Decellularized bovine small intestinal submucosa-PCL/hydroxyapatite-based multilayer composite scaffold for hard tissue repair

This study involved the development of a multilayer osteogenic tissue scaffold by assembling decellularized bovine small intestinal submucosa (bSIS) layers, together with synthetic hydroxyapatite microparticles (HAp) and poly(epsilon-caprolactone) (PCL) as the binder. As a first step, the surface and mechanical properties of the developed scaffold was determined, after which the biocompatibility was evaluated through seeding with isolated rat bone marrow mesenchymal stem cells (BM-MSCs). Then, a 21-day culture study was performed to investigate the in vitro osteoinductive potential of the scaffold on BM-MSCs under standard and osteogenic culture conditions. The SEM findings indicated that a uniform multilayer and perforated structure was acquired; that the HAp microparticles were homogenously distributed within the structure; and that the PCL-bound laminar scaffold had structural integrity. Mechanical tests revealed that the scaffold maintained its mechanical stability for at least 21 days in culture, with no changes in the first-day maximum strength and maximum stress values of 625.123 +/- 70.531 N and 6.57762 +/- 0.742 MPa, respectively. MTT and SEM analyses together revealed that BM-MSCs preserved their viability and proliferated during a 14-day culture period on the multilayer scaffold. Immunofluorescence analyses indicated that cells on the scaffold differentiated into the osteogenic lineage, by the culture-time-dependent increase in osteogenic markers' expression, i.e. Alkaline phosphatase, Osteopontin, and Osteocalcin. It was also clear that, the osteoinductive effect by the composite scaffold on BM-MSCs could be achieved even without the use of any external osteogenic inducers.