Journal of Molecular Histology

, Volume 49, Issue 4, pp 357–367 | Cite as

CGRP gene-modified rBMSCs show better osteogenic differentiation capacity in vitro

  • Xijiao Yu
  • Shuang Liu
  • Hui Chen
  • Xinyu Zhao
  • Xue Chen
  • Yi Du
  • Shu LiEmail author
Original Paper


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.


Calcitonin 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.


  1. Chen J, Liu W, Zhao J, Sun C, Chen J, Hu K et al (2017) Gelatin microspheres containing calcitonin gene-related peptide or substance P repair bone defects in osteoporotic rabbits. Biotechnol Lett 39(3):465–472CrossRefPubMedGoogle Scholar
  2. Colter DC, Class R, DiGirolamo CM, Prockop DJ (2000) Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci USA 97(7):3213–3218CrossRefPubMedGoogle Scholar
  3. Deng Y, Bi X, Zhou H, You Z, Wang Y, Gu P et al (2014) Repair of critical-sized bone defects with anti-miR-31-expressing bone marrow stromal stem cells and poly(glycerol sebacate) scaffolds. Eur Cells Mater 27(1):13CrossRefGoogle Scholar
  4. Dong Y, Long T, Wang C, Mirando AJ, Chen J, O’Keefe RJ et al (2014) NOTCH-mediated maintenance and expansion of human bone marrow stromal/stem cells: a technology designed for orthopedic regenerative medicine. Stem Cells Transl Med 3(12):1456–1466CrossRefPubMedPubMedCentralGoogle Scholar
  5. 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
  6. Imai S, Matsusue Y (2002) Neuronal regulation of bone metabolism and anabolism: calcitonin gene-related peptide-, substance P-, and tyrosine hydroxylase-containing nerves and the bone. Microscopy Res Tech 58(2):61–69CrossRefGoogle Scholar
  7. 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
  8. Kon E, Filardo G, Roffi A, Di Martino A, Hamdan M, De Pasqual L et al (2012) Bone regeneration with mesenchymal stem cells. Clin Cases Miner Bone Metab 9(1):24–27PubMedPubMedCentralGoogle Scholar
  9. Lam WL, Guo X, Leung KS, Kwong KS (2012) The role of the sensory nerve response in ultrasound accelerated fracture repair. J Bone Joint Surg Br 94(10):1433–1438CrossRefPubMedGoogle Scholar
  10. Li J, Kreicbergs A, Bergstrom J, Stark A, Ahmed M (2007) Site-specific CGRP innervation coincides with bone formation during fracture healing and modeling: A study in rat angulated tibia. J Orthopaedic Res 25(9):1204–1212CrossRefGoogle Scholar
  11. Liang W, Zhuo X, Tang Z, Wei X, Li B (2015) Calcitonin gene-related peptide stimulates proliferation and osteogenic differentiation of osteoporotic rat-derived bone mesenchymal stem cells. Mol Cell Biochem 402(1–2):101–110CrossRefPubMedGoogle Scholar
  12. 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
  13. Pang P, Shimo T, Takada H, Matsumoto K, Yoshioka N, Ibaragi S et al (2015) Expression pattern of sonic hedgehog signaling and calcitonin gene-related peptide in the socket healing process after tooth extraction. Biochem Biophys Res Commun 467(1):21–26CrossRefPubMedGoogle Scholar
  14. Parekkadan B, Milwid JM (2010) Mesenchymal stem cells as therapeutics. Annu Rev Biomed Eng 12:87–117CrossRefPubMedPubMedCentralGoogle Scholar
  15. Salah RA, Mohamed IK, El-Badri N (2018) Development of decellularized amniotic membrane as a bioscaffold for bone marrow-derived mesenchymal stem cells: ultrastructural study. J Mol Histol. PubMedCrossRefGoogle Scholar
  16. Schinke T, Liese S, Priemel M, Haberland M, Schilling AF, Catala-Lehnen P et al (2004) Decreased bone formation and osteopenia in mice lacking alpha-calcitonin gene-related peptide. J Bone Miner Res 19(12):2049–2056CrossRefPubMedGoogle Scholar
  17. Sekiya I, Larson BL, Smith JR, Pochampally R, Cui JG, Prockop DJ (2002) Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yields of early progenitors and evaluate their quality. Stem cells 20(6):530–541CrossRefPubMedGoogle Scholar
  18. Sun XK, Zhou J, Zhang L, Ma T, Wang YH, Yang YM, Tang YT, Li H, Wang LJ (2017) Down-regulation of Noggin and miR-138 coordinately promote osteogenesis of mesenchymal stem cells. J Mol Histol 48(5–6):427–436CrossRefPubMedGoogle Scholar
  19. Truong TH, Moorjani R, Dewey D, Guilcher GMT, Prokopishyn NL, Lewis VA (2016) Adverse reactions during stem cell infusion in children treated with autologous and allogeneic stem cell transplantation. Bone Marrow Transplant 51(5):680CrossRefPubMedGoogle Scholar
  20. Uslu S, Irban AG, Gereli A, Aydinlar EI, Elpen P, Ince U (2016) The effect of femoral nerve block on fracture healing via expressions of growth factors and beta-catenin. Folia Histochem Cytobiol 54(3):151–158CrossRefPubMedGoogle Scholar
  21. Wang L, Lin Z, Shao B, Zhuge Q, Jin K (2013) Therapeutic applications of bone marrow-derived stem cells in ischemic stroke. Neurol Res 35(5):470–478CrossRefPubMedGoogle Scholar
  22. 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
  23. Xian L, Bao C, Xu HHK, Jian P, Jing H, Ping W et al (2016) Osteoprotegerin gene-modified BMSCs with hydroxyapatite scaffold for treating critical-sized mandibular defects in ovariectomized osteoporotic rats. Acta Biomater 42:378–388CrossRefGoogle Scholar
  24. Xiang L, Ma L, Wei N, Wang T, Yao Q, Yang B et al (2017) Effect of lentiviral vector overexpression alpha-calcitonin gene-related peptide on titanium implant osseointegration in alpha-CGRP-deficient mice. Bone 94:135–140CrossRefPubMedGoogle Scholar
  25. Yang Z, Zhu L, Li F, Wang J, Wan H, Pan Y (2014) Bone marrow stromal cells as a therapeutic treatment for ischemic stroke. Neurosci Bull 30(3):524–534CrossRefPubMedPubMedCentralGoogle Scholar
  26. Yu X, Lv L, Zhang J, Zhang T, Xiao C, Li S (2015) Expression of neuropeptides and bone remodeling-related factors during periodontal tissue regeneration in denervated rats. J Mol Histol 46(2):195–203CrossRefPubMedGoogle Scholar
  27. Zhang Y, Xu J, Ruan YC, Yu MK, O’Laughlin M, Wise H et al. (2016). Implant-derived magnesium induces local neuronal production of CGRP to improve bone-fracture healing in rats. Nat Med 22(10):1160–1169CrossRefPubMedPubMedCentralGoogle Scholar
  28. 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
  29. Zou D, Zhang Z, He J, Zhu S, Wang S, Zhang W et al (2011) Repairing critical-sized calvarial defects with BMSCs modified by a constitutively active form of hypoxia-inducible factor-1α and a phosphate cement scaffold. Biomaterials 32(36):9707–9718CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Xijiao Yu
    • 1
    • 2
  • Shuang Liu
    • 1
  • Hui Chen
    • 2
  • Xinyu Zhao
    • 1
  • Xue Chen
    • 1
  • Yi Du
    • 2
  • Shu Li
    • 1
    Email author
  1. 1.Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Department of Periodontology, School and Hospital of StomatologyShandong UniversityJinanPeople’s Republic of China
  2. 2.Department of EndodonticsJinan Stomatological HospitalJinanPeople’s Republic of China

Personalised recommendations