Characterization of Biomimetically Enhanced Anorganic Graft Material for Bone Regenerative Applications

Clefts of the lip and palate are the most prevalent congenital craniofacial birth defects. Several surgeries are usually required to correct these defects and they involve extensive bone grafting procedures. From an orthodontic perspective, the aim of bone grafting at the alveolar cleft is to provide continuity and stabilization of the maxillary arch and to permit tooth eruption and orthodontic tooth movement. Although autologous bone graft is the gold standard, it requires a secondary surgical site and the risks of pain, morbidity, infection and scarring at donor site. Tissue engineering approaches that are aimed at improving the functionality of existing clinical materials may provide clinicians with new alternatives. Recently, biomimetic strategies that incorporate the native osteogenic extracellular matrix (ECM) within collagen-based materials have been developed to improve the osteoinductive nature of the biomaterials. This application will focus on utilizing this biomimetic strategy to integrate the osteoinductivity to a frequently used bone graft material Bio-Oss. For decade, Bio-Oss, the porous bone mineral substitute, has been widely and safely applied to dental bone grafting procedure. It is osteoconductive and functions primarily as a space maintainer. However, the clinical Bio-Oss® does not support cell attachment and is not osteoinductive. We hypothesize that: the biomimetically enhanced anorganic bone graft material will impart osteoinductivity by improving stem cell attachment, proliferation and osteogenic differentiation and, ultimately, facilitating new bone formation and remodeling. The goal of this research project was to establish a stable 3D pro-osteogenic ECM coating on Bio-Oss®, and investigate the improved osteoinductive capacity of the biomimetically enhanced Bio-Oss (BE Bio-Oss)by a series of in vitro and in vivo tests. Upon the SEM comparison, the particle size and surface morphologies of BE Bio-Oss demonstrated no difference compared to control but exhibited ECM fibers deposition. However, the HMSC proliferation and the expression of osteogenic marker genes, such as Runx2, Bmp2, colI, and OCN were increased significantly on BE-Bio-Oss. Applied the BE Bio-Oss and control Bio-Oss in the rat critical-sized calvarial bone defects, comparing the bone healing during 4-, 8- and 12-week periods, the µ-CT analysis showed that the bony content (BV/TV), bone structures and bone mineral density were different in BE-Bio-Oss group compare to the control. Under the histological analysis, the BE Bio-Oss demonstrated increased osteogenic cell infiltration and attachment on particle surface as well as enhanced particle remodeling and collagen deposition. Osteogenic markers proteins, DMP1, fibronectin, BMP2, TGFβ and osteocalcin, were strongly expressed in the experimental group compared to Bio-Oss controls. Together, our data indicate existing anorganic bone graft material, Bio-Oss, possess poor osteoinductive properites. The biomimetically enhanced Bio-Oss could promote better cell attachment, proliferation and osteogenic differentiation in vitro, and facilitate stem cell attachments, differentiation and mineralized tissue remodeling in vivo. Our results show a methodology to enhance existing anorganic clinical bone graft materials for improved osteoinductive ability.