Calcitonin gene related peptide gene-modified rat bone mesenchymal stem cells are effective seed cells in tissue engineering to repair skull defects

Abstract

The study is to verify whether Calcitonin gene related peptide (CGRP) gene-modified rat bone mesenchymal stem cells (BMSCs) are promising seed cells with great potential to repair bone defect. A recombinant lentiviral vector overexpressing CGRP peptide, pLenO-DCE-CGRP, was constructed and transfected into rBMSCs to obtain stable CGRP expression. Differently transfected rBMSCs: CGRP- modified rBMSCs (CGRP group), empty vector- modified rBMSCs (Vector group) and normal rBMSCs (Control group) were transferred to collagen scaffold to repair rat skull defects (n=6). Skull specimens were obtained at 4 and 8 weeks after operation. Expressions of osteogenesis-related indexes OCN (osteocalcin) and BMP-2 (Bone morphogenetic protein-2) were detected by immunohistochemistry and western blotting. Histological observation, Masson trichrome staining and Micro-computed tomography (Micro-CT) were applied to evaluate defect repair. Tartrate-resistant acid phosphatase (TRAP) was applied to observe bone remodeling at 4 weeks. PGP9.5 was detected to observe nerve fibers formation at 8 weeks. Osteogenic markers OCN and BMP-2 were significantly higher in CGRP -modified BMSCs at 4 weeks, compared with the vector and control groups. Few TRAP-positive expressions and scaffolds were observed in the CGRP group. More new bone formation and mineralization in defects were observed in the CGRP group at 4 and 8weeks. Many PGP9.5-positive expressions were found in the CGRP group. CGRP promoted osteogenic differentiation of BMSC and scaffold resorption in vivo. Repair effect of CGRP group was significantly better than the other two groups. CGRP gene-modified BMSCs are effective seed cells in tissue engineering to repair bone defects.