Osteogenic Effect of Magnesium and Its Potential Application for Fracture Healing Enhancement in Ovariectomized Rats

In the rodent femur, almost 99% of all sensory nerves are distributed densely in the periosteum. Neuropeptides encapsulated in the synaptic vesicles are located at the axon terminals and released through exocytosis after being stimulated at the sensory nerve endings. The neuropeptides released from nerve endings have an osteo-anabolic effect on osteoblasts. Among the many kinds of neuropeptides, which include alpha-calcitonin gene-related peptide (CGRP), substance P, and other amino molecules, CGRP is the classical and dominantly distributed peptide in sensory nerve endings. In aged animals, decreased serum CGRP and loss of bone Mg content may be the factors inhibiting fracture healing.;In this study, Mg was found to significantly promote new bone formation in the subperiosteal cortical region after it was intramedullarily implanted in the rat femur canal. Histomorphological analysis revealed that the newly formed bone grew from periosteum, a fibrous membrane constituted of blood vessels, sensory serves, and mesenchymal stem cells, and did not form any cartilage-like tissue, the latter of which is a feature of intramembranous ossification. Observation that Mg-induced new bone formation disappeared at the periosteum-stripped region revealed the existence of an interaction between the periosteum and Mg ions.;Based on previous findings, this study examined the following hypotheses: (1) Mg ions from Mg implanted in the rat femur canal act on sensory nerve endings in the periosteum and promote neuropeptide CGRP release, (2) mass CGRP release in the periosteum promotes periosteum-derived stem cells osteoblastogenesis and leads to new bone formation. Mg ions affect synaptic replasticity in dorsal root ganglia neurons, and (3) pure Mg metal affects fracture healing in ovariectomized (OVX) rats. Neuropeptide CGRP plays a pivotal role in Mg-induced new bone formation..;This hypothesis was supported by femur bone analysis showing that CGRP content significantly increased in Mg-implanted femur bone compared to control femur bone. When rat sensory nerves were destroyed by administration of high-dose capsaicin, induction of new bone formation by Mg implantation significantly decreased, proving that sensory nerves play an important role in Mg-induced osteogenesis. Because neuropeptide CGRP from sensory nerve endings may play a pivotal role in Mg's osteogenic process, the effective CGRP antagonist BIBN4096bs was administered to Mg-implanted rats. Administration of the CGRP antagonist significantly reduced newly formed bone volume after Mg implantation. To examine whether this phenomenon is dependent on the interaction between neuropeptides and MSCs, which are richly distributed in the periosteum, periosteum-derived stem cells (PDSCs) and bone marrow-derived mesenchymal stem cells (BMSCs) were isolated from the periosteum and bone marrow, respectively. It was observed that high concentrations of CGRP significantly promoted osteogenic differentiation in both PDSCs and BMSCs while high concentrations of CGRP had an obvious chemotaxis effect on BMSCs.;Mg increases CGRP release by affecting DRG neurons. The results of immunochemical staining and ELISA CGRP quantification analysis of femur samples showed that femur CGRP content in Mg-implanted samples was almost twice that of controls. Previous studies reported that Mg ions could promote neural synaptic replasticity in hippocampus neurons in vitro. This study examined the hypothesis that Mg ions could promote synaptic replasticity in DRG neurons. The neural synaptic vesicles, which contain neuropeptides of DRG neurons, including CGRP, derived from the L3-5 dorsal root ganglion were stained in vitro. The synaptic vesicles were found to significantly increase in number when their medium was changed from Mg-free medium to Mg-rich medium of 1 mM and 2 mM and to migrate from the neuron body to its axon terminals. These results proved that Mg could facilitate neuron replasticity and prompt synaptic vesicle aggregation at axon terminals, indicating that much neuropeptide release occurs after stimulation. Real-time recording of the intracellular Mg signal revealed that DRG neuron Mg influx significantly increased after Mg medium had been added and that Mg influx into neurons was mainly through the membrane Mg ion channel MagT1. Implantation of Mg ions (MgCl2) of high concentration was found to promote stem cell (PDSCs and BMSCs) osteogenic differentiation. Although the mechanism of Mg's osteogenic effect on stem cells was not thoroughly studied, cellular Mg influx was found to increase in high-Mg medium through the membrane ion channel MagT1.;Mg accelerated bone fracture in ovariectomized rats. Mg metal is too soft to repair bone fracture in animal models. To overcome this challenge, we designed a novel intramedullary nail containing Mg to accelerate osteoporotic bone fracture healing in ovariectomized (OVX) rats. The novel nail is a hollow stainless steel needle with several interlacing arranged holes drilled midway through the needle. The Mg pin is inserted into the needle canal and Mg ions released through the holes on the needle reach the fracture line during degradation in vivo. Our findings indicate that use of this Mg-containing intramedullary nail could accelerate bone fracture healing in OVX rats. Review of post-surgery X-ray results showed that the fracture callus of the Mg-treated group was significantly larger than that of the control group at weeks 2 and 4. Review of micro-computed tomography (micro-CT) scanning images indicated that both the total volume and area of callus bone in the Mg-treated group exceeded those of the control group at week 4. However, no significant difference was found between the two groups regarding callus area and volume at week 12.;Histomorphological analysis showed a wider intramembranous ossification area and woven bone area in the Mg-treated group at weeks 2 and 4 and more cartilage tissue at the callus site in the Mg-treated group at week 4. Double fluorescence labeling staining revealed more densely stained newly formed bone in the Mg-treated group than the control group at week 4, indicating accelerated callus bone formation in the Mg-treated group. The callus was observed to be undergoing endochondral ossification and woven bone remodeling at weeks 8 and 12. Review of polarized light images showed brighter and more regularly arranged collagen fibers in the Mg-treated group compared to the control group. Biomechanical testing at week 12 revealed that the ultimate load of shaft bone in the Mg-treated group had increased 30% more than that of the control group. These results indicate that the novel Mg-containing intramedullary nail designed in this study could significantly accelerate and optimize osteoporotic fracture healing in OVX rat model.;Significance: The results of this study contribute to a thorough understanding of the osteogenic effect of Mg by explicating its bioeffect on neurons and stem cells. The novel Mg-containing intramedullary nail designed in this study appears promising in osteoporotic fracture healing and to have many potential clinical applications.