Barbieri Thesis - BioMedical Materials program (BMM)

Chapter 3 – Instructive composites: effect of filler content on osteoinduction
after etching with acidic ethanol (Merck), while unstained sections were prepared for
fluorescent microscope observations.
3.2.7. Histological and histomorphometric analysis
The stained sections were scanned (Dimage Scan Elite 5400II, Konica Minolta Photo
Imaging Inc, Tokyo, Japan) to obtain low magnification images for histomorphometric
analysis. The sections were then observed with light microscopy (Nikon Eclipse E200,
Tokyo, Japan) to analyse the tissue reaction and bone formation. Unstained sections
were observed with a fluorescent microscope (Nikon Eclipse E600, camera Nikon
FDX–35) to determine the onset of bone formation. Histomorphometry was performed
using Photoshop CS5 software (v12, Adobe Systems Benelux BV). 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
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 = 100 · B / (ROI – M)
3.3. Results
3.3.1. Nano–apatite physicochemical characterization
The XRD pattern of sintered samples showed crystallized hydroxyapatite (HA),
whereas the unsintered samples showed broader peaks indicating that the precipitate
was more amorphous (CaP, Figure 1). The 2 positions of the peaks were coincident
for both sintered and unsintered apatite. They matched those of the international
JCPDS 9–432 reference for hydroxyapatite and also those sperimentally measured
for the mineral of human cortical bone (Table 1). The FTIR spectrum of unsintered
apatite highlighted the presence of all the characteristic vibrational peaks of
hydroxyapatite (i.e. sintered apatite) (Figure 1, Table 2). When compared with
literature data, the unsintered apatite powder was chemically similar to the mineral of
human cortical bone (Table 2). It is interesting that both FTIR and XRD spectra did
not have extra peaks indicating absence of other phases or impurities. The particle
dimensions were evaluated only for the unsintered apatite since it will be used to
prepare the composites. HR–SEM and TEM observations (Figure 1) indicated
average particle dimensions of 84±19 nm width and 369±41 nm length confirming the
nano–scaled nature of the synthesized powder. Further, the morphology of apatite
particles was needle–shaped (Figure 1). It is interesting that other investigations
reported that human cortical bone apatite has mainly flake–like morphology (30–100
nm length) but needle–like minerals (over 100 nm length and larger than 10 nm) were
observed as well. [23] The Ca/P ratio of unsintered apatite, characterized by EDX,
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