CGRP gene-modified rBMSCs show better osteogenic differentiation capacity in vitro
Calcitonin gene-related peptide (CGRP) is a marked and important neuropeptide expressed in nerve fibers during bone repair. This study investigated the role of CGRP overexpression on osteogenic differentiation of rat bone mesenchymal stem cells (rBMSCs). rBMSCs were infected with viral stocks of pLenO-DCE-CGRP (CGRP group) or pLenO-DCE (Vector group), while normal rBMSCs were used as a control. Transfection efficiency of rBMSCs was analyzed by flow cytometry. Cell proliferation was examined using a Cell Counting Kit-8 and flow cytometry. Expressions of alkaline phosphatase(ALP), bone sialoprotein (BSP) and Runt-related transcription factor 2(Runx2) in rBMSCs were detected at 1 and 2 weeks after mineral induction by real-time PCR and western blotting. Alizarin Red staining was applied at 28 days. The ratio of osteoprotegerin (OPG) to receptor activator of nuclear factor kappa B ligand (RANKL) was also detected to determine the underlying mechanism. pLenO-DCE-CGRP-induced rBMSCs stably overexpressing CGRP were successfully established. Overexpression of the CGRP gene significantly promoted rBMSC proliferation (p < 0.05). In addition, expressions of osteogenesis-related indexes were upregulated in the CGRP group (p < 0.05) compared with vector and control groups, and more mineralization nodules were observed in the CGRP group (p < 0.05). CGRP gene increased OPG and reduced RANKL in rBMSCs. Hence, the OPG/ RANKL ratio was increased in the CGRP group compared with the other two groups. CGRP gene-modified rBMSCs show better osteogenic differentiation capacity compared with rBMSCs in vitro.
KeywordsCalcitonin gene-related peptide Bone mesenchymal stem cells Transfection OPG/RANKL Tissue engineering
National Natural Science Foundation of China (81271138). Natural Science Foundation of Shandong Province (ZR2017QH007). Open Foundation of Shandong Provincial Key Laboratory of Oral Tissue Regeneration (SDKQ201704).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Green SA, Jackson JM, Wall DM, Marinow H, Ishkanian J (1992) Management of segmental defects by the Ilizarov intercalary bone transport method. Clin Orthopaedics Related Res 280:136–142Google Scholar
- Kim HY, Paek JY, Park HK, Mi RC, Yoon HS, Kim KS et al (2009) Efficacy and safety of autologous bone marrow-derived mesenchymal stem cell treatment in patients with amyotrophic lateral sclerosis. J Korean Neurol Assoc 27(2):163–169Google Scholar
- Nabavi SM, Aghdami N, Arab L, Hamzeloo A, IR) (2014). Safety and efficacy of intravenous injection of autologous bone marrow-derived mesenchymal stem cell in patients with multiple sclerosis: a double blind randomized semi-crossover clinical trial: preliminary report 1—safety issues. Mult Scler:935–935Google Scholar
- Wang S, Xiao J, Li Z (2017) Role of substance P and calcitonin gene-related peptide in bone metabolism. Zhong nan da xue xue bao Yi xue ban = J Cent South Univ Med Sci 42(3):334–339Google Scholar
- Zhu LY, Feng ZY, Zheng TY, Dan L, Zhou Q, Li FG et al (2016) Autologous bone marrow stem cells injection therapy resistant multi drug tuberculosis patients efficacy and safety. J Hunan Normal Univ (Med Sci) 13(1):40–42Google Scholar