Dual effects of magnesium on bone regeneration through pro-osteogenic immunomodulation and biomineralization inhibition

Abstract

Magnesium ion (Mg2+) is integral to bone homeostasis and metabolism. The use of Mg2+-modified biomaterials in bone regeneration is a promising and cost-effective therapeutic in orthopedics and dentistry. Despite various observations on the osteogenic effects of Mg2+, the diverse roles played by Mg2+ in various cell types involved in bone healing and the underlying mechanism have not been systematically dissected. In this present study, the stage-oriented effect of Mg2+ on the new bone formation was investigated by grafting short-term Mg2+ releasing alginate gel in a tunnel defect of femur in rat at different time points after the operation. It was found that only early administration of Mg2+ achieved the best outcome of bone healing, manifested by bone volume and bone mineral density compared with the sham and control group. However, the promotive effect of Mg2+ on osteogenesis diminishes when the hydrogel is grafted at the second week or over the first two weeks after the operation. Moreover, the in vitro study demonstrates that Mg2+ supply is essential for osteogenic differentiation of human mesenchymal stem cells (hMSC), but increased extracellular Mg2+ dramatically suppressed the mineralization of extracellular matrix. Moreover, a group of CD68 positive macrophage was identified to play a vital role in the Mg2+-induced osteogenesis. Both in vivo and in vitro data shows that Mg2+ increases bone formation by promoting macrophage infiltration, activation, and polarization, leading to the formation of a pro-osteogenic immune microenvironment characterized by increased production of the interleukin-8 (IL-8) and decreased the release of interleukin-1β (IL-1β). Furthermore, the Mg2+-induced inflammatory microenvironment is determined to be dominant by, but not limited to, the anti-inflammatory activation of M2 phenotype, which promotes the proliferation, osteogenic differentiation, and mineralization of MSC. The mechanism of Mg2+-induced immunomodulation was further studied using THP1-derived macrophage as an in vitro model. Upon the increase of extracellular Mg2+, there exists a significant influx of Mg2+ in macrophage, followed by the upregulation of transient receptor potential cation channel member 7 (TRPM7) and an increase of the nuclear accumulation of the TRPM7-cleaved kinase fragments (M7CKs). On the one hand, chromosome bound M7CKs were able to trigger the phosphorylation of histone H3 at Ser 10, which locates at the promoter of inflammatory cytokines, such as IL-8. On the other hand, M7CKs-induced phosphorylated histone H3S10 also upregulates the expression of IκB, resulting in NF-κB dependent inhibition on IL-1β secretion Through biomimetic precipitation and remineralization assay, the negative effect of Mg2+ on the mineralization step of osteogenesis can be explained by the suppression of hydroxyapatite precipitation and the mineralization of collagen structure. Therefore, in the late phase of bone regeneration, the presence of Mg2+ tends to decelerate bone maturation, which can override the initial pro-osteogenic benefits of Mg2+. This study revealed a novel Mg2+ dependent mechanism in tissue regeneration through the initial pro-osteogenic immune modulation followed by the subsequent inhibition of biomineralization. It establishes that the beneficial effect of Mg2+ is achieved only when administered during the initial phase of regeneration, beyond which excessive Mg2+ is detrimental to bone healing.