Barbieri Thesis - BioMedical Materials program (BMM)

Chapter 6 – Fluid uptake as instructive factor
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. With the solution removed at every refreshment, the amounts (in mg) of
calcium and phosphate ions 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 measured (mwet). Afterwards they have been vacuum–dried at room
temperature until their weight was stable and then were weighed (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 heated at 900°C to burn the polymer phase
out and determine the final effective apatite and polymer contents. The remaining part
of the samples was dissolved in chloroform (Sigma–Aldrich) and, after separating
apatite from the polymer, we determined the inherent and intrinsic viscosities (inh and
) and weight average molecular weight (Mw) using an Ubbelohde viscometer as
explained in §6.2.3. To evaluate the hydrolysis rate of the polymer phase in the two
composites we estimated the autocatalysed hydrolytic degradation rate constant
k as (see §6.2.5.1. for theory background):
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.
The results were plotted on a graph ln(/) against time t (in days) and the
constant k was estimated with linear regression.
6.2.6. Surface mineralization in simulated body fluid
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,
NaHCO3, KCl, K2HPO4·3H20, MgCl2·6H2O, CaCl2 (calcium ion standard solution 0.1
M, Metrohm, Herisau, Switzerland) and Na2SO4. The fluid was then buffered to pH 7.4
at 36.5°C using Tris ((CH2OH)3CNH3) and 1 M HCl. The final solution had an ion
concentration (in mM) as follows: Na + , 142; K + , 5; Mg 2+ , 1.5; Ca 2+ , 2.5; Cl – , 147.8;
(HCO3) – , 4.2; (HPO4) 2– , 1; (SO4) 2– , 0.5. Granules of the composites (0.25 g) and
ceramics (0.25 g, prepared by crushing and sieving the cylinders) were soaked in 100
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