Stage Treatment Through Sequential Delivery Two Key Factors For Craniofacial Bone Regeneration

Craniofacial bone defects resulting from tumor, trauma, osteoradionecrosis, osteomyelitis, periodontitis or congenital deformities present a challenge to prosthetic reconstruction. The quality, speed and biomechanical stability of new bone formation in repair of craniofacial bone defects are closely related to dental implantation. Various methods such as allografting. xenographic bone graft, artificial bone graft, cells- or/and growth factors-based tissue engineering, guided bone regeneration and distraction osteogenesis, are applied for craniofacial bone and alveolar bone reconstruction, and achieve satisfactory effects. Recently, the concepts of bone reconstruction involve the combination of a scaffold with cells and/or biomolecules that promotes the repair and/or regeneration of tissues. Therefore, it is very important to mimic the natural environment in which bone cells grow. Bone regeneration is complexly regulated processes that involve a plethora of different growth and transcription factors which coordinate the interaction of cells and matrix in response to external or internal stimuli. Basic fibroblast growth factor (bFGF) induces angiogenesis, and stimulates mitogenesis of mesenchymal cells and osteoblasts in the early phase of bone defects repair. Sonic hedgehog (Shh) is involved in osteoblast differentiation by a mechanism involving BMPs, and may play an important role in bone formation, especially at later stages of skeletogenesis and fracture repair. In this study, bFGF and Shh were used to mimic natural bone repair processes through artificial regulation, and an optimal osteo-inductive approach for craniofacial bone regeneration was discussed.In this study, recombinant viruses rAAV2-Shh and rAAV2-tet-off-bFGF that were controlled by tetracycline regulation system were reconstructed. Rat bone marrow-derived mesenchymal stem cells (BMSCs) were routinely isolated from bone marrow, and co-infected with above two recombinant viruses in vitro. The expression of bFGF was controlled through doxycycline (Dox, a tetracycline analogue) administration, and thus sequential expression of bFGF and Shh was induced. Thereafter, the co-infected BMSCs regulated by sequential delivery factors were seeded on the biodegradable scaffold (β-tricalcium phosphate), and this gene-enhanced tissue engineering bone was cultured in a three-dimensional (3D) in vitro culture system. Cell morphology, viability, and differentiation were studied at different sequential expression periods. Osteogenic potential of BMSCs was evaluated according to the concentration of alkaline Phosphatase and the mRNA expression of osteogenic marker genes. Finally, this regulated gene-enhanced tissue engineering bone was introduced into rat critical sized calvarial defects (8 mm). The sequential expression of angiogenic and osteogenic genes, which mimics the intramembranous bone repair, was regulated through Dox administration. Histological slides, obtained from defect sites at the 2nd, 4th and 8th week, were digitized, and the total two-dimensional amount of new bone area and vascular density in defects were quantitated. It was shown that the increased bone formation accompanied with angiogenesis was induced by sequential key factors delivery in vivo, and a positive correlation between new bone area and blood vessel density was found. This study suggests that stage treatment for craniofacial bone regeneration plays a significant role in enhancing bone regeneration through sequential delivery of osteogenic and angiogenic genes.