Barbieri Thesis - BioMedical Materials program (BMM)

Chapter 5 – Alkali surface treatment effects
Twelve weeks after implantation, the animals were sacrificed and 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 and split in two
parts: about ¾ for histological observations and ¼ for degradation analysis. The parts
for histology 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 after etching with
acidic ethanol (Merck). The sections were observed with a light microscope (Nikon
Eclipse E200, Japan) to analyse the tissue reaction and bone formation. BSEM
was also performed on the sectioned samples to further evaluate the materials in
terms of degradation, mineralized surface (including quantitative analysis as
described earlier) and bone formation. Before use, the other parts of the explants
were rinsed in 1% triton X–100 (Sigma–Aldrich) in phosphate buffered saline to
completely remove the tissues from the granules. Afterwards the granules were
washed several times in distilled water and vacuum–dried at 37±1°C for at least 48
hours. Part of these granules was heated at 900°C to burn the polymer phase out and
determine the final effective apatite and polymer percentage contents. Following
the procedure described in §5.2.3, the remnant part of the samples was used to
evaluate the degradation of the polymer phase by measuring its post–implant
intrinsic viscosity after 12 weeks in vivo.
5.2.10. Statistical analysis
Two tail t–test (for populations with different variance) and post–hoc Tamhane
ANOVA test were used to evaluate differences in the results. The choice between the
two tests relied on the size of the compared data. If the data populations to be
compared were two, t–test was use. If populations were more, ANOVA was used. A
p–value lower than 0.05 was considered as significant difference in both statistical
tests. The analyses were performed using Origin software (v8.0773, OriginPro,
Northampton, MA, USA).
5.3. Results
5.3.1. Characterization of apatite
XRD showed that the synthesized powder is a calcium phosphate apatite (Figure 1a).
Calculations on the XRD data, based on the diffraction angle and the corresponding
reflection plane (hkl), led to the estimation of unit cell parameters for apatite as
100