Barbieri Thesis - BioMedical Materials program (BMM)

Chapter 5 – Alkali surface treatment effects
measures cannot be directly applied to the granules. However, as alkali surface
treatment has been the same for discs and granules, the roughness and hydrophilicity
trends seen on discs are expected also on the granules.
5.2.4. In vitro degradation
A saline physiological solution (SPS) was prepared by dissolving sodium chloride
(NaCl, Merck) (8 g L –1 ) and 4–(2–hydroxyethyl)–1–iperazineethane–sulfonic acid
(HEPES) (Sigma–Aldrich) (11.92 g L –1 ) in distilled water. The pH of the solution was
adjusted to 7.3 with 2 M NaOH (Merck) at 37°C. Sterile granules of each composite
(0.5±0.01 g) were carefully weighed before use (m0) and soaked in 200 mL SPS (in
triplicate) at 37±1°C for three months under a 3–week refreshing regime. Every three
weeks, the pH of the degrading solution was recorded with a pH–meter (Orion 4 Star,
Thermo Scientific, USA). With the solution removed at every refreshing time point, the
concentration (in M) of calcium and phosphate ions were measured using
appropriate biochemical kits (QuantiChrom TM Calcium assay kit, BioAssay Systems,
USA; PhosphoWorks TM Colorimetric Phosphate Assay kit Blue Color, BioquestInc,
USA) with the help of a spectrophotometer (AnthosZenyth 3100, AnthosLabtec
Instruments GmbH, Salzburg, Austria) and absorbance filter of 620 nm for both
assays. After 12 weeks the granules were removed from the degrading media, the
excess SPS was wiped away and they were carefully weighed (mwet). Afterwards,
they were vacuum–dried at 37±1°C until their weight was stable and then weighed
again (mdry). The mass loss and fluid uptake of the composites were determined as
follows:
mass loss = 100 · (m0 – mdry) / m0
fluid uptake = 100 · (mwet – mdry) / mdry
Part of the degraded samples 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 degraded
samples was used to evaluate the degradation of the polymer phase by measuring its
post–degradation intrinsic viscosity after 12 weeks.
5.2.5. In vitro surface mineralisation
Surface mineralisation was studied in two different solutions, i.e. basic simulated body
fluid (SBF), and simulated body fluid containing 10% (in volume) foetal bovine serum
(SBF/FBS). Dipping the samples in SBF will give an indication of their surface
mineralising potential, while SBF/FBS is a solution simulating the ‘real’ situation in the
body where proteins are present. This second experiment will indicate whether the
proteins influence on the surface mineralising potential of the materials. Simulated
96