| BeckgroundBone defects caused by trauma,infection,tumor and congenital disease are the most difficult problem in clinical treatment.Autogenous bone graft is the gold standard for the treatment of bone defects,however there is a lot of limitations in Autogenous bone graft,such as a defect in donor sites,secondary injuries,and limited sources.It is difficult to be used for the treatment of large bone defects.The allograft bone and the recombinant allograft may have the risk of disease transmission,rejection,and so on.How to cure bone defects safely and effectively is a great challenge now.Therefore,new bone substitute materials to repair bone defects has become a hot topic in clinical and basic research.ObjectiveOur study aim to study on the osteogenic effect of cerium containing calcium sulfate,a new biodegradable bone substitute material.Calcium sulfate has good biocompatibility and biodegradability.Its degradation rate is consistent with the rate of local vessel ingrowth and new bone formation,and has plasticity.It can be shaped according to the area of bone defect.However,there are shortcomings of Calcium sulfate,such as lacks of osteo-inductive activity,poor mechanical strength and so on.Cerium(Cerium,Ce)has the potential to differentiate inducing bone marrow mesenchymal stem cells into osteoblasts,promote the growth of new blood vessels,promotes cell proliferation and inhibits cell apoptosis.This research preparated new bone material that is ceriurn doped calcium sulfate.Combinated cerium with calcium sulfate might enhance bone substitute osteoinductive activity.By using SD tibial bone defect model,we verificated the osteogenic effects of cerium doped calcium sulfate in vivo.So as to provide a new method for the treatment of bone defect,and provide scientific basis for its clinical application.MethodsPartI Preparation and physical and chemical properties of cerium containing calcium sulfate bone substituteThe gradient concentration of Ce containing calcium sulfate(Ce-CS)was prepared by direct mixing.X ray diffraction(XRD)was used to evaluate the composition of elements.Fourier transform infrared scanning(FTIR)was used to evaluate its crystal structure.SEM(SEM)was used to observe the structure of the surface.The influence of Ce on mechanical strength of materials was determined by pressure test.PartII The biocompatibility of the new bone substitute materialThe extract of gradient concentration of Ce-CS were preparated;MTT assay of the cytotoxicity of extracts;CCK-8 assay of the effect of extract on BMSCs proliferation;Scratch assay of the effect of gradient extracts on BMSCs migration;fluorescence quantitative PCR detected osteogenic differentiation genes BMSCs affected by Ce-CS extracts;Western-blot detected protein level of beta-catenin,Smad/3,BMP2/7 and OPN affected by Ce-CS BMSCs;Part III The bone repair function of cerium containing calcium sulfate in vivoThe critical tibial defect model of SD rats was established.48 male SD rats were divided into 4 groups,as no graft,CS graft,5%Ce-CS graft and 10%Ce-CS graft were implanted respectly.12 rats in each group.X-ray examination and micro-CT were used to observe bone defect repair and new bone morphology.The new bone formation and material degradation were observed by the H&E staining and Goldner staining.The angiogenesis around the new bone was observed by immunofluorescence.Results1.XRD and FTIR show that the diffraction and infrared spectra of Ce-CS are similar to those of calcium sulfate.SEM shows that Ce containing materials exhibits round and blunt roughness.When Ce concentration reached 2.5%,mechanical strength is 23.45±.35MPa,which is higher than that of CS group,which is 21.25±1.31MPa.2.The proliferation rate of L929 cells was no significant difference between the concentrations of cerium containing calcium sulfate.Toxicity of extraction of calcium sulfate with different cerium concentration were I level,no cell toxicity;5%Ce-CS promotes the proliferation of bone marrow mesenchymal stem cells(Bone marrow mesenchymal stem cells,BMSCs).Scratch test showed that the scar healing rate of 5%Ce-CS was the greatest.When cultured with 1.25%Ce-CS and 5%Ce-CS extract,the expression of osteogenic markers(ALP,OST,BSP and OCN)in BMSCs cells increased,and the expression of transcription factor RUNX2 was also higher than that in control group.3.X ray showed that the stent was absorbed at 4 weeks after the operation.In the blank control group,bone defects were enlarged without new bone formation.The density of CS,5%Ce-CS and 10%Ce-CS decreased gradually,and only a few new bone appeared in CS group.5%Ce-CS and 10%Ce-CS group had new bone filling with low density.Micro-CT:In 5%Ce-CS and 10%Ce-CS group,a large number of bone defects were found,and the bone defect was close to healing.H&E staining,Goldner staining showed that the blank control group,bone defect was filled with fibrous connective tissue tissue.The bone defect in the CS control group was obviously concave,with a small amount of new bone formation.There were a lot of new bone formation around the grafts in 5%Ce-CS group.Immunofluorescence showed that a large number of endothelial vascular markers CD31+SMA+ cells were found in the bone defect areas of 5%Ce-CS and 10%Ce-CS group at 8 weeks after the operation.Conclusion1.Ce has no obvious effect on the crystal structure and chemical composition of calcium sulfate.The crystal structure of calcium sulfate becomes blunt and the mechanical strength increases with the increase of cerium concentration.2.When the concentration of cerium was<10%,the cytotoxicity of cerium doped calcium sulfate was I,and there was no obvious cytotoxicity.3.Ce-CS promoted the proliferation and migration of BMSCs cells,especially when the concentration of 5%cerium was the most obvious.4.Ce-CS promotes the expression of ALP,OCN and other genes,such as ALP and OCN,and the expression of regulatory factor Runx2.5.Compared with calcium sulfate stents,5%and 10%calcium sulfate stents have better osteogenesis in the bone defect model of the body.6.5%Ce-CS can promote the formation of peripheral blood vessels. |
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