Experimental Research Of DMOG Composite Calcined Bone Scaffold On The Repair Of Bone Defect

ObjectiveIn this experiment,dimethyloxallyl glycine（DMOG）,type I collagen and true bone ceramic（TBC）were prepared as DMOG-loaded type I collagen calcined bone scaffolds.The characterization of the composite scaffold material was studied,and the osteogenic activity and bone repair ability of the composite scaffold material were evaluated at the cellular level and the animal level.Methods1.Bone marrow mesenchymal stem cells（BMSC）were isolated from SD rats and cultured in vitro.2.The effect of DMOG on osteogenic differentiation of BMSC was detected by alkaline phosphatase（ALP）staining.3.TBC scaffolds,COL I/TBC scaffolds,and DMOG/COL I/TBC scaffolds were prepared and their characterizations were analyzed by X-ray diffraction（XRD）、scanning electron microscope（SEM）.)and DMOG release curve in composites.4.The scaffolds of each group were sterilized by ⁶⁰Co irradiation and then they were co-cultured with bone marrow mesenchymal stem cells to evaluate their cytocompatibility.5.The ability of each group of scaffold materials to differentiate BMSC into osteoblasts and promote angiogenesis was detected by RT-PCR.6.Thirty New Zealand white rabbits were randomly selected to construct femoral condylar defect models and then they were divided into four groups:blank control group;TBC scaffold group;COL I/TBC scaffold group;DMOG/COL I/TBC scaffold group.12 weeks after surgery,bone tissue pathology,bone morphometry and immunohistochemistry were used to evaluate new bone formation and molecular mechanisms.Results1.After 72h of culture,the primary BMSC cells became spindle-shaped and evenly distributed,forming colonies on the 8th day and reaching 85%fusion.2.The results of ALP staining showed that the ALP staining in the experimental group was darker than that in the control group,and the ALP staining in the 200μM DMOG experimental group was darker than that in the50μM DMOG experimental group.The three groups showed significant color differences,and the DMOG could increase the osteogenic differentiation ability of the BMSCs significantly.3.The XRD pattern shows that the main component of TBC scaffold material is hydroxyapatite（Ca10（PO4）6（OH）2）,and DMOG molecules are contained in the DMOG/COL I/TBC scaffold.4.SEM scans showed that BMSCs grew well on TBC,COL I/TBC,and DMOG/COL I/TBC scaffolds.BMSCs showed long spindles shape,with a large number of cells,which were connected together and there was no significant difference between the groups.BMSC cells have good biocompatibility in DMOG/COL I/TBC scaffold materials.5.The mRNA expression levels of ALP,OCN,VEGF,and Runx2 after co-culture of BMSCs with DMOG/COL I/TBC scaffolds were significantly higher than those of COL I/TBC scaffolds and TBC scaffolds.The COL I/TBC composite scaffold material loaded with DMOG significantly improved the osteogenic differentiation and angiogenesis of BMSCs.6.The release curve indicated that DMOG did not show burst release in the composite scaffold material,and there was still a certain amount of DMOG release after 7 days,and it continued to be released slowly.7.Bone histopathological analysis and bone morphometry indicated that there were almost no new bones in the blank group,a few new bones in the TBC and COLI/TBC groups and the new bones were mainly concentrated on the edge of the scaffold.There was no significant difference between the two groups.The new bone in the DMOG/COL I/TBC group was significantly more than other groups and there were more new bones in the center of the scaffold.Under high magnification,more capillaries were seen in the DMOG/COL I/TBC group,but less in the COL I/TBC group.8.Immunohistochemical staining indicated that Runx2 and CD31 were slightly expressed in the COL I/TBC group,whereas Runx2 and CD31 were more expressed in the DMOG/COL I/TBC group.ConclusionsThis experiment shows that DMOG can promote the osteogenic differentiation of BMSC cells effectively,and the prepared composite DMOG/COL I/TBC scaffold material has good compatibility with BMSC cells.DMOG can achieve slow release by loading it to COL I/TBC scaffold material.And loading DMOG onto COL I/TBC scaffold could promote the repair of femoral condylar defects in rabbits.