Implant-derived magnesium induces local neuronal production of CGRP to improve bone-fracture healing in rats

Abstract

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.

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Figure 1: Periosteum-dependent new-bone formation induced by magnesium in rat femur.
Figure 2: Role of CGRP receptor in magnesium-induced new-bone formation in rat femur.
Figure 3: Effect of Mg2+ on rat DRG neurons in vitro.
Figure 4: CGRP promotes osteogenic differentiation of periosteum-derived stem cells (PDSCs).
Figure 5: Innovative magnesium-containing intramedullary nail accelerates fracture healing of femoral shaft in rats with ovariectomy-induced osteoporosis.
Figure 6: Role of CGRP receptor in the beneficial effect of Mg-IMN on bone-fracture healing in rats.

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Acknowledgements

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.

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Y. Zhang, J.X., S.C., X.X., L.H., L. Zheng, S.H. and D.H.K.C. conducted animal surgery and analyzed the results. Y. Zhang, J.X., Y.C.R., and M.K.Y. conducted DRG neuron vesicles and intracellular Mg2+ experiments. Y. Zhang, J.X. and N.L. contributed to isolation and culture of DRG neurons and PDSCs. M.O'L. contributed to the conjugation of CGRP receptor antagonist BIBN4096BS with Cy5. L.T. and J.W. were responsible for needle design. D.S. contributed to the FEA modeling and analysis. J.Q.F., D.C., and L. Zhao (USA) performed the analysis of magnesium-implanted samples using confocal microscope. H.L. contributed to the scanning electron microscopy experiment. D.Z., K.L., and K.C. conducted the capsaicin-related experiments and provided invaluable support on the discussion about the clinical indications. H.W. and X.G. contributed to build the platform for CGRP study. F.W. (Germany) performed the synchrotron μXRF analysis. F.W. (Germany) and Y. Zheng (Beijing, China) contributed to magnesium biomedical engineering, and also provided insightful comments on the materials-science related field. H.C.C. provided intelligence input and supervision. Y.C.R drew the schematic pictures. Y. Zhang, J.X., Y.C.R. and L.Q. designed and supervised the project as well as wrote the manuscript.

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Correspondence to Yufeng Zheng or Ling Qin.

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Mg2+ induces transportation and aggregation of vesicles toward neuronal terminals (MOV 164 kb)

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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). https://doi.org/10.1038/nm.4162

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