BMP-2 gene-modified bone marrow mesenchymal stem cells combined with nano-hydroxyapatite/polyamide 66 implants improve atrophic nonunion of bone in rats

Acta Universitatis Medicinalis Anhui     font:big middle small

Found programs: Natural Science Foundation of Qinghai Province (No.2022-ZJ-968Q)

Authors:Huang Yong, Ma Jianwen, Li Yu, Jing Qingling, Zhang Qin, Li Changshuai

Keywords:atrophic nonunion of bone; bone morphogenetic protein-2; bone marrow mesenchymal stem cells; nano-hydroxyapatite/polyamide 66; adenovirus; bone stent material

DOI:

〔Abstract〕 (Dept of Trauma Orthopedics, Affiliated Hospital of Qinghai University Xining 810001)Abstract Objective To investigate the therapeutic effect of bone marrow mesenchymal stem cells (BMSCs) modified by bone morphogenetic protein (BMP)-2 gene combined with nano-hydroxyapatite (nHA)/polyamide 66 (PA66) on femoral nonunion in rats. Methods Rat BMSCs were isolated and divided into the stent+BMSCs group, stent+BMSCs+rhBMP-2 group,and stent+BMSCs+Ad-BMP-2 group; human recombinant BMP-2 (rhBMP-2) or adenovirus-infected BMP-2 (Ad-BMP-2) vector was transfected into BMSCs and loaded onto nHA/PA66 stent materials. Flow cytometry was used to detect that BMSCs expressed specific surface markers. Cell proliferation was detected by MTT assay, related bioactive factors [platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), osteocalcin (OCN), osteonectin (ON)] were detected by ELISA, alkaline phosphatase (ALP) activity was detected, and cell growth and adhesion were observed by scanning electron microscopy (SEM). In the atrophic nonunion model of SD rats, the stent+BMSCs+rhBMP-2 or stent+BMSCs+Ad-BMP-2 complex was implanted into the defect area, which was divided into the rhBMP-2 group and the Ad-BMP-2 group, and the therapeutic effect was evaluated by X-ray and Micro CT. Results The surface of the nHA/PA66 stent was smooth and porous, and BMSCs adhered well. Flow cytometry showed high expression of CD29 and CD90 and low expression of CD45 and CD34 in BMSCs. MTT showed that the cells proliferated rapidly after 72 h. Compared with the stent+BMSCs group and the stent+BMSCs+rhBMP-2 group, the ALP activity, the expressions of PDGF, TGF-β, VEGF,FGF, OCN, and ON in the stent+BMSCs+Ad-BMP-2 group were significantly up-regulated (P<0.05). 12 weeks after surgery, the stent complex in the Ad-BMP-2 group of rats was completely wrapped by newly formed cortical bone, and the surface was smooth and intact. X-ray showed that the complex in the Ad-BMP-2 group was completely wrapped by newly formed cortical bone, and the recovery degree was better than that in the rhBMP-2 group. Micro CT results showed that compared with the rhBMP-2 group, the bone volume fraction, trabecular thickness, trabecular number, bone mineral density, and bone mineral content in the Ad-BMP-2 group all increased 12 weeks after surgery (P<0.05), and the trabecular separation level decreased (P<0.05). Conclusion Ad-BMP-2-transfected BMSCs combined with nHA/PA66 stent material can express bioactivity more effectively, provide a continuous and good microenvironment for the proliferation and differentiation of BMSCs, which is beneficial to promoting local osteogenic activity and bone defect repair.