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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Metabolic skeletal disorders associated with impaired bone formation are a major clinical challenge. One approach to treat these defects is to silence bone-formation-inhibitory genes by small interference RNAs (siRNAs) in osteogenic-lineage cells that occupy the niche surrounding the bone-formation surfaces. We developed a targeting system involving dioleoyl trimethylammonium propane (DOTAP)-based cationic liposomes attached to six repetitive sequences of aspartate, serine, serine ((AspSerSer)(6)) for delivering siRNAs specifically to bone-formation surfaces. Using this system, we encapsulated an osteogenic siRNA that targets casein kinase-2 interacting protein-1 (encoded by Plekho1, also known as Plekho1). In vivo systemic delivery of Plekho1 siRNA in rats using our system resulted in the selective enrichment of the siRNAs in osteogenic cells and the subsequent depletion of Plekho1. A bioimaging analysis further showed that this approach markedly promoted bone formation, enhanced the bone micro-architecture and increased the bone mass in both healthy and osteoporotic rats. These results indicate (AspSerSer)(6)-liposome as a promising targeted delivery system for RNA interference-based bone anabolic therapy.