524. Injectable BMP-2 Gene-Activated Scaffold for the Repair of Mouse Cranial Bone Defect

Abstract

Introduction: A major requirement in bone tissue engineering is the persistent and robust promotion of osteogenesis, which requires sustained stimulation with osteoinductive factors, such as bone morphogenetic protein-2 (BMP-2). However, the direct application of these factors remains challenging due to their short half-life and rapid systemic clearance. Given the well demonstrated benefits of recombinant adeno-associated viral (rAAV) vector, including long-term gene transfer efficiency and relative safety, rAAV-based therapies have been developed in both basic and translational research. However, traditional ex vivo gene transduction process is time consuming and requires multiple steps of cell culture. In addition, rAAV shows inconsistent transfection efficacy in primary cells. In this study, we co-packed rAAV-BMP-2 and human bone marrow stem cells (hBMSCs) into a gelatin scaffolds through one-step visible light-based phtocorsslinking (PXL), and tested their application for bone regenerationi. We hypothesize that the concentrated and localized release of rAAV-BMP-2 from the scaffold matrix results in higher transfection efficacy and BMP-2 expression in hBMSCs seeded within the scaffolds, thus enhancing their osteogenic differentiation and bone formation upon implantation in bone defect. Methods: hBMSCs were obtained with IRB approval from total joint arthroplasty patient. The rAAV6-BMP-2 and rAAV6-GFP reporter (as control) vectors were designed and prepared using an established protocol in our laboratory. PXL was performed using 10% gelatin and 10×106 hBMSCs/ml with different amount of rAAV6-GFP. Transfection efficiency was determined by counting GFP-positive cells ratio as well as assessing cell viability. At an optimal ratio, rAAV-BMP-2 and hBMSCs were included into gelatin through PXL. At different time point, BMP-2 production and rAAV6 release kinetics were analyzed. The osteogenesis of hBMSCs was assessed at day 28 by real time PCR and histological staining. In animal study, a 0.5 cm diameter critical size of cranial bone defect model was generated in SCID mice and then the constructs described above were transplanted. Micro-CT imaging and histological staining were used to evaluate the skull bone healing process up to 6 weeks. Results: Both rAAV and hBMSCs survived during the scaffold fabrication process. 10×104 rAAV/hBMSCs was shown to be the optimal ratio in maintaining cell viability and achieving transduction efficiency. After in situ transfection within scaffolds, hBMSCs produced higher BMP-2 amount than those subjected to traditional ex vivo transduction process, and underwent robust osteogenesis as well. In animal study, micro-CT imaging results indicated that rAAV BMP-2 & hBMSCs-activated gelatin scaffolds effectively promoted bone regeneration in mice cranial bone defect, in a manner significantly superior to that of scaffolds loaded with hBMSCs/BMP-2 protein, and was remarkably compatible to scaffolds loaded with hBMSCs previously ex vivo transduced by rAAV-BMP-2. The histological staining further confirmed the efficacious bone formation from rAAV BMP-2 & hBMSCs loaded scaffold. The results shown here thus demonstrate the feasibility of combining gene and cell therapy with biomaterial engineering via a single-step fabrication and its application for the repair of bone defect.