Reinforced Sintered Cancellous Bovine Bone as a Potential Bone Replacement Material

摘要

This research was undertaken in order to investigate the possibilities of transforming a widely available waste bone resource into a viable human bone replacement biomaterial. Large quantities of bovine bone are available as a resource in New Zealand due to the presence of an extensive meat industry. Cancellous bone was harvested from within the condyle regions of bovine femoral bone by cutting with either band saw or lapidary saw. Portions of cut cancellous bovine bone were significantly defatted and partially deproteinated through repeated autoclaving. The material was then sintered at 1000°C for 3 hours to give a pristine white sintered cancellous bovine bone (SCBB) material, that although weakened in comparison to its original form, retained the porous trabecular architecture of the original cancellous bone. This material was characterised by means of Fourier transform infrared (FT-IR) spectroscopy and powder X-ray diffraction (XRD), while also being subjected to a washing experiment in distilled water with pH monitoring to show that the material was hydroxyapatite (HAp) (Ca10(PO4)6(OH)2) with small quantities of CaO. Scanning Electron Microscopy (SEM) showed that the SCBB material had retained its macroporous architecture, with higher magnification micrographs revealing a microporosity with channels through crystallites of the HAp of approximately 500 – 700 nm in diameter. Organic infiltrating solutions were prepared by dissolving chitosan in hydrochloric acid in addition to calcium phosphate, such as HAp or CaHPO4, to act as a mineral reinforcing material. Due to the limited solubility of HAp, the maximum concentration that could be achieved involving a 50 : 50 w/w combination of chitosan and HAp was 6 g L-1 for both materials in ~ 0.1 mol L-1 HCl. ε Polycaprolactone (PCL) dissolved in tetrahydrofuran (THF) was also trialled as an infiltrating solution, with concentrations up to 14 % w / v dissolved in the THF. Solutions were infiltrated into the porous SCBB material by means of vacuum, vacuum and pressure or a pressure only method. The extent of penetration of the infiltrate into the SCBB was followed by labelling the chitosan molecule with the fluorophore, Fluorescein Isothiocyanate (FITC) and then examining the infiltrated SCBB by fluorescence microscopy. After infiltration into the SCBB material, chitosan and mineral reinforcing were precipitated by an increase in pH. Several methods for increasing solution pH were trialled, including; the addition of urea into the infiltrating solution with subsequent thermal hydrolysis, the catalysis of urea by urease, and treatment with ammonia gas. By products and excess ammonia were shown to be able to be removed from the infiltrated SCBB by repeated washing in water and buffer solution. Mechanical testing on the infiltrated SCBB material was carried out by compression testing on an Instron materials testing instrument. Due to the inherent variability in the density and strength of the starting SCBB material, a statistical approach was required. Multiple samples were used and values for ultimate stress, modulus and a modified toughness measurement obtained from the mechanical testing for non-infiltrated SCBB samples were compared against infiltrated samples. SCBB samples infiltrated with a chitosan / CaHPO4 infiltrate displayed an improvement in ultimate stress, while samples infiltrated with PCL showed an overall increase in modulus. Finally, biocompatibility of the SCBB samples infiltrated with chitosan based infiltrates was tested using L929 fibroblast cell culture testing and a Hen’s Egg Test - Chorioallantoic Membrane (HET-CAM) test. Materials with low cytotoxic response were produced when sufficient washing and buffer treatment was employed after infiltration. This research proved that SCBB could be successfully infiltrated with an organic / mineral composite matrix, which was capable of modifying the mechanical properties of the SCBB, while displaying positive biocompatibility potential.