Materials and methods by 0fKplDoV

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									                          Supplementary section
Materials and methods
Materials
Titanium and Titanium alloy were used to form biomimetic calcium phosphate
coatings (Liu et al, 2005). This technique is also possible to apply on calcium
phosphate based bone filling materials (Wernike et al). Recently, five types of
materials (table 1) were adopted with the same principle: Helistat® (Integra, USA),
a porous sponge-like material, is manufactured from natural collagen.
Polyactive® (IsoTis B.V., Bilthoven, the Netherlands), a porous scaffold, is a
synthetic co-polymer of ethyleneoxide terephthalate and butylene terephthalate.
EthisorbTM (Johnson & Johnson), a dense, fibrous material, is a co-polymer of
glactin and -dioxanon. PLGA (undergoing clinical trials by Smith and Nephew,
UK), a fibrous material, is a co-polymer of lactic and glycolic acids. Bio-Oss®
(Geistlich Pharma AG, Switzerlands) granules, deproteinized bone, are bovine
derived. 1-cm-diameter discs of the former four polymeric materials and 0.15g
Bio-Oss® granules were used as one sample for in vivo study.


Biphasic biomimetic calcium phosphate coating and protein incorporation
For the first step, the biomaterial to be coated is immersed in a 5-fold-
concentrated simulated body fluid (684mM NaCl, 12.5mM CaCl2.2H2O, 5mM
Na2HPO4.2H2O, and 21mM NaHCO3) in the presence of 7.5mM MgCl2.6H2O, to
inhibit crystal growth, for 24 hours at 37°C. A fine and dense layer of amorphous
calcium phosphate (ACaP) thereby formed and served as a seeding substratum
for the deposition of a crystalline layer. The crystalline layers were produced by
immersing the ACaP-coated biomaterials into a supersaturated calcium
phosphate solution (40mM HCl, 4mM CaCl2.2H2O, 136mM NaCl, 2mM
Na2HPO4.2H2O, and 50mM TRIS, with a pH value at 7.4) for 48 hours at 37°C.
Bioactive proteinaous agents were incorporated into the crystalline layer when
they were introduced into the supersaturated calcium phosphate solution. For the
in vitro study, bovine serum albumin conjugated with fluorescein isothiocyanate
(FITC-BSA) was used for incorporation as a model protein. For the in vivo study,


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an osteoinductive cytokine, bone morphogenic protein-2 (BMP-2) was used. The
amount of coating-incorporated BMP-2 on each polymer was determined using
an enzyme-linked immunosorbent assay (ELISA) kit (PeproTech EC, London,
UK). The amount of coating-incorporated BMP-2 on Bio-Oss® was determined
using our well-established radio-labelled methodology (Wernike et al. 2009).


In vitro characterization
The morphology of the materials and the coatings were examined by scanning
electron microscopy (SEM). The characteristic chemical groups and chemical
composition of the uncoated/coated materials were characterized using fourier-
transform infrared spectroscopy (FTIR, Spectrum 1000, Perkin-Elmer, Germany)
and X-ray diffractionometry (XRD, X’Pert PRO, PANalytical, the Netherlands)
respectively.
The release kinetics of coating-incorporated and of adsorbed (control) FITC-BSA
was monitored spectrophotometrically in vitro. The surface-area density and the
porosity of each polymer type were estimated histomorphometrically (see the
section of in vivo study).


In vivo studies
Animal experiments were conducted with the permission of, and in accordance
with, the regulations laid down by the Animal Protection Commission of the State
of Bern (Switzerland). To evaluate the functionalized materials, a well-
established ectopic ossification model in adult rats (each weighing from 185 to
250 g) was adopted.
One experimental and three control groups were set up for each material (n=6
animals per group): (i) coated polymer bearing an incorporated depot of BMP-2
(experimental group); (ii) uncoated polymer; (iii) coated polymer; (iv) uncoated
polymer bearing an adsorbed depot of BMP-2. Two discs were implanted per rat
within the dorsal subcutaneous tissue, one on the left side and one on the right
side.




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The samples of the selected five materials were surgically implanted within the
dorsal subcutaneous tissue of the rats. Five weeks later, the encapsulated discs
were retrieved, chemically fixed and embedded in methyl-methacrylate. The
material was cut into 10 slices, 600 m in thickness and 1mm apart, using a
diamond saw, according to a systematic random sampling protocol (Gundersen
and Jensen 1987). The slices were mounted on plexiglas holders, polished and
surface-stained with McNeal’s Tetrachrome, basic Fuchsine and Toluidine Blue
O (Schenk RK 1984).
The specimens were photographed in a light microscope. The photomicrographs
were used to estimate the volume of bone and the volume of foreign-body giant
cells (to gauge the inflammatory response) within the subcapsular space
(reference volume) according to state-of-the-art stereological methodologies (Liu
et al. 2005).
The following biological parameters were evaluated using stereological principles
(Howard 2005). The volume of each material at time 0 and the reference volume
(subcapsular space) at 5 weeks were estimated by the point-counting technique
using Cavalieri's methodology (Cavalieri 1635. Reprinted as Geometria degli
Indivisibili. Torino: Unione Tipografico-Editorice Torinese, 1966). The reference
volume was used for estimating the total volume foreign-body giant cells and
bone. The surface-area densities of each polymer type at time 0 and of Bio-Oss®
at 5 weeks were determined using a cycloid test system (Baddeley et al. 1986).
The volume densities of bone, foreign-body giant cells, fibrous capsule, and
remained materials within the subcapsular space were estimated 5 weeks after
implantation by the point-counting technique.


Statistical analysis
The data are presented as mean values together with the standard deviation.
Data pertaining to each group were compared using a one-way analysis of
variance (ANOVA). The level of significance was set at p<0.05. SPSS statistical
software (version 15.0 for windows) was used for this evaluation. Post-hoc
comparisons were made using Bonferroni corrections.



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References:


Baddeley A. J., Gundersen H. J. and Cruz-Orive L. M. 1986 Estimation of surface area
       from vertical sections. J Microsc 142(Pt 3): 259-276.
Cavalieri B. (1635. Reprinted as Geometria degli Indivisibili. Torino: Unione
       Tipografico-Editorice Torinese, 1966). Geometria Indivisibilibus Continuorum.
       Bononi: Typis Clemetis Feronij.
Gundersen H. J. and Jensen E. B. 1987 The efficiency of systematic sampling in
       stereology and its prediction. J Microsc 147(Pt 3): 229-263.
Howard C. V., Reed, M. G. (2005). Unbiased Stereology (Second edition).
Liu Y., de Groot K. and Hunziker E. B. 2005 BMP-2 liberated from biomimetic implant
       coatings induces and sustains direct ossification in an ectopic rat model. Bone
       36(5): 745-757. (DOI: S8756-3282(05)00048-7 [pii]10.1016/j.bone.2005.02.005).
Schenk RK O. A., Herrmann W. (1984). Preparation of calcified tissues for light
       microscopy. Methods of Calcified Tissue Preparation. GR D. Amsterdam,
       Elsevier Science Publishers B.V.: 1-56.
Wernike E., Hofstetter W., Liu Y., Wu G., Sebald H. J., Wismeijer D., Hunziker E. B.,
       Siebenrock K. A. and Klenke F. M. 2009 Long-term cell-mediated protein release
       from calcium phosphate ceramics. J Biomed Mater Res A 92(2): 463-474. (DOI:
       10.1002/jbm.a.32411).




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