BIOMATERIAUX

Chapitre 4 – Ciments

Chapitre 4D – Qq applications du R.S.

D. Bazin

Laboratoire de Physique des Solides UMR 8502,

Université Paris Sud, Bât 510 91405 Orsay Cedex, France.

PLAN Chapitre 4 Les ciments phosphocalciques

Chapitre 4A L’adhésion cellule – biomatériaux

Chapitre 4B Les ciments phosphatiques

Chapitre 4C Quelques généralités sur le rayonnement synchrotron

Chapitre 4D Quelques applications du R. S.

Chapitre 4D.1 Expériences de µdiffraction

Chapitre 4D.1.a Expérience de µdiffraction

Chapitre 4D.1.b Newly formed bone at prosthesis interface

Chapitre 4D.1.c Interface bone and a SiO2-stabilized TCP bioceramic

Chapitre 4D.2 Expériences de µfluorescence X

Chapitre 4D.2.a Montage expérimental

Chapitre 4D.2.b Imagerie cellulaire

Chapitre 4D.2.c Imagerie d’une dent

Chapitre 4D.2.d Cartographie du Pb et du Ca dans l’os

Chapitre 4D.3 Tomography Chapitre 4D.3.a 4D chemical imaging based on a third-generation S.R.

Chapitre 4D.3.b Synchrotron X-ray microtomography

Chapitre 4E La spectroscopie d’absorption X (Xanes – Exafs)

Chapitre 4D.1 .1 Expériences de µdiffraction

Chapitre 4D.1.a µdiffraction studies of bone tissues using S.R.1

This is the first report on a series of studies of the crystallinity of bone

tissues. The measurements were done at the microfocused diffraction beamline

at ERSF (European Synchrotron Radiation Facility) as a feasibility test on

various aspects on microdiffraction analysis. Beside the crystal structure,

crystallite size distribution and preferential orientation were also studied, with a

spatial resolution of 7 µm.

1. A. Rindby et al., Microdiffraction studies of bone tissues using synchrotron

radiationBiomaterials 19 (1998) 2083-2090

The samples were longitudinal and transversal cuts from human femoral shafts.

A polarized light micrograph of the transversal cut. The central osteon

was selected for the analysis. The individual lamellae are visible as alternating

black/white rings surrounding the Haversian canal. The arrow indicates the

position and the direction of the linescan.

The results show that the bone crystallites seem to be oriented in two

orthogonal directions,

- one parallel with the Haversian system

- and the other perpendicular following the lamella’s curvature.

Peak width analysis shows that the crystallites are about 25-40 nm along

the c-axis with a width of about 10 nm. The result also shows that the peak

width is somewhat larger close to the haversian canal compared with the outer

region of the osteon.

Chapitre 4D.1.b Newly formed bone at prosthesis interface2

Events leading to the integration of an implant into a bone, determining

the performance of the device, take place largely at the tissue/implant

interface3,4,5. After implantation, reactions occur at the tissue/implant interface

that lead to time-dependent changes in the tissues and in the surface

characteristics of the implant material.

6

2. Cedola et al., High spatial resolution X-ray microdiffraction applied to biomaterial studies

and archeometry, Spectrochimica Acta Part B 59 (2004) 1557– 1564 3. A. Cedola, V. Stanic, M. Burghammer, S. Lagomarsino, F. Rustichelli, R. Giardino, N.

Nicoli Aldini, M. Fini, V. Komlev, S. Di Fonzo, Phys. Med. Biol. 48 (2003) N37–N48. 4. M. Muller,M. Burghammer, D. Flot, C. Riekel, C. Morawe, B. Murphy, A. Cedola, J. Appl.

Crystallogr. 33 (2000) 1231– 1240. 5. P. Ducheyne, Q. Qiu, Biomaterials 20 (1999) 2287– 2303.

6. http://www.labrha.com/arthrose-de-la-hanche-coxarthrose.aspx

The major goal of the following experiment using the setup of Fig. 2 is to

clarify the processes involved in bone formation at the interface with the

implant. In particular, the study involves two different samples with different

substrates, a Yttria-stabilized Tetragonal Zirconia Polycrystal (Y-TZP) coated

with bioactive glass (RKKP bioglazeR) (sample A) and Y-TZP uncoated device

(sample B). The samples were obtained from highly pure, medical grade

powders (Yttria-stabilised ZrO2, Y-PSZ stabilised with 3 mol% Y2O3, or

5.5wt.%; Tosoh TZ3YB, Japan).

Chapitre 4D.1.c Interface between bone and a SiO2-stabilized

TCP bioceramic7

The scaffolds used in this investigation were SkeliteTM (Millenium

Biologix Corp., Kingston, Canada) which is a bone graft substitute containing

silicon in the form of Si-TCP, and consisting of approximately 67% Si-TCP

and 33% HA/B-TCP8,9.

SkeliteTM presents an interconnected, open pore structure similar to

human cancellous bone and has a porosity level of approximately 60% with a

pore size range between 200 and 500 µm. The scaffolds used in the ovine

portion of this study were cylinders of 40mm length with a central canal (18mm

ODx6mm IDx50mm, approx.) and in the case of the immunodeficient mouse

model 4x4x4mm cubes.

Image acquisitions were carried out at the European Synchrotron

Radiation Facility (ESRF, Grenoble, France) using the ID-19 beam-line, which

provides a tunable high-photon flux X-ray beam. The acquisition setup was

based on three-dimensional (4D) parallel tomography, which is described in

detail elsewhere10

.

7. Mastrogiacomo et al., Engineering of bone using bone marrow stromal cells and a silicon-

stabilized tricalcium phosphate bioceramic: Evidence for a coupling between bone formation

and scaffold resorption Biomaterials 28 (2007) 1376–1384 8. Sayer M, et al. Structure and composition of silicon-stabilized tricalcium phosphate.

Biomaterials 2003;24:369–82. 9. Reid JW, Pietak A, Sayer M, Dunfield D, Smith TJ. Phase formation and evolution in the

silicon substituted tricalcium phosphate/apatite system. Biomaterials 2005;26:2887–97. 10

. Cedola A, Mastrogiacomo M, Burghammer M, Komlev K, Giannoni P, Cancedda R, et al.

Structural study with advanced X-ray microdiffraction technique of bone regenerated by bone

marrow stromal cells. Phys Med Biol 2006;51:6N109.

Fig. 4. X-ray diffraction analysis of the 2-month-implanted sample. The X-ray

radiography of the area of the analyzed implant is reported in the inset, while the

large panel shows the corresponding collection of the measured X-ray

diffraction patterns.

Fig. 5. Quantitative analysis of the diffraction patterns along the red arrow indicated in Fig. 4:

the plot represents the variation in the amount of the three phases of interest: HA of the newly

formed bone (bone: squared points), HA of the scaffold (HA: open circles) and TCP of the

scaffold (TCP: triangle points). Three different zones can be distinguished: from left to right:

the bone-only zone, the zone where both scaffold and bone are present, and the scaffold-only

zone.

Chapitre 4D.2 Expériences de µfluorescence X

Chapitre 4D.2.a Montage expérimental In Situ Imaging of Metals in Cells and Tissues

11

Approximately one third of the human proteome contains metal cations,

either in the form of cofactors with catalytic functions or as structural support

elements12

. To guarantee a proper maintenance of this metal ion pool, both at the

cellular and at whole organism levels, nature has evolved a highly sophisticated

machinery comprised of a complex interplay between DNA, proteins, and

biomolecules13

.

Schematic diagram illustrating the components of an X-ray fluorescence

microscope. A crystal monochromator is used to select the energy of the

incident X-ray beam, which is focused with a Fresnel zone plate on the

specimen. The emitted X-rays are collected with an energy-dispersive detector,

thus allowing for simultaneous multielement analysis.

11

. McRae et al., Chem. Rev. 2009, 109, 4780–4827. 12

. Li, Y. F.; Chen, C. Y.; Qu, Y.; Gao, Y. X.; Li, B.; Zhao, Y. L.; Chai, Z. F. Pure Appl.

Chem. 2008, 80, 2577. 13

. Finney, L. A.; O’Halloran, T. V. Science 2003, 300, 931.

Chapitre 4D.2.b Imagerie cellulaire

Cartographie à l’échelle cellulaire : MicroXRF imaging of

mouse fibroblast cells grown on 200 mesh EM gold grids

coated with a Formvar-carbon thin film: (A) DIC image of in-

air-dried cell; (B) raster-scanned microXRF topographical

maps for selected elements

(excitation at 10 keV, pixel size 0.3 μm, integration time 1

s/pixel).

Reprinted with permission from ref 265. Copyright Elsevier

2006.

Chapitre 4D.2.c Imagerie d’une dent

Optical micrograph (top left) and X-ray fluorescence (XRF) elemental

distribution images of a 100-μm-thick section of an unrestored carious

human tooth. Three-color overlay (top left; linear scale; Ca red, Cu

green, Zn blue) and individual XRF elemental distribution images of a

representative area (shown as a red square) of the carious front of the

section.

Maximum concentrations, in micrograms per gram, are shown

below the elemental symbol for the XRF images.

Reprinted with permission from ref 424. Copyright 2008 Springer

Berlin.

Chapitre 4D.2.d Cartographie du Pb et du Ca dans l’os Applications of Synchrotron Radiation-Induced X-Ray Emission (SRIXE)

14

Synchrotron radiation-induced x-ray emission (SRIXE) is now a proven

analytical method for the determination of trace elements.

It is notable for spatial resolutions of less than 10 µm and

minimum detection limits that can be as low as 1 ppm by weight. The approach to SRIXE measurements in use at the Brookhaven National

Synchrotron Light Source is summarized to illustrate the experimental approach

to SRIXE.

Application of SRIXE to several different questions are then discussed.

The particular examples presented deal with the detection of trace amounts of

toxic metals in marine worms, tadpole brains and chick tibia.

14

. Jones et al., X-RAY SPECTROMETRY, VOL. 26, 350-358 (1997).

For SRIXE, bones were sliced sagittally, fixed, embedded in methyl

methacrylate and one-ground to a thickness of 60 lm and then mounted on 7.6

µm polyimide film for analysis. Two-dimensional maps were made over

significant areas of the bones so that significant physiological regions were

covered including both cartilage, trabecular and compact bone.

Pb (figure haut) and Ca (figure bas) are similarly distributed in the

columns of calcifying cartilage. These results indicate that Pb and Ca deposition

in bone are similar and that disruption of bone growth and mineralization by Pb

influences the distribution of both cations. Interestingly, the lead distribution is

relatively uniform in contrast to the surface peaking for lead observed for human

bones and for another element, Ga in mice.

Chapitre 4D.3 Tomography

Chapitre 4D.3.a 4D chemical imaging based on a third-generation S.R.15

Tomography was born in 1979 with the Nobel Prize of Cormack and

Hounsfield. Since that time, great progress in X-ray production and detection

gave rise to seven generations of computed tomography (CT) medical scanners,

now conventionally using 2D stationary detectors and helical X-ray source paths

[1].

In medical imaging, diagnoses typically include (i) retrieval of tumor

shape, (ii) bone imaging, (iii) traumatology or (iv) organ malfunctioning or

malformation, which all require 4D information about the organ.

Apart from the medical applications, CT is also used in industries for non-

destructive testing (NDT) of artificial samples (e.g., for failure or defect

detection, solder-joint quality assessment or in situ fatigue testing), usually at

millimeter (mm) or submm resolution. As in medical imaging, the 4D shape is

essential in NDT.

15

. P. Bleuet et al., Trends in Analytical Chemistry, Vol. 29, No. 6, 2010

Chapitre 4D.3.b Synchrotron X-ray microtomography (on a

micron scale) provides three-dimensional imaging representation of bone

ingrowth in calcium phosphate biomaterials16

The BCP blocks usedwere Macroporous Biphasic Calcium Phosphate

ceramic (MBCPt, Biomatlante, Vigneux, France) with a 60/40 HA/bTCP (w/w)

ratio. All implants had macropore diameters of 300–565 µm, with 40–50%

macroporosity.

Biphasic structure and ceramic purity, as checkedrespect ively by X-ray

diffraction and Fourier transform infrared (FTIR) spectroscopy, confirmedthe

HA/bTCP ratio and the absence of carbonate andpyroph osphate impurities.

Macro and micro porosity were determined by image analysis using

scanning electron microscopy (SEM) observations. Microporosity was

equivalent in all implants, representing 33%±0.5 of the ceramic surface17

.

Total micro- and macroporosity was 70%.

16

. P. Weiss et al., Synchrotron X-ray microtomography (on a micron scale) provides three-

dimensional imaging representation of bone ingrowth in calcium phosphate biomaterials,

Biomaterials 24 (2003) 4591-4601 17

. Gauthier O, Bouler JM, Aguado E, Pilet P, Daculsi G. Macroporous biphasic calcium

phosphate ceramics: influence of macropore diameter and macroporosity percentage on bone

in growth. Biomaterials 1998;19:133-9.

Fig. 1. The microtomographic stage at ID 22 (measurements in

millimeters). A sample stage (foreground) and detector system (behind)

consisting of a visible-light microscope with a three-objective revolver

magnifying the scintillation image from a doped YAG or LAG singlecrystal

screen, anda FreLon 2000 CCD camera. Sample-to-scintillator distance can be

set from less than 1mm (as shown in the figure) to almost 1m, thereby allowing

both absorption-contrast andphasecontrast imaging in different regions of

wavefront propagation.

Fig. 2. Tomographic 2D reconstructed section of the rods perpendicular to the

long axis.

- Bone in gray,

- BCP granules in white,

- and soft tissue in black:

(A) IBS 40–80 µm after 3 weeks of implantation into a rabbit model

(B) IBS 80–200 µm after 3 weeks of implantation into a rabbit model.

Fig. 3. Tomographic 4D representation of trabecular rabbit bone near

implantedbiomaterial :

(A) Osteoplasts (osteocyte lacunae),

(B) vascular canals

(C) large soft tissue areas.