Effects of Sterilization and Hydrolytic Degradation on the Structure, Morphology and Compressive Strength of Polylactide-Hydroxyapatite Composites

Abstract

Composites based on polylactide (PLA) and hydroxyapatite (HA) were prepared using a thermally induced phase separation method. In the experimental design, the PLA with low weight-average molar mass (M_w) and high M_w were tested with the inclusion of HA synthesized as whiskers or hexagonal rods. In addition, the structure of HA whiskers was doped with Zn, whereas hexagonal rods were mixed with Sr salt. The composites were sterilized and then incubated in phosphate-buffered saline for 12 weeks at 37 °C, followed by characterization of pore size distribution, molecular properties, density and mechanical strength. Results showed a substantial reduction of PLA M_w for both polymers due to the preparation of composites, their sterilization and incubation. The distribution of pore size effectively increased after the degradation process, whereas the sterilization, furthermore, had an impact on pore size distribution depending on HA added. The inclusion of HA reduced to some extent the degradation of PLA quantitatively in the weight loss in vitro compared to the control without HA. All produced materials showed no cytotoxicity when validated against L929 mouse skin fibroblasts and hFOB 1.19 human osteoblasts. The lack of cytotoxicity was accompanied by the immunocompatibility with human monocytic cells that were able to detect pyrogenic contaminants.

Keywords: PLA; biocomposites; hydrolytic degradation; hydroxyapatite; sterilization.

Figure A1

XRD analysis of whiskers.

Figure A2

XRD analysis of hexagonal rods.

Figure A3

XRD analysis of whiskers with Zn.

Figure A4

XRD analysis of hexagonal rods with Sr.

Figure A5

DSC curves for PLA under investigation (pure granulates).

Figure A6

DSC curves for Low M_w PLA at different stage of studies—after preparation, after sterilization and degradation.

Figure A7

DSC curves for High M_w PLA at different stages of studies—after preparation, sterilization and degradation.

Figure A8

DSC curves for Low M_w PLA + HA whiskers composites at different stages of studies—after preparation, sterilization and degradation.

Figure A9

DSC curves for Low M_w PLA + Zn-HA whiskers composites at different stage of studies—after preparation, after sterilization and degradation.

Figure A10

DSC curves for Low M_w PLA + HA rods composites at different stage of studies—after preparation, after sterilization and degradation.

Figure A11

DSC curves for Low M_w PLA + Sr-HA rods composites at different stage of studies—after preparation, after sterilization and degradation.

Figure A12

DSC curves for High M_w PLA + HA whiskers composites at different stage of studies—after preparation, after sterilization and degradation.

Figure A13

DSC curves for High M_w PLA + Zn-HA whiskers composites at different stage of studies—after preparation, after sterilization and degradation.

Figure A14

DSC curves for High M_w PLA + HA rods composites at different stage of studies—after preparation, after sterilization and degradation.

Figure A15

DSC curves for High M_w PLA + Sr-HA rods composites at different stage of studies—after preparation, after sterilization and degradation.

Figure 1

Microstructure of HA: (a) whiskers (b) whiskers doped with Zn, (c) hexagonal rods, (d) hexagonal rods mixed with Sr. Scale bar 50 µm.

Figure 2

SEM images of the prepared, sterilized and degraded composites made of Low M_w PLA. Scale bar, 500 µm.

Figure 3

SEM images of the prepared, sterilized and degraded composites made of High M_w PLA. Scale bar, 500 µm.

Figure 4

Distribution of pore size in the composites (%). X-axis indicates the compartment of pore size as such; I, 0–50 µm; II, 50–100 µm; III, 100–150 µm; IV, 150–200 µm; V, 200–250 µm; VI, 250–300 µm; VII, 300–350 µm; VIII, 350–600 µm.

Figure 5

Compression strength of low and high M_w PLA composites.

Figure 6

Glass transition temperature recorded for the tested composites.

Figure 7

Weight loss of specimen at particular time of degradation.

Figure 8

Weight loss of polymer inside composites at particular time of degradation.

Figure 9

Cytobiocompatibility assessment with cells cultured on examined composites. Mouse skin L929 fibroblasts (A) or human hFOB1.19 osteoblasts (B) were co-cultured with the sections of composites according to ISO-10993-5:2009 and the viability was estimated in the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay. Dotted line indicates the cytotoxicity threshold as cell viability of 70%, respective to control cells cultured without the composite (100%). The quantitative measurement of activation of the nuclear factor NF-κB in THP1-Blue™ monocytes co-cultured with composites for 24 h (C), in comparison to lipopolysaccharide-treated (LPS Escherichia coli O55:B5, positive control) or non-treated cells (culture medium, negative control). The toll-like receptor-mediated activation of NF-κB resulted in the production of secreted embryonic alkaline phosphatase (SAEP) which was assayed in a colorimetric reaction (absorbance at 650 nm) with QUANTI-Blue™ substrate solution. Data are presented as mean ± SD of three separate experiments.