Effects Of High Mobility Group Box 1 Immobilized Nanofibrous Scaffold On MSCs Osteogenic Differentiation And Bone Regeneration

Bone is a highly vascularised tissue and vascularization plays a pivotal role in skeletal development and large bone defects repair. The repair of large bone defects remains a major clinical orthopaedic challenge. Currently, the clinical approaches to achieve complete healing are autograft or allograft transplantations for large defects treatment in surgery. However, the use of them can not induce vascularization and are insufficient due to limited availability of suitable donor tissue, donor site morbidity, previous harvesting and pathogen transfer. Tissue engineering scaffolds avoid these shortages. However, largely due to a lack of functional vascular supply, clinical applications of tissue engineering bone grafts have been inadequate and sometimes failure due to the inability to provide oxygen, nutrients transport and to clear byproducts of metabolism. Thus fabricating scaffolds that can enhance vascularization as well as osteogenesis is of great significance.After implantation of scaffolds, the blood vessels ingrowth from the host tissue and vascularization from stem cells are induced by inflammatory response. After injury, pro-inflammatory cytokine high mobility group box 1(HMGB 1) is released from necrotic cells as one of the first tissue damage signals to participate in inflammatory response and tissue regeneration. HMGB 1 acts as a multiple functional signal to induce the the migration of stem cells as well as differentiation of mesenchymal stem cells(MSCs), which suggests HMGB 1 has the capability to induce osteogenesis. HMGB 1 induces differentiation of cardiac and angiogenic stem/progenitor cells, which implies HMGB 1 is a potent angiogenic cytokine. In this study, a scaffold with the abilities to promote osteogenesis and vascularization simultaneously was fabricated by using HMGB 1 as a signal molecule. HMGB 1 was immobilized on electrospun nanofibrous scaffold via heparin. The effects of this scaffold on vascularization and osteogenesis were researched as well as its effects on bone regeneration. The main research contents and results of this study are as follows:① Isolation, cultivation and identification of mesenchymal stem cells(MSCs).Primary cultured MSCs had a fibroblast-like morphology, good adhesion and good proliferative capacity in vitro. The induced differentiation experiment was performed to in identify the multiple differentiation potential of MSCs. The results showed MSCs could differentiatiate in to osteoblasts, chondrocytes and adipocytes.② Preparation and characterization of HMGB 1 immobilized nanofibrous bone tissue engineering scaffold.Nanofibrous scaffold with interconnected porous structure, increased mechanical properties and the ability to mimic extracellular matrix was successfully prepared. Via heparin, more HMGB 1 was immobilized onto scaffold and its stability was improved.③ In vitro response of MSCs to HMGB 1 immobilized scaffold.In vitro experiments showed HMGB 1 immobilized scaffold had no significant influences on MSCs proliferation in 7 days and had no significant influences on MSCs survival in 14 days. The scaffold had no cytotoxicity. HMGB 1 immobilized scaffold increased cell adhesion. The MSCs cultured on HMGB 1 immobilized scaffold showed a slenderer morphology and was aligned. HMGB 1 immobilized nanofibrous scaffold induced MSCs differentiation along osteoblastic pathway in vitro.④ Subcutaneous implantation of HMGB 1 immobilized scaffold to evaluate effects of scaffold on cell infiltration, osteogenesis and vascularization in vivo.H-E staining was performed to examine cell infiltration. The results show cell infiltrated into the scaffold, which suggest HMGB 1 immobilized scaffold had a good biocompatibility. The results of CD31 and CD34 immunofluorescence staining showed HMGB 1 immobilized scaffold increased the number of vascular endothelial cells. The results of osteocalcin(OCN) immunofluorescence staining showed HMGB 1 immobilized scaffold enhanced osteogenesis. Further tests showed HMGB 1 immobilized scaffold recruited MSCs into scaffold by up regulating the expression of Stromal cell-derived factor 1(SDF 1). The exprsssion of CD68 indicated inflammatory response of HMGB 1 immobilized scaffold was not risen compared with the scaffolds without HMGB 1.⑤ The effects of HMGB 1 immobilized scaffold on rat calvarial defects repair and the effects of scaffold on osteogenesis and vascularization in rat calvarial defect animal model.H-E staining showed HMGB 1 immobilized scaffold significantly improved the speed and quality of rat calvarial defects regeneration. CD34 and OCN staining results showed HMGB 1 immobilized scaffold enhanced osteogenesis and vascularization in bone environment.In summary, this study successfully fabricated a bone tissue engineering scaffold with HMGB 1 as a tissue engineering signal. The scaffold simultaneously realized multiple functions, including enhancing vascularization, inducing osteogenesis,recruiting stem cells and as a result bone regeneration was accelerated.