Barbieri Thesis - BioMedical Materials program (BMM)

Chapter 7 – Polymer molecular weight and instructive composites
recommended that such tests, to be valid, should be performed at temperatures lower
than the glass transition temperature (i.e. Tg) of the considered polymer to avoid
unwanted effects due to polymer state transitions. [354–357, 368] A phosphate buffered saline
(PBS; Invitrogen, Darmstadt, Germany) solution having initial pH of 7.5±0.05 was used.
Sterile granules of each composite (75±0.5 mg) were carefully weighed before use (m0)
and soaked in 30 mL PBS at 55±0.5°C for five weeks. The temperature was set
referring to the polymers Tg of the copolymers used here (i.e. 55–60°C). For each of the
three time points considered, i.e. 1, 3 and 5 weeks, three samples of each material were
used. Every day the pH of the degrading solution was recorded with a pH–meter (Orion
4 Star, Thermo Scientific, USA) and the medium was refreshed in case the pH fell below
7.0. With the solution removed at every refreshment, the amounts (in M) of calcium
and phosphate ion released from the samples were measured using appropriate
biochemical kits (QuantiChrom TM Calcium assay kit, BioAssay Systems, USA;
PhosphoWorks TM Colorimetric Phosphate Assay kit Blue Color, Bioquest Inc, USA) with
the help of a spectrophotometer (AnthosZenyth 3100, Anthos Labtec Instruments
GmbH, Salzburg, Austria) and absorbance filter of 620 nm for both assays. At each of
the considered time points, i.e. 1, 3 and 5 weeks, the granules were removed from the
degrading media, the excess PBS was wiped away and their wet weight was carefully
weighed (mwet). Afterwards they have been vacuum–dried at room temperature until their
weight was stable and then were weighed again (mdry). The mass loss and fluid uptake
of the composites was determined as:
mass loss = 100 · (m0 – mdry) / m0
fluid uptake = 100 · (mwet – mdry) / mdry
Part of the degraded samples were then was heated at 900°C to burn the polymer
phase out and determine the final effective apatite and polymer percentage contents
(in %wt., pap and ppol respectively). The remaining part of the samples was dissolved in
chloroform (Sigma–Aldrich) and, after separating apatite from the polymer, we
determined the intrinsic viscosity () using an Ubbelohde viscometer as explained in
§7.2.2. As already mentioned in Chapter 6 (i.e. §6.2.5. for theory background), to
evaluate the hydrolysis rate of the polymer phase in the two composites we estimated
the autocatalysed hydrolytic degradation rate constant k as:
k · t = –ln( / )
where is the initial intrinsic viscosity and is the intrinsic viscosity at the
considered time point t (t=0, 7, 21 and 35 days) for the considered polymer phase. For
convenience, the results were plotted on a graph ln(/) against time t (in days).
7a.2.4. Dynamic mechanical analysis
It has been done following the protocol described in §7.2.7.
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