Barbieri Thesis - BioMedical Materials program (BMM)

Chapter 3 – Instructive composites: effect of filler content on osteoinduction
the viscosity of the polymer as supplied and the polymer phase in the prepared
materials. In the case of composites, we dissolved the samples in chloroform (CHCl3,
Sigma–Aldrich, Steinhem, Germany) at the concentration of 4 mg mL –1 . We then
separated apatite particles from the polymer component through vacuum filtering with
glass funnels (ROBU, Germany; borosilicate 3.3 with glass filter having porosity #5)
and PTFE membrane filter (Toyo Roshi Kaisha, Advantec, Japan; pore size 0.1 m)
and let chloroform completely evaporate to obtain the polymer samples. Afterwards,
we dissolved again the filtered polymers in chloroform (Sigma–Aldrich) at a
concentration of 0.1 g dL –1 and measured the relative viscosity (rel) using an
Ubbelohde (ASTM) viscometer (0C, PSL–Rheotek, Burnham on Crouch, United
Kingdom) at 25±0.1°C. From rel we could determine the inherent (inh) and
intrinsic () viscosity (Solomon–Ciută equation). [356] Then, by using Mark–
Houwink equation, the weight average molecular weight (Mw) of the polymers was
calculated, as follows: [337]
rel = tpol / tchlor
sp = rel – 1
inh = ln(rel) / ĉ
= [sqrt(2 · (sp – ln(rel) ))] / ĉ
ln(inh) = ln(K) + ā · ln(Mw) from which Mw = exp[ (ln(inh) – ln(K)) / ā ]
where tpol and tchlor are the measured time for the solution of polymer and of pure
chloroform to flow in the viscometer respectively, and ĉ is the polymer concentration
in chloroform. The used Mark–Houwink constants related to inherent viscosity for
poly(D,L–lactide) were K=1.8 · 10 –4 dL g –1 and ā=0.72. [337]
3.2.4. In vitro calcium ion release
A simulated physiological solution (SPS) was prepared by dissolving NaCl (Merck;
c=8 g L –1 ) and 4–(2–hydroxyethyl)–1–piperazineethane–sulfonic acid (HEPES)
(Sigma–Aldrich; c=11.92 g L –1 ) in distilled water. The pH of the solution was adjusted
to 7.3 with 2M NaOH (Sigma–Aldrich). Calcium ion release was evaluated by soaking
the irregularly shaped composite granules (v=0.13 cc) in 100 mL of SPS at 37±1°C
for eight hours. While carefully stirring at 150±5 rpm without touching the samples,
the calcium ion concentration in SPS was recorded every minute using a calcium
electrode (Metrohm 692 ISE meter, Ag/AgCl reference electrode, Metrohm, Herisau,
Switzerland).
3.2.5. In vitro surface mineralization
Simulated body fluid (SBF) was prepared according to Kokubo [247] by dissolving
reagent grade chemicals (Merck) in distilled water strictly in the following order: NaCl,
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