Abstract
Hydroxyapatite (HA) forms the inorganic component of bones and teeth, has been a popular choice of bone substitute due to its bioactivity, biocompatibility and osteoconductivity. Bovine bone derived HA generated wider interest due to its abundant availability as meat wastes and similarity in terms of morphology and mineral composition to the human bone. Previous research from our group developed an inexpensive and reproducible method to prepare HA from bovine bone (BHA) using a subcritical-water based extraction process. NZ sourced bovine bones are advantageous since there is no risk of bovine spongiform encephalopathy (BSE) in the country. The process ensured the production of phase pure prion-free HA with satisfactory biological properties but lacked the mechanical properties required for biomedical applications.
The aim of this study was to achieve significant improvement in the physicochemical, mechanical and biological properties of the BHA. This was achieved in two steps, 1) Co-substitution of Mg and Sr ions into the BHA lattice using a sol-gel based method, 2) Infiltrating the prepared scaffolds with a polymer: chitosan (CS) or its water-soluble variant, succinyl chitosan (SCS), in order to compensate for loss of organic (collagen) phase during the bone processing. SCS proved to be the better choice of polymer than CS due to its better chemical stability at physiological pH while positively influencing the mechanical properties of the BHA scaffold. Three scaffolds, BHA (bovine HA), SHA (SCS infiltrated bovine HA) and XHA (Mg-Sr co-substituted SCS infiltrated BHA) were prepared to evaluate the combined effect of co-substitution and polymer infiltration on the overall properties of BHA.
The prepared bone scaffolds were subject to physical, chemical and thermal characterisation. Infrared spectroscopy confirmed the presence of HA phase, co-substitution of Mg-Sr ions and infiltration of SCS. The results of Energy Dispersive X-ray and Inductively Coupled Plasma-Mass Spectroscopic analyses ascertained the presence of Ca, P, Mg, Sr, SCS phases and Ca/P ratios, complementing the results of the IR spectroscopy. X-ray diffraction data confirmed the crystallinity of the scaffolds and the phase transformation resultant of Mg-Sr co-substitution in HA. Thermogravimetric analysis highlighted the thermal stability of the scaffolds at temperatures as high as 1000ºC. All three scaffolds showed excellent chemical stability and biodegradation behaviour in simulated body fluid (SBF). XHA scaffolds presented better chemical stability and rate of biodegradation compared to BHA. An Instron Universal Testing Machine was used to evaluate the compressive mechanical properties of the scaffolds. XHA produced the higher Young’s modulus and nearly three-fold increase in the compressive strength compared to BHA scaffolds.
The in vitro biocompatibility of the scaffolds towards Saos-2 cells was evaluated. All three scaffolds exhibited excellent cell viability and XHA scaffolds showed significantly higher cell proliferation of Saos-2 cells compared to BHA and SHA. The XHA scaffolds showed excellent antibacterial activity against E. coli and S. aureus when compared to BHA. The SHA scaffolds produced no antibacterial activity. Finally, a pilot HET-CAM (Hen’s egg test - chorioallantoic membrane) study was conducted to determine the in vivo performance of BHA, SHA and XHA scaffolds. The results revealed promising signs suggestive of the osteogenic potential of the scaffolds which could be explored further.