Barbieri Thesis - BioMedical Materials program (BMM)

Chapter 6 – Fluid uptake as instructive factor
mL SBF (in triplicate) at 37±1°C for two days. The granules were then removed from
SBF, carefully rinsed with distilled water, dried, gold–sputtered and observed with
SEM in secondary electron modality.
6.2.7. Animal experiments
With the permission of the local animal care committee (Animal Center, Sichuan
University, Chengdu, China; protocol #P11029), granules of the three composites (1
cc) and cylinders of the two tricalcium phosphate ceramics were implanted in the
dorsal muscles of five sheep (female, 2–3 years old) for six months. Surgeries were
performed under general anaesthesia (Isoflurane, Merck, Darmstadt, Germany; 2% in
oxygen, oxygen flow rate of 4 L min –1 ) and sterile conditions. The muscles were
exposed by longitudinal skin incisions and two lines of independent muscle pockets
were created by blunt dissection and the samples were inserted. The pockets were
then closed individually with non–resorbable suture material and the wound was
closed using silk sutures. Six months after implantation, the animals were sacrificed
with a lethal injection of pentobarbital (Vetanarcol ® , Merck; 60 mg kg –1 ).The samples
were harvested with surrounding tissues and fixed in 4% buffered formaldehyde
solution (pH=7.4) at 4°C for one week. After rinsing with phosphate buffer saline
(PBS, Invitrogen), the samples were trimmed from surrounding soft tissues. Then the
samples were dehydrated in a series of ethanol solutions (70%, 80%, 90%, 95% and
100% ×2) and embedded in methyl metacrylate (MMA, LTI Nederland, the
Netherlands). Non–decalcified histological sections (10–20 m thick) were made
using a diamond saw microtome (Leica SP1600, Leica Microsystems, Germany).
Sections for light microscopy observations were stained with 1% methylene blue
(Sigma–Aldrich) and 0.3% basic fuchsin (Sigma–Aldrich) solutions
The stained sections were scanned using a slide scanner (Dimage Scan Elite 5400II,
Konica Minolta Photo Imaging Inc, Tokyo, Japan) to obtain low magnification images
for histomorphometric analysis. The sections were observed with a light microscope
(Nikon Eclipse E200, Tokyo, Japan) to analyse the tissue reaction and bone
formation. Histomorphometry was performed using Adobe Photoshop Elements 4.0
software. Firstly the whole sample was selected as a region of interest and the
corresponding number of pixels was read as ROI. Then the material and mineralized
bone were pseudo–coloured, and their respective pixels were counted as M and B
respectively. The percentage of bone in the available space of the explants (Bp) was
then determined as Bp = B · 100 / (ROI – M).
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