Enhance osteoinductivity of DBM with Noggin suppression in 3D hydrogel system
Bone defects have become a challenging health issue after extensive trauma, osteoporosis, and tumor extirpation worldwide. To enhance bone healing, bone grafts have been employed in clinical treatment for many years. Autologous grafts have been considered as the “gold standard” for the bone graft material, but their application is limited by the donor site morbidity. Demineralized bone matrix (DBM), one of the alternations of autologous graft, has been widely used for craniofacial bone repair. However, the application of DBM is compromised due to its rapid dispersion after the implantation and low osteoinductivity. In order to deliver and localize DBM, carriers with high bioactivity are in need to be developed.
Chitosan has been proved to be attractive for researchers in the field of tissue bioengineering due to its high biocompatibility, biodegradability, and low immunogenicity. Besides, heparin, a highly sulfated polysaccharide in the extracellular matrix, possesses the typical structure which can interact with multiple osteoinductive factors to enhance and stabilize the biological activities of growth factors. Therefore, we synthesized a chitosan-based hydrogel with heparinized conjugation (Hep-MeGC), which can not only deliver DBM but also enhance its osteogenic activity. Moreover, upon the stimulation of BMPs (major osteogenic factors in DBM), BMP efficacy is significantly reduced due to increased expression of BMP antagonists like Noggin. Thus, the potency of BMPs in DBM may be enhanced through Noggin suppression. This study aims to investigate the new method by using Noggin suppression and Hep-MeGC hydrogel to enhance DBM-mediated bone repair.
To suppress Noggin, human bone marrow stem cells (hBMSCs) were transfected with lentiviral particles that encode Noggin-shRNA. The methacrylate glycol chitosan (MeGC) and heparinized chitosan hydrogel (Hep-MeGC) hydrogel, were then prepared to deliver DBM and BMSCs with Noggin-suppression. The effect of DBM and Noggin suppression on osteogenesis was evaluated in vitro, and the ability of DBM and Noggin suppression in Hep-MeGC hydrogels to bone regeneration was investigated through in vivo study.
Noggin suppression + DBM greatly enhanced the osteogenesis of BMSCs in Hep-MeGC hydrogels, as shown by the increased ALP production, calcium deposition, and expression of osteogenic genes. The mechanistic study showed that Noggin suppression enhanced the DBM-based osteoinductivity through the stimulation of BMP-Smad signals. Detected by microCT and histological/immunohistochemical analysis, the combinational treatment further displayed a robust bone healing capacity in the critical-sized mouse calvarial defect model. To conclude, the incorporation of DBM and Noggin suppression in the Hep-MeGC hydrogel significantly promoted bone healing.