Orthopedic implants containing biodegradable magnesium have been used for fracture repair with considerable efficacy; however, the underlying mechanisms by which these implants improve fracture healing remain elusive. Here we show the formation of abundant new bone at peripheral cortical sites after intramedullary implantation of a pin containing ultrapure magnesium into the intact distal femur in rats. This response was accompanied by substantial increases of neuronal calcitonin gene-related polypeptide-α (CGRP) in both the peripheral cortex of the femur and the ipsilateral dorsal root ganglia (DRG). Surgical removal of the periosteum, capsaicin denervation of sensory nerves or knockdown in vivo of the CGRP-receptor-encoding genes Calcrl or Ramp1 substantially reversed the magnesium-induced osteogenesis that we observed in this model. Overexpression of these genes, however, enhanced magnesium-induced osteogenesis. We further found that an elevation of extracellular magnesium induces magnesium transporter 1 (MAGT1)-dependent and transient receptor potential cation channel, subfamily M, member 7 (TRPM7)-dependent magnesium entry, as well as an increase in intracellular adenosine triphosphate (ATP) and the accumulation of terminal synaptic vesicles in isolated rat DRG neurons. In isolated rat periosteum-derived stem cells, CGRP induces CALCRL- and RAMP1-dependent activation of cAMP-responsive element binding protein 1 (CREB1) and SP7 (also known as osterix), and thus enhances osteogenic differentiation of these stem cells. Furthermore, we have developed an innovative, magnesium-containing intramedullary nail that facilitates femur fracture repair in rats with ovariectomy-induced osteoporosis. Taken together, these findings reveal a previously undefined role of magnesium in promoting CGRP-mediated osteogenic differentiation, which suggests the therapeutic potential of this ion in orthopedics.
Subscribe to Journal
Get full journal access for 1 year
only $18.75 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Cheung, W.H., Chin, W.C., Qin, L. & Leung, K.S. Low intensity pulsed ultrasound enhances fracture healing in both ovariectomy-induced osteoporotic and age-matched normal bones. J. Orthop. Res. 30, 129–136 (2012).
Hayes, J.S. & Richards, R.G. The use of titanium and stainless steel in fracture fixation. Expert Rev. Med. Devices 7, 843–853 (2010).
Gu, X.N., Xie, X.H., Li, N., Zheng, Y.F. & Qin, L. In vitro and in vivo studies on a Mg-Sr binary alloy system developed as a new kind of biodegradable metal. Acta Biomater. 8, 2360–2374 (2012).
de Baaij, J.H., Hoenderop, J.G. & Bindels, R.J. Magnesium in man: implications for health and disease. Physiol. Rev. 95, 1–46 (2015).
Li, F.Y. et al. Second messenger role for Mg2+ revealed by human T-cell immunodeficiency. Nature 475, 471–476 (2011).
Staiger, M.P., Pietak, A.M., Huadmai, J. & Dias, G. Magnesium and its alloys as orthopedic biomaterials: a review. Biomaterials 27, 1728–1734 (2006).
Castiglioni, S., Cazzaniga, A., Albisetti, W. & Maier, J.A. Magnesium and osteoporosis: current state of knowledge and future research directions. Nutrients 5, 3022–3033 (2013).
Zberg, B., Uggowitzer, P.J. & Löffler, J.F. MgZnCa glasses without clinically observable hydrogen evolution for biodegradable implants. Nat. Mater. 8, 887–891 (2009).
Witte, F. The history of biodegradable magnesium implants: a review. Acta Biomater. 6, 1680–1692 (2010).
Witte, F. et al. In vivo corrosion of four magnesium alloys and the associated bone response. Biomaterials 26, 3557–3563 (2005).
Gu, X. et al. Corrosion of, and cellular responses to Mg-Zn-Ca bulk metallic glasses. Biomaterials 31, 1093–1103 (2010).
Li, H.F. et al. In vitro and in vivo studies on biodegradable CaMgZnSrYb high-entropy bulk metallic glass. Acta Biomater. 9, 8561–8573 (2013).
Tang, J. et al. Surface coating reduces degradation rate of magnesium alloy developed for orthopaedic applications. J. Orthop. Translat. 1, 41–48 (2013).
Kuhlmann, J. et al. Fast escape of hydrogen from gas cavities around corroding magnesium implants. Acta Biomater. 9, 8714–8721 (2013).
Meunier, P.J. et al. The effects of strontium ranelate on the risk of vertebral fracture in women with postmenopausal osteoporosis. N. Engl. J. Med. 350, 459–468 (2004).
Wang, J. et al. Surface modification of magnesium alloys developed for bioabsorbable orthopedic implants: a general review. J. Biomed. Mater. Res. B Appl. Biomater. 100, 1691–1701 (2012).
Seeman, E. The periosteum—a surface for all seasons. Osteoporos. Int. 18, 123–128 (2007).
Zebaze, R.M. et al. Intracortical remodelling and porosity in the distal radius and post-mortem femurs of women: a cross-sectional study. Lancet 375, 1729–1736 (2010).
Castañeda-Corral, G. et al. The majority of myelinated and unmyelinated sensory nerve fibers that innervate bone express the tropomyosin receptor kinase A. Neuroscience 178, 196–207 (2011).
Zhang, X., Awad, H.A., O'Keefe, R.J., Guldberg, R.E. & Schwarz, E.M. A perspective: engineering periosteum for structural bone graft healing. Clin. Orthop. Relat. Res. 466, 1777–1787 (2008).
Wang, X.Y., Guo, X., Qu, S.X., Weng, J. & Cheng, C.Y. Temporal and spatial CGRP innervation in recombinant human bone morphogenetic protein induced spinal fusion in rabbits. Spine 34, 2363–2368 (2009).
Ding, Y., Arai, M., Kondo, H. & Togari, A. Effects of capsaicin-induced sensory denervation on bone metabolism in adult rats. Bone 46, 1591–1596 (2010).
Niedermair, T. et al. Absence of substance P and the sympathetic nervous system impact on bone structure and chondrocyte differentiation in an adult model of endochondral ossification. Matrix Biol. 38, 22–35 (2014).
McDonald, A.C., Schuijers, J.A., Shen, P.J., Gundlach, A.L. & Grills, B.L. Expression of galanin and galanin receptor-1 in normal bone and during fracture repair in the rat. Bone 33, 788–797 (2003).
Kavalali, E.T. The mechanisms and functions of spontaneous neurotransmitter release. Nat. Rev. Neurosci. 16, 5–16 (2015).
Matteoli, M. et al. Differential effect of α-latrotoxin on exocytosis from small synaptic vesicles and from large dense-core vesicles containing calcitonin gene-related peptide at the frog neuromuscular junction. Proc. Natl. Acad. Sci. USA 85, 7366–7370 (1988).
Brenner, S.L. & Korn, E.D. The effects of cytochalasins on actin polymerization and actin ATPase provide insights into the mechanism of polymerization. J. Biol. Chem. 255, 841–844 (1980).
Feske, S., Skolnik, E.Y. & Prakriya, M. Ion channels and transporters in lymphocyte function and immunity. Nat. Rev. Immunol. 12, 532–547 (2012).
Zhou, H. & Clapham, D.E. Mammalian MagT1 and TUSC3 are required for cellular magnesium uptake and vertebrate embryonic development. Proc. Natl. Acad. Sci. USA 106, 15750–15755 (2009).
Goytain, A. & Quamme, G.A. Identification and characterization of a novel mammalian Mg2+ transporter with channel-like properties. BMC Genomics 6, 48 (2005).
Chokshi, R., Fruasaha, P. & Kozak, J.A. 2-aminoethyl diphenyl borinate (2-APB) inhibits TRPM7 channels through an intracellular acidification mechanism. Channels (Austin) 6, 362–369 (2012).
Voets, T. et al. TRPM6 forms the Mg2+ influx channel involved in intestinal and renal Mg2+ absorption. J. Biol. Chem. 279, 19–25 (2004).
Zhang, Z.H. et al. Calcitonin gene-related peptide enhances CREB phosphorylation and attenuates tau protein phosphorylation in rat brain during focal cerebral ischemia/reperfusion. Biomed. Pharmacother. 64, 430–436 (2010).
Nishio, Y. et al. Runx2-mediated regulation of the zinc finger Osterix/Sp7 gene. Gene 372, 62–70 (2006).
Koga, T. et al. NFAT and Osterix cooperatively regulate bone formation. Nat. Med. 11, 880–885 (2005).
Fu, S.C., Cheuk, Y.C., Hung, L.K. & Chan, K.M. Limb Idleness Index (LII): a novel measurement of pain in a rat model of osteoarthritis. Osteoarthritis Cartilage 20, 1409–1416 (2012).
Zhen, G. et al. Inhibition of TGF-β signaling in mesenchymal stem cells of subchondral bone attenuates osteoarthritis. Nat. Med. 19, 704–712 (2013).
Li, J., Ahmad, T., Spetea, M., Ahmed, M. & Kreicbergs, A. Bone reinnervation after fracture: a study in the rat. J. Bone Miner. Res. 16, 1505–1510 (2001).
Minardi, S. et al. Evaluation of the osteoinductive potential of a bio-inspired scaffold mimicking the osteogenic niche for bone augmentation. Biomaterials 62, 128–137 (2015).
Weizbauer, A. et al. Magnesium-containing layered double hydroxides as orthopaedic implant coating materials-An in vitro and in vivo study. J. Biomed. Mater. Res. B Appl. Biomater. 104, 525–531 (2016).
Zhao, D. et al. Vascularized bone grafting fixed by biodegradable magnesium screw for treating osteonecrosis of the femoral head. Biomaterials 81, 84–92 (2016).
Chaya, A. et al. Fracture healing using degradable magnesium fixation plates and screws. J. Oral Maxillofac. Surg. 73, 295–305 (2015).
Cadosch, D. et al. Humoral factors enhance fracture-healing and callus formation in patients with traumatic brain injury. J. Bone Joint Surg. Am. 91, 282–288 (2009).
Boes, M. et al. Osteogenic effects of traumatic brain injury on experimental fracture-healing. J. Bone Joint Surg. Am. 88, 738–743 (2006).
Naot, D. & Cornish, J. The role of peptides and receptors of the calcitonin family in the regulation of bone metabolism. Bone 43, 813–818 (2008).
Komori, T. Signaling networks in RUNX2-dependent bone development. J. Cell. Biochem. 112, 750–755 (2011).
Mayr, B. & Montminy, M. Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat. Rev. Mol. Cell Biol. 2, 599–609 (2001).
Tian, G., Zhang, G. & Tan, Y.H. Calcitonin gene-related peptide stimulates BMP-2 expression and the differentiation of human osteoblast-like cells in vitro. Acta Pharmacol. Sin. 34, 1467–1474 (2013).
Mrak, E. et al. Calcitonin gene-related peptide (CGRP) inhibits apoptosis in human osteoblasts by β-catenin stabilization. J. Cell. Physiol. 225, 701–708 (2010).
Rude, R.K. & Gruber, H.E. Magnesium deficiency and osteoporosis: animal and human observations. J. Nutr. Biochem. 15, 710–716 (2004).
Madsen, J.E. et al. Fracture healing and callus innervation after peripheral nerve resection in rats. Clin. Orthop. Relat. Res. (351), 230–240 (1998).
Zaidi, M. Skeletal remodeling in health and disease. Nat. Med. 13, 791–801 (2007).
Li, J., Kreicbergs, A., Bergström, J., Stark, A. & Ahmed, M. Site-specific CGRP innervation coincides with bone formation during fracture healing and modeling: A study in rat angulated tibia. J. Orthop. Res. 25, 1204–1212 (2007).
Qin, L., Mak, A.T., Cheng, C.W., Hung, L.K. & Chan, K.M. Histomorphological study on pattern of fluid movement in cortical bone in goats. Anat. Rec. 255, 380–387 (1999).
Xia, W. & Springer, T.A. Metal ion and ligand binding of integrin α5β1. Proc. Natl. Acad. Sci. USA 111, 17863–17868 (2014).
Stürmer, E.K. et al. Standardized bending and breaking test for the normal and osteoporotic metaphyseal tibias of the rat: effect of estradiol, testosterone, and raloxifene. J. Bone Miner. Res. 21, 89–96 (2006).
Li, X. et al. Targeted deletion of the sclerostin gene in mice results in increased bone formation and bone strength. J. Bone Miner. Res. 23, 860–869 (2008).
Zhang, G. et al. A delivery system targeting bone formation surfaces to facilitate RNAi-based anabolic therapy. Nat. Med. 18, 307–314 (2012).
Salomé, M. et al. The ID21 scanning X-ray microscope at ESRF. J. Phys. Conf. Ser. 425, 182004 (2013).
Tian, S.F. et al. Mechanisms of neuroprotection from hypoxia-ischemia (HI) brain injury by up-regulation of cytoglobin (CYGB) in a neonatal rat model. J. Biol. Chem. 288, 15988–16003 (2013).
Suen, P.K. et al. Sclerostin monoclonal antibody enhanced bone fracture healing in an open osteotomy model in rats. J. Orthop. Res. 32, 997–1005 (2014).
Zheng, L.F. et al. Calcitonin gene-related peptide dynamics in rat dorsal root ganglia and spinal cord following different sciatic nerve injuries. Brain Res. 1187, 20–32 (2008).
Ng, K.Y., Wong, Y.H. & Wise, H. Glial cells isolated from dorsal root ganglia express prostaglandin E(2) (EP(4)) and prostacyclin (IP) receptors. Eur. J. Pharmacol. 661, 42–48 (2011).
Ng, K.Y., Yeung, B.H., Wong, Y.H. & Wise, H. Isolated dorsal root ganglion neurones inhibit receptor-dependent adenylyl cyclase activity in associated glial cells. Br. J. Pharmacol. 168, 746–760 (2013).
Henkel, A.W., Lübke, J. & Betz, W.J. FM1-43 dye ultrastructural localization in and release from frog motor nerve terminals. Proc. Natl. Acad. Sci. USA 93, 1918–1923 (1996).
Ren, Y., Lin, S., Jing, Y., Dechow, P.C. & Feng, J.Q. A novel way to statistically analyze morphologic changes in Dmp1-null osteocytes. Connect. Tissue Res. 55 Suppl 1, 129–133 (2014).
Xu, J.K. et al. Optimal intensity shock wave promotes the adhesion and migration of rat osteoblasts via integrin β1-mediated expression of phosphorylated focal adhesion kinase. J. Biol. Chem. 287, 26200–26212 (2012).
He, Y.X. et al. Impaired bone healing pattern in mice with ovariectomy-induced osteoporosis: A drill-hole defect model. Bone 48, 1388–1400 (2011).
We acknowledge Li Ka Shing Institute of Health Sciences (LiHS) for providing a harmonious working environment. This work was supported by Hong Kong RGC Collaborative Research Fund (2014/2015, C4028-14GF to L.Q.), General Research Fund (no. 14112714, 14114415 to L.Q.), NSFC/RGC (N_CUHK449/13 to L.Q.; 51361165101 to Y.Z.), Innovation and Technology Fund (no. ITS/350/13 to L.Q.), SMART Program to K.C., L.Q., H.C.C. and Y.C.R. (Lui Che Woo Institute of Innovative Medicine in Faculty of Medicine, the Chinese University of Hong Kong), National Basic Research Program of China (973 Program, no. 2012CB619102 to Y.Z., no.2012CB944903 to H.C.C. and Y.C.R. and no. 2013CB967401 to H.C.C.), National Natural Science Foundation of China (no. 51225101; no. 51431002 to Y.Z.). We thank B. Hesse for collecting and analyzing the μXRF data. We thank G. Wu and J. Lu for help with scanning electron microscopy analysis.
The authors declare no competing financial interests.
About this article
Cite this article
Zhang, Y., Xu, J., Ruan, Y. et al. Implant-derived magnesium induces local neuronal production of CGRP to improve bone-fracture healing in rats. Nat Med 22, 1160–1169 (2016) doi:10.1038/nm.4162
Metal-Based Nanostructures/PLGA Nanocomposites: Antimicrobial Activity, Cytotoxicity, and Their Biomedical Applications
ACS Applied Materials & Interfaces (2020)
Comparative in vitro study on binary Mg-RE (Sc, Y, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu) alloy systems
Acta Biomaterialia (2020)
Journal of Materials Science & Technology (2020)
Materials Science and Engineering: C (2020)