Function Of Magnesium In Bone Biomineralization

Objective: To clarify the role of magnesium in natural skull development and biomineralization,and to explore the impact of excessive magnesium on bone biomineralization from a micro-perspective.Materials and methods: Female and male C57BL/6 mice were caged,and mice embryos at different time points were collected every other day.The skull was separated by a stereo-microscope.After freeze-drying,the calcium and magnesium contained in the skull were quantitatively determined by ICP-OES.Samples were obtained from the frontier of mineralization,fixed with glutaraldehyde,and analyzed the mineralization process inside and outside the cells in vivo by transmission electron microscopy.The semi-quantitative determination of the local element content of the skull sample was performed using scanning transmission electron microscopy mode.Bone marrow mesenchymal stem cells were extracted from C57BL/6 mice and differentiated into osteoblasts in vitro by osteogenic-induced medium.Magnesium chloride was added at various concentration gradients and Alizarin red staining was used to detect the mineralization effect.Total RNA was extracted at each mineralization time point and transcription levels of osteogenic markers after the addition of 1 m M magnesium ion were detected by rt-q PCR.The morphological changes of the cells during mineralization process were observed and analyzed using transmission electron microscopy and scanning electron microscopy at various stages of cell mineralization.Juvenile female mice were fed with a high-magnesium diet.After adulthood,they were caged with normal male mice.Embryonic skulls at specific time points were collected and analyzed by transmission electron microscopy.Embryo mice fed with high magnesium were born,and continue to be given high magnesium diet after lactation until adulthood.Skulls,femurs,tibia,uterus,ovaries and other organs were collected.And after freeze-drying,ICP-OES was used to quantitatively determine calcium and magnesium content.Point-type EDS and SAED were used for micro-element and crystallization analysis of mineralized precursors and collagen,respectively.Results and discussion: The skull of C57BL/6 mice began to be enriched with inorganic elements on day E13.5,and magnesium began to decline on day E15.5,while calcium continued to increase.Additionally,semiquantitative determination of mitochondrial elements showed the same trend.EDS and SAED tests showed that the content of magnesium ions was lower in well-mineralized collagen,higher in poorlymineralized collagen,higher in intracellular mineralized precursors,and lower in extracellular mineralized precursors.The addition of 0.25 m M magnesium ions into mineralization-inducing medium of bone marrow mesenchymal stem cells promoted the mineralization slightly,and then,as the concentration increased,they all showed inhibition of mineralization.After the beginning of mineralization,it was replaced with osteogenic-induced medium containing 1 m M magnesium at different times.It was found that the earlier the addition,the stronger the inhibitory effect on mineralization.The m RNA level of each osteogenic marker in the early stage of mineralization indicated that high concentrations of magnesium ions can inhibit Alp and Col1,but had less effect on osteogenic differentiation indicators(Runx2 and Osterix).Scanning electron microscopy showed that after adding 1 m M magnesium ion,collagen content of the extracellular matrix decreased and the morphology of hydroxyapatite changed.Transmission electron microscopy showed that the extracellular matrix collagen was immature and weakened in mineralization.Simulated collagen mineralization experiments also showed that collagen mineralization became worse under magnesium treatment.Collagen mineralization were deteriorated determined by transmission electron microscope of skull samples obtained after high magnesium diet feeding.ICPOES results also showed that the calcium content of the skull and long bones increased while the calcium content of the skull and long bones increased after high magnesium feeding.Biomechanical measurements showed that bones of mice became brittle after high-magnesium feeding.Conclusion: It is important to regulate the normal concentration of magnesium ions for normal biomineralization.As the development process progresses,the bone tissue is enriched with sufficient magnesium.The presence of magnesium can protect the amorphous calcium phosphate from crystallizing in advance,and ensure the normal accumulation of mineralized precursors on collagen to complete the mineralization.In the later stages of development,magnesium begins to fall in case the wrong morphology of hydroxyapatite is formed.If high concentrations of magnesium ions are present in the early stages of development,it will inhibit the production of collagen and other raw materials(such as calcium phosphate)required for bone formation,thereby affecting the degree of bone mineralization.At the same time,if given a high magnesium diet to female mice,it will affect the normal development of bones in the offspring of mice.