Stromal cell-derived factor-1/bone morphogenetic protein 2-loaded chitosan hydrogel controlled-release microbody promoting osteogenesis differentiation of rabbit bone marrow mesenchymal stem cells

Abstract

Objective To prepare stromal cell-derived factor-1 (SDF-1)/bone morphogenetic protein-2 (BMP-2)-loaded chitosan hydrogel controlled-release microbody in combination with the rabbit bone marrow mesenchymal stem cells (BMSCs) separated and cultured in vitro,and investigate the feasibility as a tissue engineering scaffold material to repair bone defect.Methods BMSCs were separated from rabbits,and their growth in different generations was observed.The growth curve was drawn using cell counting kit-8 (CCK-8) method.Flow cytometry was used to detect the stem cell surface marker expression of CD44,and CD90.BMSCs were induced to cartilage differentiation in vhro,and authenticated by toluidine blue staining.Chemotaxis of SDF-1 in rabbit BMSCs was detected by Transwell method.SDF-1/BMP-2loaded chitosan hydrogel controlled-release microbody was prepared by the method of freeze drying and emulsion crosslinking.Scanning electron microscope was used to observe the slow-release characteristics,and enzyme linked immunosorbent assay (ELISA) method was used to detect the cytokines slow-release function.The rabbit BMSCs were inoculated to chitosan hydrogel controlled-release microbody,and their prolteration and osteogenesis differentiation were tesed.The secretion differences of osteogenesis related indicators [alkaline phohatase (ALP),osteopontin (OPN) and type Ⅰ collagen protein] were compared.Results The density gradient centrifugation combined with differential adherent method could obtain rabbit BMSCs characterized by high purity,high proliferation capability and multi-directional differentiation potential.The CD44 expression rate in the third generation of rabbit BMSCs was (86.3 ± 6.5) ％,and CD90 expression rate was (73.50-± 4.85) ％.SDF-1 could drive rabbit BMSCs to migrate to chitosan hydrogel controlled-release microbody,and promote their growth and adhesion.Meanwhile,SDF-1/BMP-2-loaded chitosan hydrogel controlled-release microbody could promote osteogenesis differentiation of rabbit BMSCs and the osteogenesis related protein secretion was increased.Conclusion SDF-1/BMP-2 chitosan hydrogel controlled-release microbody can be used as an ideal tissue engineering bone repairing materials. Key words: Rabbit; Bone marrow mesenchymal stem cells; Stromal cell derived factor-1 ; Bone morphogenetic protein-2 ; Chitosan; Osteogenesis