A Magnesium-enriched 3D Culture System That Mimics The Bone Development Microenvironment For Vascularized Bone Regeneration

ObjectiveA magnesium-enriched environment is taken to mimic the bone development microenvironment.Based on this,a stem cell 3D culture system is prepared by using hydrogel.Such magnesium-enriched cell delivery is used to achieve vascularized bone regeneration in bone defects.We hope that our biomaterial engineering strategy could provide new ideas for further biomimetic design of biomaterials for bone regeneration.MethodsThe correlation between magnesium transporter subtype 1?MagT1?and bone development was evaluated in vitro and in vivo by taking immunofluorescence staining and other techniques.Then the optimum concentration of magnesium ions(Mg²⁺)was screened to mimic the bone development microenvironment using MagT1 expression as guidance.Western Blot,CRISPR/Cas9,and other methods were taken to investigate the possible molecular basis underlying the osteoinductivity of Mg²⁺on stem cells.Moreover,a novel magnesium-enriched stem cell 3D culture system was fabricated using hydrogel.The osteogenic and angiogenic effects of the magnesium-enriched environment were evaluated by PCR,EdU staining,immunofluorescence staining,and other methods.Finally,the rat skull defect model was used to evaluate the effect of such stem cell deliveries on vascularized bone regeneration.Results1.The high expression of MagT1 was observed both in the endochondrol ossification region of mouse embryos and osteogenic differentioation of stem cells.The magnesium-enriched environment could significantly up-regulated the expression of stem cells in vitro.The similar trend in the expression of ostegenic-related proteins was also observed.2.The MAPK/ERK pathway was stimulated by Mg²⁺.The knockout of MagT1 had the inhibitory effects on the phosphorylation of ERK protein,resulting in a significantly suppression on osteogenic differentiation of stem cells.3.A magnesium-enriched stem cell culture system was successfully fabricated.The magnesium-enriched microenvironment not only promoted the osteogenic differentiation of stem cells encapsulated,but also exerted obvious angiogenic effects.After implantation of such cell delivery vehicles to the rat skull defects,obvious vascularized bone regeneration was achieved.ConclusionsThe MagT1 is well correlated with the osteogenic differentiation capacity of stem cells.The Mg²⁺could mimic the bone development microenvironment via high MagT1expression.Based on these results,we fabricated a novel magnesium-enriched 3D culture system for cell delivery and bone regeneration.Obvious vascularized bone regeneration was achieved by using such cell delivery vehicles.