR 55:: DDIISSCCUUSSSSIIOONN

5.1 TRAUMA MODEL

The aim of the first part of this project was to create a suitable and reliable trauma

model to enable us to perform experimental studies on large animals (in this case

sheep) and to examine and answer clinically relevant research questions in the field of

orthopaedic trauma. A specific trauma model provided the inspiration towards this

doctoral project. A car bumper hit to a pedestrian leg causes a severe injury to the limb.

This type of injury contains severe soft tissue damage as well as a complex fracture.

Therefore, to investigate the trauma model further, the region of interest was defined to

be the distal part of the thigh and femur. Based on the information from previous

research studies and pilot experiments, an all-in-one trauma model to create such a

complex injury was abandoned. Consequently, the trauma model was developed,

validated and implemented in two separate parts: the severe soft tissue damage model

classified according to Tscherne 2 to be of grade III and the multi-fragmentary fracture

model classified according as AO type C-fracture 3

Many challenges had to be overcome throughout the development stage of both the

models. The validation process involved the replicating each newly developed step

several times to ensure reliability and consistency of the results. Further adjustments to

both the components of the trauma model were made as required. This finally led to the

following optimised protocol. Under general anaesthesia, the combined trauma was

achieved in one single session, with approximately 20 minutes required for the entire

procedure. The pendulum impact device created the desired degree of severity of soft

tissue injury in a reproducible manner. The consistency of the experiments was

confirmed by post-mortem macroscopic dissection and post-mortem CT scans, as well

as the consistent increase in serum breakdown markers (CK and LDH). The appropriate

positioning of the sheep’s femur in the device is simple and guarantees that the injury is

limited to the soft tissues only. The steps for creating the fracture consisted of partial

osteotomies, drill holes, blade bar insertion and chisel hits to initiate the fracture lines.

. Finally, in the in vivo experiments,

both parts were carried out in sequence and hence, collectively turned out to be the

novel, combined trauma model.

125

All these procedures were carried out with considerable consistency and created AO

C3-type fractures 3

. All these steps could be performed through a small, trans-muscular

approach, thereby minimising further iatrogenic damage to the soft tissues. Finally, all

fractures could be stabilised subsequently with the same configuration of the external

fixator frame, as well as internal fixator construct, resulting in a comparable mechanical

environment for all animals, which further minimises variability during the course of

fracture healing.

Soft tissue damage and recovery

Local monitoring of the soft tissue damage and its recovery with the compartment

probe clearly showed changes of the compartment pressure in the traumatised rectus

femoris muscle. The compartment pressure peaked post-operatively within a few hours

up to 6 mmHg (maximally 12 mmHg), despite a ‘mini’-fasciotomy performed during

the small approach for the fracture creation. This corresponds with a moderate damage

and muscle swelling. Over the following two days the compartment pressure recovered,

while the partial oxygen pressure reached its highest value in this period. These

physiological changes may be explained by the hyper-perfusion reaction of the

inflammation phase during the early recovery of the muscle damage. This observation

is consistent with the measurements by Epari et al. (2008) 114, the only published report

on partial oxygen pressure in a fracture healing study to date. They used the same

sensor to monitor hydrostatic pressure, partial oxygen pressure and temperature, in a

simple, tibia osteotomy model in sheep. In their experiments, measurements could only

be taken from seven of the 16 implanted sensors, whilst the other sensors were

damaged during the experimental course. These circumstances including the small

sample size and the high variations of the values from day three onwards, did not allow

them to draw further conclusions. The challenges of using this highly sensitive sensor

in sheep have been discussed by Epari et al. (2008) 114

The systemic soft tissue monitoring with serum markers for tissue breakdown

started with pre-operative baseline values. Some of those values were above the normal

level, which may be explained by pre-operative physical stress and soft tissue

contusions caused by falls during the preparation stage. All animals showed the highest

.

126

CK and LDH levels on the first post-operative day. These peak values reached 2610 U/l

for CK (normal range from 50 to 180 U/l) and 1010 U/l for LDH (normal range from

150 to 400 U/l), which reflects significant, severe soft tissue and muscle damage,

especially with this localised injury. The analysis of daily blood tests showed steadily

decreasing values and all animals including those with high serum marker

concentrations recovered within three to four days. This short recovery time highlights

the healthy general condition of the sheep. However, as the macroscopic dissection in

the ex vivo experiments and the visual functional observation of the animals in the in

vivo experiments confirmed, the injury was limited to the anatomical structure of the

muscles only. Gait analysis or weight bearing studies were not performed, however, the

limb function recovered well from the initial reduced weight bearing and limping. At

any time, all injured limbs were adequately perfused and moved, ruling out any damage

to the vital structures, such as the main blood vessels and the nerves.

The amount of muscle mass in the area of trauma which was measured by the

circumference of the distal thigh, did not vary significantly between the individual

animals (32 ± 1.1 cm (mean ± SD), range from 30.5 to 34.5 cm) or between the

experimental groups. The serum markers, LDH and CK, to measure the muscle damage

presented a relatively wide distribution of the values. This outlines a certain limitation

for the use of these serum markers to quantify muscle damage. These findings are

supported by the clinical experience with the inter-individual variability of those

markers. To determine and validate the amount of muscle damage morphological

assessments such as in vivo CT scans with intravenous contrast, in vivo MRI and early

histological investigations, could have been included. The use of these methods was

evaluated, but unfortunately they were not accessible for this study for logistical

reasons.

The newly developed soft tissue trauma device was successfully implemented and

used in this in vivo ovine study. Severe soft tissue injury was achieved without any

local or general complication, such as nerve damage, main blood vessel damage, the

development of a compartment syndrome or a crush syndrome with renal failure. The

severity of the damage was consistent within a range of physiological and inter-

individual variability and reflected quite realistically a clinical scenario.

127

Fracture creation model

The primary goal was to create a realistic and natural multi-fragmentary fracture

with a consistent pattern. In order to strike a balance between reproducibility and close

similarity to clinical, high impact fractures with multiple fragments, the fracture

configuration was defined to be an AO C-type 3 pattern with two intermediate

fragments. High forces are required to break cortical bone in sheep, especially in certain

anatomical areas, such as the anterior cortex of the distal femur. On the other hand,

ovine bones tend to split, mainly in longitudinal direction, which can cause

inconsistency in the length of the fractures. This could potentially result in differences

in mechanical conditions of the fracture site as well as manifest as problems to stabilise

them with the same mechanical construct. These challenges were overcome by a few

methods as detailed below. Firstly, bones were weakened in pre-determined areas. Then

the fracture lines were initiated in a sequential manner as published by Baumgaertel et

al. (1994) 39. Partial anterior osteotomies were required to weaken the strong anterior

cortical bone of the distal femur. This procedure was performed carefully by cooling

the blade and the bone edges with continuous rinsing with sterile saline. This

precaution was undertaken due to the possibility of the development of heat necrosis at

the adjacent bone fragments. However, a disadvantage of this fracture model was the

creation of smooth fracture lines at the osteotomy sites. These smooth osteotomy sites

behaved mechanically different to the spiky fragments observed in clinical fractures

with more contact surfaces. All procedures carried out towards bone weakening and

fracture initiation were performed through a small, consistent trans-muscular surgical

approach. This procedure did not replicate the most common clinical scenario with

either an open compound or a closed blunt fracture mechanism. The open approach

thereby influenced the development of the compartment pressure by releasing, at least

partially, the intra-muscular pressure by performing a ‘mini-fasciotomy’. However,

high-impact injuries often result in lacerations of the soft tissue envelope which would

have the same effect 6

The analysis of the fracture configuration of the 24 in vivo fractures showed a high

consistency in fracture classification with 100% AO C3-type fractures, in fracture

pattern and fracture length. 87.5% of fractures were with two intermediate cortical

.

128

fragments. The fracture lengths were with constant values of 30.1 ± 2.7 mm (mean ±

SD). Additionally all fractures could be stabilised with the same implant configuration

(external fixator and internal fixator). This provided comparable mechanical conditions

between the specimens and emphasized the reliability of the achieved fracture model.

5.2 MINIMALLY INVASIVE VERSUS OPEN PLATE OSTEOSYNTHESIS

Recently, there has been a trend in clinical practice towards less invasive surgical

procedures that preserves the integrity of the tissues. This way, the biological potential

for optimal healing conditions is maximised. This ‘damage reduction’ concept has also

been applied to the operative treatment of fractures. The aim of minimally invasive

plate osteosynthesis is to minimise the iatrogenic damage including any disturbance to

the vascularity of the fracture zone and the surrounding soft tissues. From the clinical

experience, this new technique seems to contribute to an advanced fracture healing in

terms of shorter recovery times with less pain and discomfort for the patient. There are

also fewer complications that occur such as infections and delayed-unions. Despite

these promising experiences, there is a lack of solid clinical and experimental evidence

supporting the advantages for MIPO 1; 76. Furthermore, there are concerns that the

perceived benefits of MIPO may be outweighed by the demanding surgical technique,

the possibility for mal-reduction and mal-alignment as well as the increased exposure to

radiation during visualisation 63

This animal model was designed to replicate a clinical situation of a multi-

fragmentary fracture and associated severe soft tissue injury that typically results from

a high-energy accident. The treatment modalities were chosen according to clinical

practices. The initial treatment of this severe combined injury involved the preliminary

fracture reduction and stabilisation with the application of an external fixator frame.

The external fixator immobilised the injured area and aided the recovery process of the

soft tissues. In our study, this recovery could be demonstrated by the restoration of

normal levels of local and systemic parameters after an initial increase. After this

. The goal of the present study was therefore to compare

the healing course of fractures treated either with a standard open surgical approach or

by the minimally invasive plate osteosynthesis technique, using the newly developed

trauma model in sheep.

129

recovery period of four days, the second surgical procedure with fracture reduction,

randomised, definitive internal plate fixation, as well as removal of the external fixator

was carried out safely. This minimised the risk of reaching critical inflammation levels

of the soft tissues.

The demographic data of the sheep were normally distributed in terms of

differences in age, body weight and muscle mass of the right thigh. All operative

procedures were performed without any intra-operative complications. Despite the

more demanding percutaneous technique, the duration of the minimally invasive

surgery was significantly shorter. This difference in operation time could be even larger

in the clinical environment, where the image intensifier is also routinely used in the

ORIF method and not just the MIPO technique and further prolongs the surgery time.

Shorter procedure times could potentially impact the patient’s morbidity in terms of a

slightly lower peri-operative complication risk and lower rates of wound infections,

especially if the operation exceeds the two hour time span. Furthermore, shorter use of

the operating theatre could have a positive logistical and health-economical impact.

All except one sheep presented uneventful recoveries with adequate wound healing

and timely return of limb function and full weight-bearing status. The infected sheep

presented a wound infection at one of the proximal external fixator pin sites. The site of

infection was not related to either of the randomised procedures and the sheep was

consequently excluded from the study. Such a pin track infection especially during the

hot and humid weather conditions of Brisbane was not a surprise. However of

particular relevance, this event did not influence the outcome of the study.

Following the second operation, the enzyme levels (CK and LDH) were distinctly

different between the two groups. In the ORIF group, both enzyme levels peaked at a

similar magnitude to that caused by the soft tissue injury. In contrast, only a small

increase was determined in the MIPO group. In addition, higher values of compartment

pressure occurred following internal fixation with the open approach, although in both

groups the measured hydrostatic pressures in the tissue were below those indicating in a

potential compartment syndrome (< 20 mmHg). These in vivo measurements of intra-

muscular pressure and the serum markers associated with tissue breakdown clearly

demonstrate that a higher degree of tissue injury is coupled to the open surgical

130

approach. This finding was certainly expected from the clinical experience, however,

the question that remains to be answered is whether these ‘biological’ differences

around the fractured bone significantly influence the fracture healing outcome or not.

The course and outcome of fracture healing was assessed with monitoring the callus

progress by fortnightly x-rays, post-mortem morphological evaluations based on CT

scans as well as post-mortem mechanical testing to determine the mechanical properties

of the healed femurs. The healing of the femur was monitored at two defined time

points, four and eight weeks post-operatively. To sum it up, in this severe animal

trauma model, the fracture healing showed advanced progress with significant

differences in the early stage (four weeks) and smaller differences at the later stage

(eight weeks) for the minimally invasive plate osteosynthesis.

The evaluation of the biweekly conventional radiographs (x-rays) taken in two

perpendicular planes, presented at all time points further progressed callus healing with

more connecting callus bridges (significantly higher value after four weeks) in the

MIPO group compared to the ORIF group. A limitation of the analysis and

interpretation of conventional radiographs originates from the two-dimensional

projections of the three-dimensional fracture callus. This represents a summation of the

mineralised areas, which may potentially cover and hide non-calcified regions.

Furthermore, the analysis is highly dependent on the quality of the pictures and the

exact leg position during the radiographic procedure. In the presented series, the quality

of the radiographs was assured with constant radiation levels and a standardised

positioning of the image intensifier and the sheep hind limb. Nevertheless, some intra-

and inter-individual differences with respect to rotation and angulation in both planes

(antero-posterior and latero-medial) could not be avoided.

Quantitative analysis of the CT data was performed to specifically investigate the

callus morphology. No significant differences were found in the total mineralised callus

volume, total callus length and the proximal and distal cross-sectional callus areas

through the two transverse fracture planes. This may be partly explained by the large

variability in these measures, which is common and attributable to inter-individual

differences, as well as due to the complex fracture pattern used in this trauma model.

However, the total mineralised callus volume and length tended to be larger in the

131

ORIF group, while both cross-sectional callus areas showed higher values after four

weeks for the MIPO group and after eight weeks for the ORIF group. These results

indicate that the quality of callus, in terms of callus distribution, location of new bone

formation, mineral density of the callus organisation of the callus and possibly, even

early signs of callus remodelling must have been superior in the MIPO group to explain

the greater mechanical competence determined in that group. The results of the semi-

quantitative callus morphology analysis with the callus bridging score attempted to

capture some of those previously mentioned characteristics. The significantly higher

bridging score at four weeks for the MIPO technique with high correlation to the

mechanical properties (torsional stiffness) supported the advanced functional integrity

of the new bone formation from the morphological side.

The fracture healing was further quantified by determining the mechanical

competence assessed by torsional stiffness and torsional moment at failure of the

healing femurs. At the early time point of four weeks, a significantly higher torsional

stiffness was found in the group that underwent the minimally invasive plating

procedure. The torsional strength was also higher in this group, but not statistically

significant. Chehade et al. (1997) 125 investigated the relationship between stiffness and

strength changes during fracture healing and its clinical implications. They concluded

that the testing of stiffness is valuable primarily in assessing the progress towards

fracture union, while the ultimate strength more accurately describes the recovery of the

mechanical integrity until the fracture is clinically healed and the bone is fully

functional. Taking these findings into account, the significant difference in torsional

stiffness in the early healing phase gives evidence of a more advanced fracture healing

progress with MIPO. This enhanced mechanical competence could be explained by the

callus which has matured with more areas of highly mineralised callus bridges and

better positioned callus sites linking the fracture gaps. This observation is supported by

the higher callus bridging score of the CT scan evaluation. Especially the three-

dimensional analysis showed a more homogenous callus and more mineralised callus

bridges linking the cortical fragments for the bones of the MIPO group. From the

clinical point of view, at this stage of fracture healing the focus lies on the

demonstration of callus formation and maturation, indicating the successful progression

132

of the fracture healing towards union of the fracture fragments. The strength of the

fracture callus at this stage is secondary, as the bone is mechanically protected by the

internal fixator. Therefore, in light of the consistent mechanical conditions for the

bones of both groups as designed in this study, the increased stiffness in the healing

bones of the MIPO group at four weeks indicates an advanced healing rate that may

lead to an earlier fracture union. However, at eight weeks, the torsional stiffness and the

ultimate torque at failure tended to be higher in the MIPO group, but were no longer

significant. It has been demonstrated that once the fracture fragments are linked

together, the differences in the mechanical integrity are expected to be smaller (Schell

et al. (2005) 122

While the superior mechanical competence of the femurs that were stabilised by

means of MIPO can be explained partly through morphological advantages of the callus

of the healing bones, there are several possible explanations as to why the healing was

improved in this group. From the experimental design with exactly the same implant

construct, no difference in the overall mechanical stability was introduced. However,

differences were found in the degree of the reduction of the intermediate bone

fragments, with larger maximal displacement in the MIPO group. This may have

caused more micro-movements within the fracture zone and potentially increased the

mechanical stimuli for callus induction. Furthermore, the accelerated fracture healing

associated with the minimally invasive procedures may be attributed to a better

biological environment with less disruption of the bone vascularity and less irritation of

the healing process. As part of the open surgical approach, the fracture fragments were

cleaned and the fracture haematoma was washed out. It is possible that this procedure

caused a setback to the fracture healing process through possible renewal of the fracture

haematoma formation and inflammatory processes. This could have delayed the healing

process in comparison to the minimally invasive technique.

. At eight weeks, all bones in our study showed solid bridging of the

fracture fragments, which might explain the smaller differences of the mechanical

properties observed at that time.

In the single previous experimental study 1 performed to compare the less invasive

technique with the open approach, no differences in healing were found. That study was

carried out on sheep tibiae and the difference in the anatomical locations of the two

133

studies may have been the cause for the inability to detect a difference in healing.

Because the tibiae have minimal soft tissue coverage on the antero-medial aspect, the

bone can be approached just by a simple longitudinal, sharp dissection of the skin and

the subcutaneous layers without the need for a trans-muscular approach. Hence, even

the open approach would have caused minimal damage to the surrounding soft tissues.

To address this clinically relevant problem, it was necessary to use a suitable

experimental animal model. Hence, this study was developed in a novel trauma model

to mimic the conditions of a significant impact causing both a severe soft tissue injury

and a multi-fragmentary fracture. Whilst every effort was made to perform this in a

reproducible manner, there is more inherent variability in such a model as compared to

a simple osteotomy model studying bone healing. This variability made the analysis of

callus morphology more challenging. However, the benefits of a clinically relevant

trauma model outweighed this disadvantage.

Before discussing the clinical relevance of these results, it is pertinent to firstly

highlight the following exposition: The surgical procedures carried out in the present

study were performed on healthy, middle-aged sheep. Healing in sheep, in spite of their

early and occasionally excessive weight bearing, is observed to be very robust as

indicated by the extent that one must go to in creating models of non-union 126

This experimental, animal study and the previously discussed outcomes can lead

into the following possible outcomes in relation to clinical perspective and practice: In

general, soft tissue management and the biological handling of fracture care influences

the fracture healing process. Consequently, the soft tissues adjacent to and around the

fracture areas are to be treated cautiously to minimise additional, iatrogenic damage in

both, open and minimally invasive, techniques of fracture management. Fractures fixed

by MIPO may unite in a shorter timeframe than the ORIF, especially in anatomical

regions with larger surrounding soft tissues, such as the femur or the humerus. A faster

.

Therefore, it may be interpreted that the deficit of the blood supply to surrounding

tissues caused by open surgical approaches may be more pronounced in the clinical

situation than observed here, particularly in cases where a deficit in the biological

healing potential already exists through a pre-existing medical condition, such as

diabetes, smoking, vascular diseases or just through old age.

134

recovery from these injuries would definitely benefit the individual patients, but also,

when viewed at a larger scale, have further socioeconomic implications, with faster

return to work after trauma.

CCHHAAPPTTEERR 66:: CCOONNCCLLUUSSIIOONN AANNDD FFUUTTUURREE DDIIRREECCTTIIOONNSS

The newly developed trauma model, with consistent and reproducible results of

severe soft tissue injury and multi-fragmentary distal femur fractures, was successfully

implemented in a clinically relevant, in vivo experimental ovine model. Consequently,

this trauma model represents a significant contribution to the tools available for the

study of complex long bone fractures and severe soft tissue injuries, as well as the

combination thereof, in large laboratory animals.

The comparative study to investigate the effects of different surgical approaches on

the healing process demonstrated that the minimally invasive technique reduces the

additional soft tissue damage and leads to an advanced fracture healing outcome, at

least in the early healing stages, when compared to the conventional open reduction and

internal fixation method.

Further assessments of the present study:

The radiological methods used for the initial evaluation of the specimens in this

study (clinical CT scanning and planar x-rays) allowed for characterisation of the gross

callus morphology and a basic quantification of callus dimensions. However, for a

detailed analysis of callus morphology, and the progress of the callus formation on a

micro-structural level, other evaluation methods are necessary. The specimens from the

research for this study are available and while it was not possible to include other

measurements in the scope of this thesis, further investigations are planned to be

continued. Micro CT scanning and histological assessments will aim to more

specifically examine the integrity of the callus region. Firstly, micro CT scans of the

shaft region of the femurs will be performed to determine the detailed callus

morphology and bone density measurements for specific areas of interest within the

fracture zone. Such regions may include areas of superior callus apposition or on the

contrary, other regions where bridging or callus formation was delayed. After the

135

micro CT scans are completed, the specimens will be processed for histological

assessments. The results of the micro CT scan analysis will aid in the subsequent

histological evaluation in terms of making decisions as to which areas of the callus

need to be focused on or which directions the sectioning and preparation of the samples

should be performed. Histomorphometric evaluation of the callus and temporal callus

development will be performed using fluorescence labelling techniques. During the

healing period, all four week animals were injected with the fluorochromes Xylenol-

orange in week two and with Calcein-green in week four, while all the eight week

animals were injected Xylenol-orange in week six and Calcein-green in week eight.

The fortnightly injected fluorochromes stain areas where callus mineralisation is active

and therefore will allow for a dynamic observation of the callus formation and

mineralisation. All these further assessments aim to gain further insights of the

temporal and spatial development of the mineralised callus in this complex facture

situation.

Further studies using the new trauma model:

For future applications of this newly developed trauma model, further

characterisation of the different aspects of the model is necessary.

First, the soft tissue damage created with the pendulum impactor needs to be

evaluated in terms of the detailed structural and histo-pathologic changes in the affected

tissues and the recovery thereof. This evaluation should lead to a more detailed grading

of the severity of the soft tissue injury. Suitable methods for this evaluation include in

vivo imaging methods, such as contrast CT scans and MRI scans as well as histological

assessments at early time points. The most significant period of time with regard to soft

tissue healing, as outlined by the present study, is within the first seven to ten days post

trauma. Once the characterisation of the soft tissue injury is established, the trauma

model would be even more appropriate to study the influence of different severities of

the damage on the recovery process. The validated trauma model could then be used to

compare the fracture healing process in presence and absence of a soft tissue injury, or

the soft tissue recovery could be analysed in the presence or absence of a fracture.

A further limitation of the present experimental design comparing the two surgical

approaches is the delayed definitive fracture treatment, which potentially influenced the

136

open method more than the MIPO technique. The fracture area was thoroughly cleaned

during the ORIF procedure and may have led to a ‘delayed’ start of the fracture healing

process with renewal of the fracture haematoma. This topic could be addressed by

setting up a new experimental design with an initial, definitive fracture stabilisation

with an internal fixator. The study of the soft tissue damage and its recovery could be

included to add valuable further information on the clinically relevant question as to

when and in what manner severe fractures should be stabilised.

Another research topic for further investigation would be the study of the very early

stage of fracture healing including the characterisation and the development of the

fracture haematoma. Within the present study, a pilot experiment was carried out, in

which small volume extra-cellular fluid samples were collected by a small probe

housing a semi-permeable membrane directly from the fracture haematoma using

microdialysis methods. This was, to our knowledge, the first application of

microdialysis in the field of orthopaedic traumatology. The analysis of the samples

using this proteomics method is still ongoing and is hopeful that this will provide a

profile of the interstitial proteins and other constituents, whose expression is mostly

affected by the combined trauma. Depending on the outcome of this pilot study, this

method could be used for the future characterisation of the biochemical changes within

the fracture haematoma.

Further studies in the field of minimally invasive plate osteosynthesis:

The presented study was conducted primarily to assess the influence of the two

different surgical methods ORIF and MIPO on fracture and soft tissue healing directly.

However, there are other perceived advantages of the minimally invasive technique for

plate osteosynthesis that can be examined further:

• Reduced rate of infections: In large animals, such as sheep, it is logistically

difficult to perform a conventional experimental infection study. Such studies

usually examine the effect of different surgical methods on the onset of an

infection after exposure of the implant or animal (wound) to a controlled

number of bacteria. While small animal models would be more suitable for such

experiments, the application of reproducible fractures and the fixation thereof is

challenging in these cases, as outlined earlier. Alternatively, prospective clinical

137

studies can be conducted to establish statistical evidence for or against the

minimally invasive technique. Particularly in developing countries, where the

overall infection risk is increased due to compromised aseptic conditions in

operation theatres and the general hospital environment, the differences between

the two surgical methods could be highlighted and can significantly impact on

patient outcomes. Therefore, clinical study designs could include comparisons

of infection risks between the two surgical approaches in hospitals of developed

and developing countries.

• Better functional outcome: Gait analysis and weight-bearing measurements on

sheep during fracture healing using instrumented treadmills and force platforms

are established techniques and could be used to better assess limb function post-

operatively. Clinically, randomised trials could be designed to compare the time

of fracture union, delayed and non-union rates, the early and the final functional

outcome, as well as even socio-economic aspects, such as the time to return to

work, hospital and even overall costs.

Clinical impact of the present study:

Most importantly, the outcome of this study highlights that the different surgical

approaches do have an effect on the healing process of the soft tissues and the bones.

Consequently, in clinical practice, any surgical approach should be performed by

minimising the additional damage so that it does not compromise the healing process

and helps to create the optimal environment towards a successful outcome.

Based on the results of this study, MIPO can be considered as a safe and valid

option to treat certain long bone fractures. However, to be able to achieve good results,

a more challenging technique should be thoroughly planned and carefully performed.

Furthermore, in order to facilitate the practise of MIPO, as well as to reduce the

learning curve and complication rates, educational tools such as virtual skill stations

and cadaver workshops should be developed and implemented for surgeons and

trainees. Finally, the surgical technique of MIPO needs to be refined for special

anatomical regions and the implants and instruments need to be developed to support its

uneventful application.

138

BBIIBBLLIIOOGGRRAAPPHHYY

1. Schuetz, M, Schmeling, A, Kääb, M, et al. 1999. Effect of surgical approach on

fracture healing: comparison of minimal invasive approach (MIS) to conventional

open reduction and internal fixation technique (ORIF) in a sheep tibial shaft

fracture. 15th Annual Meeting OTA. Charlotte, USA.

2. Tscherne, H, Oestern, HJ. 1982. [A new classification of soft-tissue damage in open

and closed fractures (author's transl)]. Unfallheilkunde 85: 111-115.

3. Mueller, ME, Nazarian, S., Koch, P., etal. 1990. The Comprehensive Classification

of Fractures of Long Bones. Berlin Heidelberg New York: Springer-Verlag.

4. Bradley, C, Harrison, J. 2004. Descriptive epidemiology of traumatic fractures in

Australia. Injury Research and Statistics Series Number 17. Australian Institute of

Health and Welfare (AIHW), Adelaide: (AIHW cat no. INJCAT 57).

5. Gustilo, RB, Mendoza, RM, Williams, DN. 1984. Problems in the management of

type III (severe) open fractures: a new classification of type III open fractures. J

Trauma 24: 742-746.

6. Court-Brown, CM, Rimmer, S, Prakash, U, McQueen, MM. 1998. The epidemiology

of open long bone fractures. Injury 29: 529-534.

7. Tu, YK, On Tong, G, Wu, CH, et al. 2008. Soft-tissue injury in orthopaedic trauma.

Injury 39 Suppl 4: 3-17.

8. Giannoudis, PV, Einhorn, TA, Marsh, D. 2007. Fracture healing: a harmony of

optimal biology and optimal fixation? Injury 38 Suppl 4: S1-2.

9. Einhorn, TA. 1998. The cell and molecular biology of fracture healing. Clin Orthop

Relat Res: S7-21.

10. Dimitriou, R, Tsiridis, E, Giannoudis, PV. 2005. Current concepts of molecular

aspects of bone healing. Injury 36: 1392-1404.

11. Perren, SM. 2008. Fracture healing. The evolution of our understanding. Acta

chirurgiae orthopaedicae et traumatologiae Cechoslovaca 75: 241-246.

12. Ruedi, TP, Buckley, RE, Moran, CG. 2007. AO Principles of Fracture

Management. 2nd edition, Thieme New York, USA.

139

13. McKibbin, B. 1978. The biology of fracture healing in long bones. J Bone Joint

Surg Br 60-B: 150-162.

14. Phillips, AM. 2005. Overview of the fracture healing cascade. Injury 36 Suppl 3:

S5-7.

15. Brighton, CT, Hunt, RM. 1991. Early histological and ultrastructural changes in

medullary fracture callus. J Bone Joint Surg Am 73: 832-847.

16. Bhandari, M, Tornetta, P, 3rd, Sprague, S, et al. 2003. Predictors of reoperation

following operative management of fractures of the tibial shaft. J Orthop Trauma

17: 353-361.

17. Chao, EY, Inoue, N, Elias, JJ, Aro, H. 1998. Enhancement of fracture healing by

mechanical and surgical intervention. Clin Orthop Relat Res: S163-178.

18. Farouk, O, Krettek, C, Miclau, T, et al. 1999. Minimally invasive plate

osteosynthesis: does percutaneous plating disrupt femoral blood supply less than the

traditional technique? J Orthop Trauma 13: 401-406.

19. Gopal, S, Majumder, S, Batchelor, AG, et al. 2000. Fix and flap: the radical

orthopaedic and plastic treatment of severe open fractures of the tibia. J Bone Joint

Surg Br 82: 959-966.

20. Tielinen, L, Lindahl, JE, Tukiainen, EJ. 2007. Acute unreamed intramedullary

nailing and soft tissue reconstruction with muscle flaps for the treatment of severe

open tibial shaft fractures. Injury 38: 906-912.

21. Mueller, ME, Allgoewer, M, Willenegger, H. 1965. Technique of Internal Fixation

of Fractures. Berlin Heidelberg New York: Springer-Verlag.

22. Rhinelander, FW. 1974. Tibial blood supply in relation to fracture healing. Clin

Orthop Relat Res: 34-81.

23. Chalmers, J, Gray, DH, Rush, J. 1975. Observations on the induction of bone in soft

tissues. J Bone Joint Surg Br 57: 36-45.

24. Claes, L, Maurer-Klein, N, Henke, T, et al. 2006. Moderate soft tissue trauma

delays new bone formation only in the early phase of fracture healing. J Orthop Res

24: 1178-1185.

25. Utvag, SE, Grundnes, O, Rindal, DB, Reikeras, O. 2003. Influence of extensive

muscle injury on fracture healing in rat tibia. J Orthop Trauma 17: 430-435.

140

26. Melnyk, M, Henke, T, Claes, L, Augat, P. 2008. Revascularisation during fracture

healing with soft tissue injury. Arch Orthop Trauma Surg 128: 1159-1165.

27. Hansmann, C. 1886. Eine neue Methode der Fixierung der Fragmente bei

komplizierten Frakturen. Verh.Dtsch.Ges.Chir. 158.

28. Lambotte, A. 1907. L'intervention operatoire dans les fractures recentes et

anciennes. Maloine, Paris.

29. Lane, WA. 1914. The operative treatment of fractures. Medical Publishing, London.

30. Danis, R. 1947. Theorie et pratique de l'Osteosynthese. Masson et Cie, Paris.

31. Mueller, ME, Allgoewer, M, Schneider, R, Willenegger, H. 1992. Manual der

Osteosynthese.

32. Kuentscher, G. 1940. Die Marknagelung von Knochenbruechen. Arch Klin Chir

200:443.

33. Gautier, E, Ganz, R. 1994. [The biological plate osteosynthesis]. Zentralblatt fur

Chirurgie 119: 564-572.

34. Mast, JW, Jakob, RP, Ganz, R. 1989. Planning and reduction technique in fracture

surgery. Berlin, Heidelberg: Springer.

35. Perren, SM, Cordey, J, Rahn, BA, et al. 1988. Early temporary porosis of bone

induced by internal fixation implants. A reaction to necrosis, not to stress

protection? Clin Orthop Relat Res: 139-151.

36. Alexander, AH, Cabaud, HE, Johnston, JO, Lichtman, DM. 1983. Compression

plate position. Extraperiosteal or subperiosteal? Clin Orthop Relat Res: 280-285.

37. Gautier, E, Rahn, B, Perren, S. 1995. Vascular remodelling. Injury 26 Suppl.

38. Haasnoot van, FE, Muench, WH, Matter, P, Perren, S. 1995. Radiological

sequences of healing in internal plates and splints of different surface to bone (DCP,

LC-DCP and PC-Fix). Injury 26 Suppl.

39. Baumgaertel, F, Perren, SM, Rahn, B. 1994. [Animal experiment studies of

"biological" plate osteosynthesis of multi-fragment fractures of the femur].

Unfallchirurg 97: 19-27.

40. Perren, SM. 2001. Evolution and rationale of locked internal fixator technology.

Introductory remarks. Injury 32 Suppl 2: B3-9.

141

41. Tepic, S, Remiger, AR, Morikawa, K, et al. 1997. Strength recovery in fractured

sheep tibia treated with a plate or an internal fixator: an experimental study with a

two-year follow-up. J Orthop Trauma 11: 14-23.

42. Tepic, S, Perren, S. 1995. The biomechanics of the PC-Fix internal fixator. Injury

26 Suppl.

43. Kregor, PJ, Senft, D, Parvin, D, et al. 1995. Cortical bone perfusion in plated

fractured sheep tibiae. J Orthop Res 13: 715-724.

44. Seibold, R, Schlegel, U, Cordey, J. 1995. [A method for inducing standardized

spiral fractures of the tibia in the animal experiment]. Unfallchirurg 98: 376-378.

45. Wullschleger, ME, Schmeling, A, Steck, R, et al. 2006. Comparison of fracture

healing treated with an internal fixator or plate in an ovine tibial model. 12th

Annual Meeting, ANZORS, Canberra, Australia.

46. Arens, S, Hansis, M, Schlegel, U, et al. 1996. Infection after open reduction and

internal fixation with dynamic compression plates--clinical and experimental data.

Injury 27 Suppl 3: SC27-33.

47. Haas, N, Schuetz, M, Hoffmann, R, Suedkamp, NP. 1997. LISS - Less Invasive

Stabilization System - ein neuer Fixateur intern fuer distale Femurfakturen. OP-

Journal 13:340.

48. Schavan, R, Angst, M. 1997. LISS - Ein neuartiges minimal invasives

extramedullaeres Fixationssystem fuer metaphysaere Frakturen der unteren

Extremitaet. Hefte zu der Unfallchirurg. 702-706.

49. Fankhauser, F, Gruber, G, Schippinger, G, et al. 2004. Minimal-invasive treatment

of distal femoral fractures with the LISS (Less Invasive Stabilization System): a

prospective study of 30 fractures with a follow up of 20 months. Acta orthopaedica

Scandinavica 75: 56-60.

50. Kolb, W, Guhlmann, H, Windisch, C, et al. 2008. Fixation of distal femoral

fractures with the Less Invasive Stabilization System: a minimally invasive

treatment with locked fixed-angle screws. J Trauma 65: 1425-1434.

51. Kregor, PJ, Stannard, JA, Zlowodzki, M, Cole, PA. 2004. Treatment of distal femur

fractures using the less invasive stabilization system: surgical experience and early

clinical results in 103 fractures. J Orthop Trauma 18: 509-520.

142

52. Markmiller, M, Konrad, G, Sudkamp, N. 2004. Femur-LISS and distal femoral nail

for fixation of distal femoral fractures: are there differences in outcome and

complications? Clin Orthop Relat Res: 252-257.

53. Wong, MK, Leung, F, Chow, SP. 2005. Treatment of distal femoral fractures in the

elderly using a less-invasive plating technique. Int Orthop 29: 117-120.

54. Frigg, R. 2003. Development of the Locking Compression Plate. Injury 34 Suppl 2:

B6-10.

55. Wagner, M. 2003. General principles for the clinical use of the LCP. Injury 34

Suppl 2: B31-42.

56. Sommer, C, Gautier, E. 2003. [Relevance and advantages of new angular stable

screw-plate systems for diaphyseal fractures (locking compression plate versus

intramedullary nail]. Ther Umsch 60: 751-756.

57. Gerber, C, Mast, JW, Ganz, R. 1990. Biological internal fixation of fractures. Arch

Orthop Trauma Surg 109: 295-303.

58. Claudi, BF, Oedekoven, G. 1991. ["Biological osteosynthesis"]. Der Chirurg;

Zeitschrift fur alle Gebiete der operativen Medizen 62: 367-377.

59. Baumgaertel, F, Gotzen, L. 1994. [The "biological" plate osteosynthesis in multi-

fragment fractures of the para-articular femur. A prospective study]. Unfallchirurg

97: 78-84.

60. Wenda, K, Runkel, M, Rudig, L. 1995. [The "inserted" condylar plate].

Unfallchirurgie 21: 77-82.

61. Krettek, C, Schandelmaier, P, Miclau, T, Tscherne, H. 1997. Minimally invasive

percutaneous plate osteosynthesis (MIPPO) using the DCS in proximal and distal

femoral fractures. Injury 28 Suppl 1: A20-30.

62. Helfet, DL, Shonnard, PY, Levine, D, Borrelli, J, Jr. 1997. Minimally invasive plate

osteosynthesis of distal fractures of the tibia. Injury 28 Suppl 1: A42-47; discussion

A47-48.

63. Sommer, C, Bereiter, H. 2005. [Actual relevance of minimal invasive surgery in

fracture treatment]. Ther Umsch 62: 145-151.

143

64. Zhiquan, A, Bingfang, Z, Yeming, W, et al. 2007. Minimally invasive plating

osteosynthesis (MIPO) of middle and distal third humeral shaft fractures. Journal

Of Orthopaedic Trauma 21: 628-633.

65. Imatani, J, Noda, T, Morito, Y, et al. 2005. Minimally invasive plate osteosynthesis

for comminuted fractures of the metaphysis of the radius. J Hand Surg [Br] 30: 220-

225.

66. Schuetz, M, Muller, M, Kääb, M, Haas, N. 2003. Less invasive stabilization system

(LISS) in the treatment of distal femoral fractures. Acta chirurgiae orthopaedicae et

traumatologiae Cechoslovaca 70: 74-82.

67. Ricci, WM, Loftus, T, Cox, C, Borrelli, J. 2006. Locked plates combined with

minimally invasive insertion technique for the treatment of periprosthetic

supracondylar femur fractures above a total knee arthroplasty. J Orthop Trauma 20:

190-196.

68. Apivatthakakul, T, Chiewcharntanakit, S. 2009. Minimally invasive plate

osteosynthesis (MIPO) in the treatment of the femoral shaft fracture where

intramedullary nailing is not indicated. Int Orthop 33: 1119-1126.

69. Cole, PA, Zlowodzki, M, Kregor, PJ. 2004. Treatment of proximal tibia fractures

using the less invasive stabilization system: surgical experience and early clinical

results in 77 fractures. J Orthop Trauma 18: 528-535.

70. Oh, JK, Oh, CW, Jeon, IH, et al. 2005. Percutaneous plate stabilization of proximal

tibial fractures. J Trauma 59: 431-437.

71. Helfet, DL, Suk, M. 2004. Minimally invasive percutaneous plate osteosynthesis of

fractures of the distal tibia. Instructional course lectures 53: 471-475.

72. Krackhardt, T, Dilger, J, Flesch, I, et al. 2005. Fractures of the distal tibia treated

with closed reduction and minimally invasive plating. Arch Orthop Trauma Surg

125: 87-94.

73. Wullschleger, ME, Walliser, M, Jenni, R, et al. 2006. Distal tibia shaft fractures:

First results of minimally invasive plate osteosynthesis. 10th Conference of

International Society for Fracture Repair (ISFR), Adelaide, Australia.

144

74. Collinge, C, Kuper, M, Larson, K, Protzman, R. 2007. Minimally invasive plating

of high-energy metaphyseal distal tibia fractures. Journal Of Orthopaedic Trauma

21: 355-361.

75. Hasenboehler, E, Rikli, D, Babst, R. 2007. Locking Compression Plate with

Minimally Invasive Plate Osteosynthesis in diaphyseal and distal tibial fracture: A

retrospective study of 32 patients. Injury 38: 365-370.

76. An, Z, Zeng, B, He, X, et al. 2009. Plating osteosynthesis of mid-distal humeral

shaft fractures: minimally invasive versus conventional open reduction technique.

Int Orthop.

77. Baumgaertel, F, Buhl, M, Rahn, BA. 1998. Fracture healing in biological plate

osteosynthesis. Injury 29 Suppl 3: C3-6.

78. Hurme, T, Kalimo, H, Lehto, M, Jarvinen, M. 1991. Healing of skeletal muscle

injury: an ultrastructural and immunohistochemical study. Med Sci Sports Exerc 23:

801-810.

79. Szczesny, G, Veihelmann, A, Nolte, D, Messmer, K. 2001. Changes in the local

blood and lymph microcirculation in response to direct mechanical trauma applied

to leg: in vivo study in an animal model. J Trauma 51: 508-517.

80. Smith, TL, Curl, WW, Smith, BP, et al. 1993. New skeletal muscle model for the

longitudinal study of alterations in microcirculation following contusion and

cryotherapy. Microsurgery 14: 487-493.

81. Kalicke, T, Schlegel, U, Printzen, G, et al. 2003. Influence of a standardized closed

soft tissue trauma on resistance to local infection. An experimental study in rats. J

Orthop Res 21: 373-378.

82. Schaser, KD, Vollmar, B, Menger, MD, et al. 1999. In vivo analysis of

microcirculation following closed soft-tissue injury. J Orthop Res 17: 678-685.

83. Gierer, P, Mittlmeier, T, Bordel, R, et al. 2005. Selective cyclooxygenase-2

inhibition reverses microcirculatory and inflammatory sequelae of closed soft-tissue

trauma in an animal model. J Bone Joint Surg Am 87: 153-160.

84. Dixon, CE, Lighthall, JW, Anderson, TE. 1988. Physiologic, histopathologic, and

cineradiographic characterization of a new fluid-percussion model of experimental

brain injury in the rat. J Neurotrauma 5: 91-104.

145

85. Lighthall, JW, Dixon, CE, Anderson, TE. 1989. Experimental models of brain

injury. J Neurotrauma 6: 83-97.

86. Goodman, JC, Cherian, L, Bryan, RM, Jr., Robertson, CS. 1994. Lateral cortical

impact injury in rats: pathologic effects of varying cortical compression and impact

velocity. J Neurotrauma 11: 587-597.

87. Marmarou, A, Foda, MA, van den Brink, W, et al. 1994. A new model of diffuse

brain injury in rats. Part I: Pathophysiology and biomechanics. J Neurosurg 80:

291-300.

88. Eskelund, V, Plum, CM. 1949. Experimental investigations into the healing of

fractures. I. Healing of fractures in the femoral diaphysis in rats. Acta orthopaedica

Scandinavica: 433-475.

89. Herbsman, H, Kwon, K, Shaftan, GW, et al. 1966. The influence of systemic factors

on fracture healing. J Trauma 6: 75-85.

90. Jackson, RW, Reed, CA, Israel, JA, et al. 1970. Production of a standard

experimental fracture. Can J Surg 13: 415-420.

91. Oni, OO, Gregg, PJ, Morrison, C, Ponter, AR. 1988. An investigation of the

fracture characteristics of the tibia of mature rabbits. Injury 19: 172-176.

92. Herzog, L, Huber, FX, Meeder, PJ, et al. 2002. Laser doppler flow imaging of open

lower leg fractures in an animal experimental model. J Orthop Surg (Hong Kong)

10: 114-119.

93. Bak, B, Jensen, KS. 1992. Standardization of tibial fractures in the rat. Bone 13:

289-295.

94. Oszwald, M, Westphal, R, O'Loughlin, PF, et al. 2008. A rat model for evaluating

physiological responses to femoral shaft fracture reduction using a surgical robot. J

Orthop Res 26: 1656-1659.

95. Harry, LE, Sandison, A, Paleolog, EM, et al. 2008. Comparison of the healing of

open tibial fractures covered with either muscle or fasciocutaneous tissue in a

murine model. J Orthop Res 26: 1238-1244.

96. Ashhurst, DE, Hogg, J, Perren, SM. 1982. A method for making reproducible

experimental fractures of the rabbit tibia. Injury 14: 236-242.

146

97. Park, SH, Cassim, A, Llinas, A, et al. 1994. Technique for producing controlled

closed fractures in a rabbit model. J Orthop Res 12: 732-736.

98. Holstein, JH, Menger, MD, Culemann, U, et al. 2007. Development of a locking

femur nail for mice. J Biomech 40: 215-219.

99. Schoen, M, Rotter, R, Schattner, S, et al. 2008. Introduction of a new interlocked

intramedullary nailing device for stabilization of critically sized femoral defects in

the rat: A combined biomechanical and animal experimental study. J Orthop Res

26: 184-189.

100. Grongroft, I, Heil, P, Matthys, R, et al. 2009. Fixation compliance in a mouse

osteotomy model induces two different processes of bone healing but does not lead

to delayed union. J Biomech 42: 2089-2096.

101. Schenk, R, Willenegger, H. 1963. [on the Histological Picture of So-Called

Primary Healing of Pressure Osteosynthesis in Experimental Osteotomies in the

Dog.]. Experientia 19: 593-595.

102. Lanyon, LE, Goodship, AE, Baggott, DG. 1976. The significance of bone strain

"in vivo". Acta Orthop Belg 42 Suppl 1: 109-122.

103. Ozaki, A, Tsunoda, M, Kinoshita, S, Saura, R. 2000. Role of fracture hematoma

and periosteum during fracture healing in rats: interaction of fracture hematoma and

the periosteum in the initial step of the healing process. J Orthop Sci 5: 64-70.

104. Heitemeyer, U, Claes, L, Hierholzer, G, Korber, M. 1990. Significance of

postoperative stability for bony reparation of comminuted fractures. An

experimental study. Arch Orthop Trauma Surg 109: 144-149.

105. Dumont, C, Kauer, F, Bohr, S, et al. 2008. Long-term effects of saw osteotomy

versus random fracturing on bone healing and remodeling in a sheep tibia model. J

Orthop Res.

106. Sarmiento, A, Mullis, DL, Latta, LL, et al. 1980. A quantitative comparative

analysis of fracture healing under the influence of compression plating vs. closed

weight-bearing treatment. Clin Orthop Relat Res: 232-239.

107. Macdonald, W, Skirving, AP, Scull, ER. 1988. A device for producing

experimental fractures. Acta orthopaedica Scandinavica 59: 542-544.

147

108. Schmeling, A, Schuetz, M, Kääb, M, et al. 2000. A realistic experimental model of

shaft fractures and concomitant soft tissue trauma in the sheep tibia. In, ORS 46th

Annual Meeting. Orlando, Fl, USA.

109. Bonnarens, F, Einhorn, TA. 1984. Production of a standard closed fracture in

laboratory animal bone. J Orthop Res 2: 97-101.

110. An, Y, Friedman, RJ, Parent, T, Draughn, RA. 1994. Production of a standard

closed fracture in the rat tibia. J Orthop Trauma 8: 111-115.

111. Tatari, H, Fidan, M, Erbil, G, et al. 2007. A new device to produce a standardized

experimental fracture in the rat tibia. Saudi Med J 28: 866-871.

112. Seekamp, A, Van Griensven, M, Blankenburg, H, Regel, G. 1997. Intramuscular

partial oxygen tension monitoring in compartment syndrome--an experimental

study. Eur J Emerg Med 4: 185-192.

113. Steckeler, S, Botel, U, Warninghoff, V. 1994. [Hyperbaric oxygen therapy--an

adjuvant therapeutic procedure with problem cases in reconstructive bone surgery].

Fortschr Kiefer Gesichtschir 39: 164-167.

114. Epari, DR, Lienau, J, Schell, H, et al. 2008. Pressure, oxygen tension and

temperature in the periosteal callus during bone healing--an in vivo study in sheep.

Bone 43: 734-739.

115. Strecker, W, Gebhard, F, Rager, J, et al. 1999. Early biochemical characterization

of soft-tissue trauma and fracture trauma. J Trauma 47: 358-364.

116. Oni, OO, Fenton, A, Iqbal, SJ, Gregg, PJ. 1989. Prognostic indicators in tibial

shaft fractures: serum creatinine kinase activity. J Orthop Trauma 3: 345-347.

117. Holstein, JH, Garcia, P, Histing, T, et al. 2009. Advances in the establishment of

defined mouse models for the study of fracture healing and bone regeneration. J

Orthop Trauma 23: S31-38.

118. Holstein, JH, Matthys, R, Histing, T, et al. 2009. Development of a stable closed

femoral fracture model in mice. J Surg Res 153: 71-75.

119. Claes, L, Augat, P, Suger, G, Wilke, HJ. 1997. Influence of size and stability of

the osteotomy gap on the success of fracture healing. J Orthop Res 15: 577-584.

148

120. Hente, R, Fuchtmeier, B, Schlegel, U, et al. 2004. The influence of cyclic

compression and distraction on the healing of experimental tibial fractures. J Orthop

Res 22: 709-715.

121. Kaspar, K, Schell, H, Seebeck, P, et al. 2005. Angle stable locking reduces

interfragmentary movements and promotes healing after unreamed nailing. Study of

a displaced osteotomy model in sheep tibiae. J Bone Joint Surg Am 87: 2028-2037.

122. Schell, H, Epari, DR, Kassi, JP, et al. 2005. The course of bone healing is

influenced by the initial shear fixation stability. J Orthop Res 23: 1022-1028.

123. Hente, R, Cordey, J, Rahn, BA, et al. 1999. Fracture healing of the sheep tibia

treated using a unilateral external fixator. Comparison of static and dynamic

fixation. Injury 30 Suppl 1: A44-51.

124. Klein, P, Schell, H, Streitparth, F, et al. 2003. The initial phase of fracture healing

is specifically sensitive to mechanical conditions. J Orthop Res 21: 662-669.

125. Chehade, MJ, Pohl, AP, Pearcy, MJ, Nawana, N. 1997. Clinical implications of

stiffness and strength changes in fracture healing. J Bone Joint Surg Br 79: 9-12.

126. Schell, H, Thompson, MS, Bail, HJ, et al. 2008. Mechanical induction of critically

delayed bone healing in sheep: radiological and biomechanical results. J Biomech

41: 3066-3072.

149

AAPPPPEENNDDIICCEESS

APPENDIX A:

List of publications (2005 – 2009)

Schmutz B, Wullschleger ME, Kim H, Noser H, Schuetz MA: Fit assessment of anatomic plates for the distal medial tibia. J Orthop Trauma, 22:258-263, 2008. Reichert JC, Saifzadeh S, Wullschleger ME, Epari DR, Schuetz MA, Duda GN, Schell H, van Griensven M, Redl H, Hutmacher DW: The challenge of large animal models for segmental bone defect research. Biomaterials 30:2149-2163, 2009. Lutton C, Sugiyama S, Wullschleger ME, Williams R, Cambpell J, Crawford R, Goss B: Transplanted abdominal granulation tissue induced bone formation – an in vivo study in sheep. Connective Tissue Research, accepted in February 2009. Nijboer JMM, Wullschleger ME, Nielsen SE, McNamee AM, Lefering R, ten Duis HJ, Schuetz MA: A comparison of severely injured trauma patients admitted to level one trauma centers in Queensland and Germany. ANZ J Surg, accepted in March 2009. Reichert JC, Epari DR, Wullschleger ME, Saifzadeh S, Steck R, Lienau J, Schuetz MA, Duda GN, Hutmacher DW: Establishment of a Preclinical Ovine Model for Tibial Segmental Bone Defect Repair by Applying Bone Tissue Engineering Strategies. Tissue Engineering, accepted in September 2009. Chen G, Schmutz B, Wullschleger ME, Pearcy M, Schuetz MA: Computational Investigations of Mechanical Failures of Internal Plate Fixation. J Engineering in Medicine, accepted in October 2009. Schmutz B, Rathnayaka K, Wullschleger ME, Meek J, Schuetz MA: Quantitative fit assessment of tibial nail designs using 3D computer modeling. Injury, accepted in October 2009. Schmutz B, Wullschleger ME, Meek J, Noser H, Barry M, Schuetz MA: Fit optimization of a Distal Medial Tibia Plate, submitted to Computer Methods in Biomechanics and Biomedical Engineering in March 2009. Thomas C, Athanasiov A, Wullschleger ME, Schuetz MA: Current concepts in tibial plateau fractures, submitted to Acta Chir Orthop Trauma Cechoslovaca in August 2009.

150

Wullschleger ME, Steck R, Matthys R, Webster J, Wilson K, Ito K, Schuetz MA: Ovine model allowing the study of bone fracture healing with closed, severe soft tissue trauma, rejected by J Orthop Res in September 2009, in revision process.

APPENDIX B:

List of conference papers (2005 - 2009)

Sommer C, Jenni R, Bircher HP, Wullschleger ME: Minimally invasive plate osteosynthesis (MIPO) with locking compression plate (LCP) in proximal diaphyseal humerus fractures. Swiss knife (special edition):10, 2005. Wullschleger ME, Sommer C: Experience with the locking compression plate (LCP) in fracture treatment of osteoporotic bone. The bone and joint decade, Multidisciplinary Research Day, Brisbane, November 2005. Wilson CJ, Pettet GJ, Chen G, Mishra SK, Steck R, Wullschleger ME, Schuetz MA: Is callus formation optimised for fracture stability? A computational study. The 10th

Conference of the International Society for Fracture Repair, Adelaide, May 2006.

Wullschleger ME, Jenni R, Walliser M, Schuetz MA, Sommer C: Minimally invasive plate osteosynthesis (MIPO) with locking compression plate (LCP) in proximal diaphyseal humerus fractures. The 10th


Wullschleger ME, Walliser M, Jenni R, Schuetz MA, Sommer C: Distal tibia shaft fractures: First results of minimally invasive plate osteosynthesis. The 10th


Sommer C, Walliser M, Jenni R, Wullschleger ME: Distal tibia shaft fractures: 5 years follow-up of minimally invasive plate osteosynthesis. Annual Conference of Swiss Surgical Society, Lugano, June 2006. Wullschleger ME, Schmeling A, Steck R, Ito K, Schuetz M: Comparison of fracture healing treated with an internal fixator or plate in an ovine tibial model, Australian & New Zealand Orthopaedic Research Society (ANZORS), 12th

Annual Scientific Meeting, Canberra, October 2006.

Gongfa C, Wilson CJ, Wullschleger ME, McElwain DLS, Mishra SK, Pearcy MJ, Perren SM, Pettet GJ, Steck R, Schuetz, MA: Modelling the effects of bone fragment contact in fracture healing, ANZORS, 12th

Annual Scientific Meeting, Canberra, October 2006.

Schmutz B, Wullschleger ME, Schuetz MA: The effect of CT slice spacing on the geometry of 3D models, 6th

Biennial Australasian Biomechanics Conference, Auckland, February 2007.

151

Goss B, Sugiyama S, Wullschleger ME, Lutton C: Transplanted abdominal granulation tissue induced bone formation – an in vivo study in sheep, Spine Society of Australia Conference, Hobart, April 2007. Stillhardt P, Wullschleger ME, Furrer M, Sommer C: Management of peripheral arterial injuries, EATES conference, Graz, Austria, May 2007 (Poster). Nielsen S, Lefering R, Wullschleger ME, McNamee A, Sarai H, Carter L, Davies T, Aitkin L, Schuetz MA: Benchmarking the Trauma care in an Australian Hospital against an European Trauma Registry, Swan XY Trauma Conference, Sydney, July 2007. Wullschleger ME, Steck R, Matthys R, Toggwiler P, Wilson K, Ito K, Schuetz MA: A novel sheep model for the experimental study of severe trauma to the distal femur, 67th

AOA Scientific Meeting, Gold Coast, October 2007.

Tung JP, Fung L, Wullschleger ME, Wood P, Wilson K, Fraser J: A comparison of the oxidative responses of ovine and human neutrophils, HAA 2007 Conference, combined annual scientific meeting, Gold Coast, October 2007. Wullschleger ME, Webster J, Freeman A, Sugiyama S, Steck R, Schuetz MA: Soft tissue evaluation in a sheep model comparing minimally invasive versus open plate osteosynthesis, ANZORS, 13th Annual Scientific Meeting, Auckland, October 2007. Schmutz B, Wullschleger ME, Steck R, Schuetz MA: Optimising a periarticular fracture fixation plate: Does one shape fit all? ANZORS, 13th Annual Scientific Meeting, Auckland, October 2007. Mishra S, Collier L, Chen G, Steck R, Schmutz B, Wullschleger ME, Schuetz MA: The effect of distraction vector orientation on the regenerate tissue during bilateral mandibular distraction, ANZORS, 13th Annual Scientific Meeting, Auckland, October 2007. Klaus A, Schmutz B, Wullschleger ME, Schuetz MA, Steck R: Quantification of fracture callus volume from CT scans, ANZORS, 13th Annual Scientific Meeting, Auckland, October 2007. Schmutz B, Wullschleger ME, Schuetz MA: Optimising a periarticular fracture fixation plate: Does one shape fit all?, 6th

Combined Meeting of the Orthopaedic Research Societies, Hawaii, October 2007.

Schmutz B, Wullschleger ME, Schuetz MA: Optimising the fit of a periarticular fixation plate, Third Asian Pacific Conference on Biomechanics, AP Biomech 2007, Tokyo, November 2007.

152

Wullschleger ME, Webster J, Freeman A, Steck R, Schuetz MA: Soft tissue evaluation in a sheep model comparing minimally invasive versus open plate osteosynthesis, IHBI Postgraduate Student Research Conference, Brisbane, November 2007. Nijboer A, Wullschleger ME, Nielsen S, ten Duis HJ, Schuetz MA: Benchmarking trauma care performance of a tertiary hospital in Queensland to European Trauma Centres: Using the German Trauma Registry as a model, Austrauma, Sydney, February 2007. Lutton C, Sadahiro S, Wullschleger ME, Goss B: Transplanted abdominal granulation tissue induced bone formation – an in vivo study in sheep, Orthopaedic Research Society, San Francisco, March 2008. Gongfa C, Schmutz B, Steck R, Pearcy M, Wullschleger ME, Wilson C, Schuetz MA: Predicting the fatigue life of internal fracture fixation plates, 16th

Congress of European Society of Biomechanics, Luzern, July 2008.

Steck R, Gregory L, Minehara H, Wullschleger ME, Schuetz MA: Three-dimensional visualisation of callus geometry in new murine fracture model. ISFR’s 11th

Biennial Conference, Lake Tahoe, USA, July 2008 (poster).

Wullschleger ME, Nielsen SE, Nijboer A, McNamee A, Lefering R, Schuetz MA: Secondary referred trauma patients: Comparison to those with primary admissions to a tertiary hospital in South-East Queensland, SWAN conference, Sydney, July 2008. Schmutz B, Wullschleger ME, Steck R, Schuetz MA: Periarticular implants – optimisation of plate design with computer modelling, 68th

Annual scientific meeting of Australian Orthopaedic Association, Hobart, October 2008.

Wullschleger ME, Steck R, Wilson K, Ito K, Schuetz MA: Minimally invasive plate osteosynthesis: advanced early fracture healing in an ovine trauma model, 68th


Schuetz MA, Nijboer A, McNamee A, Wullschleger ME, Nielsen S: Development of a comprehensive trauma system in Queensland – Benchmarking trauma care performance in a tertiary hospital in Queensland to European trauma centres, 68th


Nijboer A, Wullschleger ME, Nielsen S, Duis ten HJ, Schuetz MA: Benchmarking trauma care performance of a tertiary hospital in Queensland to European Trauma Centres: using the German Trauma Registry as a model, German Orthopaedic and Trauma Conference (DGU), Berlin, October 2008. Sugiyama S, Goss B, Wullschleger ME, Wilson K, Williams R: Reliability of clinical measurement for assessing spinal fusion: an experimental sheep study, ANZORS, 14th Annual Scientific Meeting, Brisbane, November 2008 (poster).

153

Wullschleger ME, Schmutz B, Ito K, Steck R, Schuetz MA: Minimally invasive versus open plate osteosynthesis: Quantitative radiographic analysis of callus morphology of distal femur fractures in sheep using computed tomography imaging, ANZORS, 14th Annual Scientific Meeting, Brisbane, November 2008 (poster). Steck R, Gregory L, Minihara H, Wullschleger ME, Schuetz MA: Three-dimensional visualisation of callus geometry in new murine fracture model, ANZORS, 14th

Annual Scientific Meeting, Brisbane, November 2008.

King B, Wullschleger ME, Mishra S, Schuetz MA, Schmutz B: Volumetric analysis of the space between bone surfaces and fracture fixation plates using 3D computer models, ANZORS, 14th Annual Scientific Meeting, Brisbane, November 2008 (poster). Wullschleger ME, Steck R, Webster J, Freeman A, Ito K, Schuetz MA: Minimally invasive plate fixation leads to advanced early fracture healing in a sheep trauma model, IHBIinspires Conference, Gold Coast, December 2008. Schuetz MA, Wullschleger ME: Major Pelvic Trauma, Austrauma, Sydney, February 2009. Schmutz B, Wullschleger ME, Meek J, Schuetz MA: Optimized Fitting of Precontoured Implants for Fracture Care, 55th

Annual Meeting of Orthopaedic Research Society (ORS), Las Vegas, USA, February 2009.

Wullschleger ME, Steck R, Webster J, Wilson K, Ito K, Schuetz: Minimally invasive plate osteosynthesis accelerates early fracture healing in an ovine trauma model, 55th

Annual Meeting of Orthopaedic Research Society (ORS), Las Vegas, USA, February 2009.

Wullschleger ME, Steck R, Webster J, Wilson K, Ito K, Schuetz MA: Minimally invasive plate osteosynthesis: advanced early fracture healing in a sheep trauma model, 78th Annual Scientific Congress of Royal Australasian College of Surgeons, Brisbane, May 2009; published in ANZ J Surg 79.1:86, 2009. 1st

prize for free paper of Trauma sessions.

Louie B, Wullschleger ME, Mundy J, Schuetz MA, O’Rourke TR: Outcomes of emergency thoracotomies in an Australian Trauma Centre, 78th

Annual Scientific Congress of Royal Australasian College of Surgeons, Brisbane, May 2009.

Broszczak D, Wullschleger ME, Steck R, Upton Z, Parker T: A novel method to assess extra-cellular fluid using SELDI-TOF-MS, ASMR Queensland Postgraduate Student Conference / IHBI inspires conference, Brisbane, May / November 2009.

154

Wullschleger ME, Steck R, Webster J, Wilson K, Ito K, Schuetz MA: Minimally invasive plate osteosynthesis: advanced early fracture healing in a sheep trauma model, Queensland AOA Annual Meeting, Noosa, June 2009. Schmutz B, Rathnayaka K, Wullschleger ME, Meek J, Schuetz MA: Quantitative fit assessment of tibial nail designs using 3D computer modeling, Queensland AOA Annual Meeting, Noosa, June 2009. Wullschleger ME, Steck R, Webster J, Wilson K, Ito K, Schuetz MA: Minimally invasive plate osteosynthesis: advanced early fracture healing in a sheep trauma model, 96th

Annual scientific meeting of Swiss Society of Surgery (SGC), Montreux, Switzerland, June 2009 (Swiss knife, special edition, p25, 2009).

Sugiyama S, Goss B, Wullschleger ME, Wilson K, Williams R: Reliability of clinical measurement for assessing spinal fusion: an experimental sheep study, Global Spine Congress, San Francisco, USA, June 2009. Wullschleger ME, Steck R, Webster J, Wilson K, Ito K, Schuetz MA: Minimally invasive plate osteosynthesis: advanced early fracture healing in a sheep trauma model, 73rd

Annual scientific meeting of German Orthopaedic & Trauma Association (DGU), Berlin, Germany, October 2009 (poster).

Schmutz B, Rathnayaka K, Wullschleger ME, Meek J, Schuetz MA: Quantitative fit assessment of tibial nail designs using 3D computer modeling, 73rd

Annual scientific meeting of German Orthopaedic & Trauma Association (DGU), Berlin, Germany, October 2009.

Steck R, Ueno M, Gregory L, Wullschleger ME, Schuetz MA, Itoman M: Histological characterization of healing of murine femoral fractures stabilized with locking plates with two different stiffnesses, 73rd

Annual scientific meeting of German Orthopaedic & Trauma Association (DGU), Berlin, Germany, October 2009 (poster).

Wullschleger ME, Steck R, Webster J, Wilson K, Ito K, Schuetz MA: Minimally invasive plate osteosynthesis: advanced early fracture healing in a sheep trauma model. 25th

Annual Meeting of Orthopaedic Trauma Association (OTA), San Diego, USA, October 2009.

Schmutz B, Rathnayaka K, Wullschleger ME, Meek J, Schuetz MA: Quantitative fit assessment of tibial nail designs using 3D computer modeling, accepted for 69th

Annual Scientific meeting of Australian Orthopaedic Association, Cairns, October 2009.

Steck R, Gregory L, Minehara H, Wullschleger ME, Schuetz MA: Histological characterisation of a new murine fracture model with locking plate stabilisation, 69th

Annual Scientific meeting of Australian Orthopaedic Association, Cairns, October 2009.

155

APPENDIX C:

AO Research Grant approval document 05-W17 (20 June 2005)

156

APPENDIX D:

QUT Animal project approval certificate 4222A (26 October 2005)

157

APPENDIX E:

Flowchart of trauma model algorithm (June 2005)

158

APPENDIX F:

Detailed results of macroscopic dissection from trials of the soft tissue trauma

model with the pendulum device:

Sheep 346:

Right leg (comminuted distal femur fracture, Figure 1):

(body weight: 27 kg; age: 8 years; no additional impact weight)

• skin: no lacerations, but superficial bruise to the skin

• subcutaneous tissue: slight haematomas, bruise and horizontal layer disruption

• muscles: vastus lateralis: about 2/3 bruised with small haematoma

vastus intermedius and medius: only little bruised

• sciatic nerve and femoral vessels: intact, but superficial bruise of epineural layer

Left leg (Figure 2):

• skin: closed, but slightly bruised skin

• subcutaneous tissue: small haematoma and horizontal layer disruption

• muscles: vastus lateralis: about 2/3 bruised with haematoma

vastus intermedius and medius: some bruises in all layers

• sciatic nerve and femoral vessels: intact, but superficial bruise of epineural layer

Figure 1: Fractured femur with Figure 2: Sub-fascial bruise of muscles

muscle haematoma with small haematomas

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Sheep 347:

Right leg (Figure 3):

(body weight: 30 kg; age: 8 years; 2 kg additional impactor weight)

• skin: no wounds, but skin bruise with haematomas

• subcutaneous tissue: haematomas, bruises and horizontal layer disruption

• muscles: extensive muscle damage with large intramuscular haematomas as

well as partial muscle disruption

• sciatic nerve and femoral vessels: in continuity intact, but severe nerve

contusion including areas of reduced volume


• skin: closed, but intracutaneous haematoma

• subcutaneous tissue: haematomas and horizontal layer disruption

• muscles: extensive damage including partial disruption and multiple

haematomas

• femoral vessels (artery and vein): intact

• sciatic nerve: significant damage with deep bruises and reduced volume, but

continuity intact

Figure 3: Extensive muscle damage with partial disruption and large haematomas

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Figure 4: Nerve damage: epineural bruises with small haematomas

Sheep 336:

Right leg (Figure 5):

(body weight: 27 kg; age: 8 years; 0.5 kg additional impactor weight)

• skin: no wounds, but superficial skin bruises

• subcutaneous tissue: bruise and horizontal layer disruption

• muscles: all muscles bruised with large intramuscular haematomas (lateral >

medial)

• femoral vessels: intact

• sciatic nerve: areas of superficial contusions with little haematomas and reduced

volume (not only epineurium)


• skin: closed, but bruised areas

• subcutaneous tissue: small haematomas and horizontal layer separation

• muscles: severe bruising of all the muscles with partial muscle disruption and

some lateral haematoma

• sciatic nerve and femoral vessels: intact, but nerve superficially slightly

damaged

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Figure 5: Severe damage including nerve lesion

Figure 6: Severe muscle bruise with large haematomas (cross-section)

Sheep 344:

Left leg (Figure 7 and 8):

(body weight: 27 kg; age: 9 years; no additional impactor weight)

• skin: no wounds, but internal skin bruise

• subcutaneous tissue: small haematoma and horizontal layer disruption

• muscles: extensive bruise incl. partial disruption (lateral vastus) (Figure 7)

• sciatic nerve and femoral vessels: intact, but epineural contusion with small

haematomas (Figure 8)

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Figure 7: Extensive muscle damage (lateral vastus muscle)

Figure 8: Epineural contusion with small haematomas

Right leg (Figure 9 and 10):

• skin: no wound, but several bruised areas

• subcutaneous tissue: bruise and horizontal layer separation

• muscles: reasonable amount of bruises and haematomas of all muscles, but no

disruption (Figure 9)

• sciatic nerve and femoral vessels: intact, but epineurium with superficial

contusion marks (Figure 10)

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Figure 9: Severe muscle contusion of lateral vastus muscle

Figure 10: Intact nerve, but superficial small haematomas

Sheep 389:

Right leg:

(body weight: 30 kg; age: 8 years; no additional impactor weight)

• skin: no wounds, just superficial bruise

• subcutaneous tissue: smart bruise and horizontal layer disruption (degloving)

• muscles: moderate muscle damage with small intramuscular haematomas

• sciatic nerve and femoral vessels: intact, no signs for any nerve damage

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APPENDIX G:

Trial of fracture creation model:

First test series with the new fracture model on cadaver femur bones: Step by step

sequence as illustrated below:

1st step: Distal anterior osteotomy 2nd step: Proximal anterior osteotomy

3rd step: Distal (a-p) drill hole (2.5mm) 4th step: Middle (a-p) drill hole (3.5mm)

5th step: Distal lateral drill hole (3.5mm) 6th step: Proximal lateral drill hole

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7th step: Remote cortical drill hole 8th step: Remote cortical drill hole (2.5mm) in postero-medial direction (2.5mm) in medial direction

9th step: Blade bar insertion 10th step: Distal chisel hit 11th step: Prox. chisel hit

Final result: ‚H’ type butterfly fracture (AO C-type fracture).

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APPENDIX H:

Timetable of pilot study (September 2006 – November 2006)

Sheep no 1/425 (open approach; ORIF) 2/412 (minimally invasive; MIPO) Group 6 week 6 week Body weight; age 44.9 kg; 7-8 years 45 kg; 7-8 years Operation dates 13 and 18 September 2006 20 and 25 September 2006 Date 13/09/2006 first operation 14/09/2006 1 (lab, cp) 15/09/2006 2 (lab, cp) 16/09/2006 3 (lab, cp) 17/09/2006 4 (lab, cp) 18/09/2006 2nd operation (ORIF) 19/09/2006 6 (lab, cp) 20/09/2006 7 (lab, cp) first operation 21/09/2006 8 (lab, cp) 1 (lab, cp) 22/09/2006 9 (lab, cp) 2 (lab, cp) 23/09/2006 3 (lab, cp) 24/09/2006 11 (lab, cp) 4 (lab, cp) 25/09/2006 1w 2nd operation (MIPO) 26/09/2006 6 (lab, cp) 27/09/2006 14 (lab, cp) 7 (lab, cp) 28/09/2006 8 (lab, cp) 29/09/2006 9 (lab, cp) 30/09/2006 1/10/2006 11 (lab, cp) 2/10/2006 2w x-R 1w 3/10/2006 4/10/2006 14 (lab, cp) 5/10/2006 6/10/2006 7/10/2006 8/10/2006 9/10/2006 3w 2w x-R 10/10/2006 11/10/2006 12/10/2006 13/10/2006 14/10/2006 15/10/2006 16/10/2006 4w x-R 3w 17/10/2006 18/10/2006 19/10/2006 20/10/2006 21/10/2006 22/10/2006

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23/10/2006 5w 4w x-R 24/10/2006 25/10/2006 26/10/2006 27/10/2006 28/10/2006 29/10/2006 30/10/2006 6w (Eu, x-R, CT, mech test) 5w 31/10/2006 1/11/2006 2/11/2006 3/11/2006 4/11/2006 5/11/2006 6/11/2006 6w (Eu, x-R, CT, mech test)

Legend: x-R x-rays in 2 planes lab blood samples (CK and LDH) cp compartment pressure monitoring Eu euthanasia CT CT-scan mech test mechanical testing

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APPENDIX I:

Timetable of main series study (November 2006 – October 2007)

Sheep No Group Method 1st procedure 2nd operation Euthanasia 1/421 8 weeks ORIF 15/11/2006 20/11/2006 15/01/2007 2/427 8 weeks ORIF 15/11/2006 20/11/2006 15/01/2007 3/431 8 weeks ORIF 16/11/2006 21/11/2006 29/11/2006 4/426 8 weeks MIPO 16/11/2006 21/11/2006 16/01/2007 5/432 8 weeks MIPO 29/11/2006 4/12/2006 5/12/2006 6/420 8 weeks MIPO 30/11/2006 5/12/2006 30/01/2007 7/407 8 weeks MIPO 17/01/2007 22/01/2007 19/03/2007 8/411 8 weeks MIPO 17/01/2007 22/01/2007 19/03/2007 9/430 8 weeks ORIF 18/01/2007 23/01/2007 19/03/2007 10/418 8 weeks MIPO 18/01/2007 23/01/2007 19/03/2007 11/423 8 weeks ORIF 14/02/2007 19/02/2007 17/04/2007 12/429 8 weeks MIPO 14/02/2007 19/02/2007 17/04/2007 13/416 8 weeks ORIF 15/02/2007 20/02/2007 17/04/2007 14/417 8 weeks ORIF 15/02/2007 20/02/2007 17/04/2007 15/410 4 weeks MIPO 21/03/2007 26/03/2007 23/04/2007 16/433 4 weeks ORIF 21/03/2007 26/03/2007 23/04/2007 17/424 4 weeks MIPO 22/03/2007 27/03/2007 23/04/2007 18/406 4 weeks MIPO 22/03/2007 27/03/2007 23/04/2007 19/472 4 weeks ORIF 11/04/2007 16/04/2007 14/05/2007 20/474 4 weeks ORIF 11/04/2007 16/04/2007 14/05/2007 21/414 4 weeks MIPO 30/05/2007 4/06/2007 2/07/2007 22/478 4 weeks ORIF 30/05/2007 4/06/2007 2/07/2007 23/476 4 weeks MIPO 31/05/2007 5/06/2007 2/07/2007 24/413 4 weeks ORIF 31/05/2007 5/06/2007 2/07/2007 25/471 4 weeks ORIF 4/07/2007 9/07/2007 6/08/2007 26/428 4 weeks MIPO 4/07/2007 9/07/2007 6/08/2007 27/473 4 weeks ORIF 5/07/2007 10/07/2007 6/08/2007 28/477 4 weeks MIPO 20/09/2007 25/09/2007 22/10/2007

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APPENDIX J: Table with detailed list of fracture length evaluation: This table shows the fracture length measurement of all 24 sheep out of the two conventional x-ray views (a-p and lateral), processed and measured with the AMIRA. The values are listed in mm; the last column contains the average length per fracture, the bottom row contains the average fracture length for all sheep. Sheep antero-posterior view latero-medial view average no lateral middle medial anterior middle posterior fracture length 1 / 421 26.44 29.38 29.42 26.8 27.52 12.24 25.30 2 / 427 31.07 30.96 31.52 32.08 31.73 35.54 32.15 4 / 426 31.78 30.56 33 29.57 30.27 33.06 31.37 6 / 420 33.54 31.95 32.99 30.74 33.8 40.04 33.84 7 / 407 26.52 30.44 28.1 29.14 29.85 28.48 28.76 8 / 411 22.34 25.85 22.68 24.25 23.39 36.22 25.79 9 / 430 24.77 25.5 20.57 23.6 25.08 27.38 24.48 10 / 418 29.47 30.73 30.76 28.15 30.06 30.37 29.92 11 / 423 28.15 30.21 28.86 27.05 29.21 49.07 32.09 12 / 429 31.23 35.56 35.1 33.23 31.57 24.89 31.93 13 / 416 28.21 31.01 25.83 31.76 30.86 32.92 30.10 14 / 417 29.3 31.25 30.15 31.82 30.37 29.7 30.43 15 / 410 30.83 33.46 32.49 32.3 33.88 36.12 33.18 16 / 433 30.29 30.48 29.53 24.9 26.36 21.6 27.19 17 / 424 30.1 30.12 30.16 28 29.28 20.22 27.98 18 / 406 31.04 32.42 26.18 26.78 32.12 36.57 30.85 19 / 472 31.7 30.67 29.69 28.51 32.55 18.14 28.54 21 / 414 31.98 32.41 30.33 28.21 29.75 30.49 30.53 22 / 478 31.11 36.29 39.6 33.36 29.34 29.39 33.18 23 / 476 27.7 31.23 31.3 28.4 30.77 15.15 27.43 24 / 413 30.19 30.46 33.67 32.34 30.86 29.93 31.24 25 / 471 36.17 33.08 31.62 31.38 33.71 42.11 34.68 27 / 473 32.8 33.91 29.79 30.39 31.28 32.67 31.81 28 / 477 25.78 31.26 30.5 30.15 31.83 27.66 29.53 Total average: 30.10 ± 2.7 (SD)

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APPENDIX K: Table of list of physical data from all 24 animals: Sheep No Group Age Start weight Final weight Thigh circumference years kg kg cm 4/426 MIPO 5 38.6 36.5 32 6/420 MIPO 5.5 41 40 30.5 7/407 MIPO 5 39.2 37 32 8/411 MIPO 5 33.2 36.2 30.6 10/418 MIPO 4 33.1 31.4 31 12/429 MIPO 6 35.75 35.5 31 15/410 MIPO 6.5 41.55 33.15 32.5 17/424 MIPO 4 35.65 32.2 31.5 18/406 MIPO 5 40.35 34.55 33 21/414 MIPO 5 34 33.5 30.5 23/476 MIPO 7 42 45 32 28/477 MIPO 5 42.7 41.2 34 mean ± SD 5.3 ± 0.9 38.1 ± 3.6 36.4 ± 4.0 31.7 ± 1.1 1/421 ORIF 5 35.4 36.3 32.5 2/427 ORIF 5 34.7 35 31 9/430 ORIF 6 39.5 40.7 32.5 11/423 ORIF 4 46 43.5 34.5 13/416 ORIF 5 37.2 37.45 32 14/417 ORIF 5 36.4 34.3 32.5 16/433 ORIF 7 38.4 33.1 31 19/472 ORIF 5 48.2 49.8 34 22/478 ORIF 6 42 41.2 32 24/413 ORIF 6 30.7 32.6 30.5 25/471 ORIF 6 47.75 46.45 33 27/473 ORIF 7 44 42.2 32 mean ± SD 5.6 ± 0.9 40.0 ± 5.6 39.4 ± 5.5 32.3 ± 1.2 total mean ± SD 5.4 ± 0.9 39.1± 4.7 37.9 ± 4.9 32.0 ± 1.1 This table illustrates the physical data of all 24 sheep of the main study. The animals of the two experimental groups (MIPO and ORIF) are listed separately; the final row concludes the data with the overall mean values and its standard deviations (SD).

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APPENDIX L:

Two representative X-ray series of the fracture healing progress:

Open reduction and internal fixation

(Sheep 417):

A-p view of radiographs taken at time points: postop 1, postop 2, 2 weeks, 4 weeks, 6 weeks and 8 weeks.

Lateral view of radiographs taken at time points: postop 1, postop 2, 2 weeks, 4 weeks, 6 weeks and 8 weeks.

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Minimally invasive plate osteosynthesis

(Sheep 426):

A-p view of radiographs taken at time points: postop 1, postop 2, 2 weeks, 4 weeks, 6 weeks and 8 weeks.

Lateral view of radiographs taken at time points: postop 1, postop 2, 2 weeks, 4 weeks, 6 weeks and 8 weeks.

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