Divalent metal ions enhance bone regeneration through modulation of nervous systems and metabolic pathways

The divalent metal cations promote new bone formation through modulation of sensory and sympathetic nervous systems (SNS) activities. In addition, acetylcholine (Ach), as a chief neurotransmitter released by the parasympathetic nervous system (PNS), also affects bone remodeling, so it is of worth to investigate if the divalent cations influence PNS activity. Of note, these cations are key co-enzymes modulating glucose metabolism. Aerobic glycolysis rather than oxidative phosphorylation favors osteogenesis of mesenchymal stem cells (MSCs), so it is of interest to study the effects of these cations on glucose metabolic pathway. Prior to biological function assessment, the tolerance limits of the divalent metal cations (Mg²⁺, Zn²⁺, and Ca²⁺) and their combinations were profiled. In terms of direct effects, these divalent cations potentially enhanced migration and adhesion capability of MSCs through upregulating <i>Tgf-β1</i> and <i>Integrin-β1</i> levels. Interestingly, the divalent cations alone did not influence osteogenesis and aerobic glycolysis of undifferentiated MSCs. However, once the osteogenic differentiation of MSCs was initiated by neurotransmitters or osteogenic differentiation medium, the osteogenesis of MSCs could be significantly promoted by the divalent cations, which was accompanied by the improved aerobic glycolysis. In terms of indirect effects, the divalent cations significantly upregulated levels of sensory nerve derived CGRP, PNS produced choline acetyltransferase and type H vessels, while significantly tuned down sympathetic activity in the defect zone in rats, thereby contributing to significantly increased bone formation relative to the control group. Together, the divalent cations favor bone regeneration via modulation of sensory-autonomic nervous systems and promotion of aerobic glycolysis-driven osteogenesis of MSCs after osteogenic initiation by neurotransmitters.