Ostrich Eggshell – Hydroxyapatite Composite as a Safe Scaffold with Osteoinductive Properties

Abstract: In the current study, we aimed at preparing a novel bone engineering powder scaffold composed of a natural biomaterial, i.e. hydroxyapatite (HA) and ostrich eggshell (OE), characterizing it as well as exploring its cytocompatibility and osteoconductive capability in vitro. OE powder was immersed into 1% Triton X-100 for 24 hours followed by two times autoclaving. Medical grade HA was combined with pulverized OE at different ratios to yield three different OE/HA powder scaffolds. The resultant scaffolds underwent characterization experiments by different techniques including FTIR, XRD, radiography and SEM analyses. Rabbit adipose-derived mesenchymal stem cells (RADMSCs) were successfully isolated from adipose tissues and characterized by morphologic and flow cytometry-based surface marker evaluations. Cytotoxic evaluation of the scaffolds was performed by direct contact test using L929 cells. Also, the isolated RADMSCs were cultured on the prepared scaffolds and their adhesion (SEM imaging), viability (DAPI and Annexin V/PI staining) and osteogenic differentiation (ALP assay) were investigated. The performed tests showed that the prepared scaffold is composed of HA and OE as confirmed by FTIR and XRD analyses. Cytotoxicity assay using L929 cells verified that OE/HA powder scaffold is safe and further functional experiments could be carried out. Behavioral assays, radiography, surface markers and surface topography analyses as well as viability tests yielded promising results confirming cellular acceptance of the scaffold. In the present study, we provided evidence that the prepared OE/HA scaffold is not only cytocompatible but also can enhance mesenchymal stem cells adhesion, growth and osteogenic differentiation in vitro.