Synthesis and Characterization of Hydroxyapatite-Silk Composite Scaffold for Bone Tissue Engineering

Challenges persist in the restoration of tibial and femoral defects caused by anterior cruciate ligament (ACL) reconstruction surgery. Since one of the major inorganic components of bone tissue is hydroxyapatite (HA), we hypothesize that the implantation of HA-modified silk (HA-silk) scaffolds can promote osteogenesis in the tibial and femoral defects. This study focuses on evaluating and characterizing the HA-silk scaffolds fabricated with two different techniques: coated HA (HA-coated) and cross-linked with HA-nanoparticles encapsulated microspheres (HA-microspheres), with the aim of determining the optimal form for HA-silk porous scaffolds for this application. Results show that the mean diameter of HA-microspheres ranges from 200 mu m to 280 mu m. Swelling ratio measurements reveal that all specimens can absorb 3-4 folds of physiological fluid with their form and stability maintained. In terms of mechanical attributes, HA-silk scaffolds exhibit significantly improved mechanical properties compared to pure silk scaffolds. In vitro culture results with bone marrow derived mesenchymal stem cells (BMSCs) show that the HA-silk scaffolds supported cell viability and proliferation as the HA-silk scaffolds have significantly more cells (2.20-3.45 x 10(5) cells/scaffold) than the pure silk scaffolds (1.5 x 10(5) cells/scaffold) after 14 days (p < 0.05). Bone tissue-specific gene transcription for osteonection, osteopontin and collagen (types I, II, and III) detected via quantitative real-time RT-PCR show a significant up-regulation of osteonectin and osteopontin in HA-silk scaffolds by 2.5-3.7 and 1.7-2.3 folds respectively compared to undifferentiated BMSCs (p < 0.05). Histological observations indicate uniform cellular deposition and mineralization in the HA-silk scaffolds. From the results, it is indicative that the HA-silk scaffolds, especially that with nanoparticular HA-microspheres of high HA content (500 mg), are suitable for bone tissue engineering applications.