Barbieri Thesis - BioMedical Materials program (BMM)

Chapter 3 – Instructive composites: effect of filler content on osteoinduction
diffraction peak (in radians) at half–way height between the background signal and
the peak maximum. The determination of was based on three spectra per material,
including silicon for instrumental broadening estimation (explained below). For a
better measurement of , the background noise was removed (cubic spline method)
and a pattern smoothing algorithm (parabolic Savitzky–Golay filter set at 15 points)
was applied. This value was then adjusted to consider the effect of the instrumental
broadening of the XRD machine, which can be estimated by measuring a standard
silicon sample and considering its diffraction peak related to the reflection plane (1 1
1). [312] Thus it was estimated as
= sqrtm 2 – i 2 ) (4)
where m is the observed FWHM at the chosen plane for apatite and i is the FWHM
for the standard silicon. [313] In the determination of L, we assumed that the lattice
strain is negligibly small [312] and, since in hydroxyapatite and biological apatite the
largest dimension is parallel to the c–axis, the (0 0 l) planes were considered. In
particular the (0 0 2) order of reflection is reported to be the most reliable peak for the
determination of L in apatite. [312]
3.2.3. Preparation and characterization of nano–apatite/poly(D,L–lactide) composites
Poly(D,L–lactide) with declared molecular weight Mw=52 kDa (Phusis Matériaux
Biorésorbables, Saint Ismier, France) was dissolved in acetone (c=0.33 g mL –1 ) and
used to prepare seven composites with different contents of unsintered apatite: 0%,
12.5%, 25%, 30%, 40%, 50% and 75% by weight. The apatite suspension and
polylactide solution were blended, in due proportions, for four hours in a rotational ball
milling system using glass beads (diameter 3–10 mm, total beads volume 1/3 of the
milling room volume) at room temperature and rotational speed of 12 rpm. After
evaporation of acetone we checked which apatite/polymer ratios were suitable to
have solid and not brittle composites. Sodium chloride granules (NaCl, size 300–400
μm; Merck, Darmstadt, Germany) were added to the chosen mixtures and uniformly
mixed to obtain blocks having 60%v/v. porosity. After evaporation of acetone and
leaching NaCl granules with distilled water, porous bodies were obtained (pore size
300–400 μm, 60% porosity). Regular shaped porous blocks (dimension 7×7×7 mm)
and irregularly shaped granules (dimension 2–3 mm) were manufactured and
sterilized by –irradiation (irradiation dose range 28.9–30.7 kGy, IsoTron Nederland
BV, Ede, the Netherlands) for further studies. The chemistry of the composites was
analyzed with FTIR after dissolving the materials in acetone and mixing them with KBr.
The FTIR protocol followed is described in §3.2.1., while the surface morphology and
apatite distribution in the polymer matrix were observed with scanning electron
microscopy (Philips XL 30 ESEM–FEG, Philips, Eindhoven, the Netherlands) in
secondary (SEM) and backscattered electron (BSEM) modes respectively. To
evaluate whether the polymer phase changed during manufacturing, we measured
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