Experimental Study On Repairing Bone Defect With Treated Dentin Matrix Combined With α-calcium Sulfate Hemihydrate

PurposeBone defects caused by tumor,trauma and other causes seriously affect the quality of life of patients.Autologous bone transplantation and allogeneic bone transplantation are common clinical treatment methods,but there are some problems such as big trauma and easy infection.Bone graft is an ideal treatment for large area of bone defects,but single-material artificial bone is difficult to meet all the requirements of ideal bone graft.In this study,will be treated as rich in a variety of growth factors of treated dentin matrix(TDM)and alpha calcium sulphate hemihydrate(α-CSH)with different quality than mixed,the preparation of treated dentin matrix composite alpha calcium sulphate hemihydrate(TDM/α-CSH).It provides a new idea for the preparation of bone graft materials.MethodsThis experiment is mainly composed of three parts.The first part is the preparation and characterization of the material.Clinical teeth in vitro were collected,and after gradient demineralization with EDTA,TDM particles were prepared by freeze-drying at low temperature.TDM/α-CSH composites were prepared by mixing different mass ratios of TDM andα-CSH at the liquid-solid ratio of 0.4 ml/g.Through mechanics test,scanning electron microscopy(SEM),X-ray diffractometer(XRD)and fourier transform infrared spectroscopy(FT-IR)surface functional group analysis,degradation experiment test,hemolysis test of material characterization.In the second part,the effects of composite materials on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells(BMMSCs)were studied.First,BMMSCs were extracted,and the P3 generation of BMMSCs were extracted for subsequent experiments.Cell surface markers were identified by flow cytometry.Osteogenesis and adipogenesis were induced to identify the multidirectional differentiation ability of cells.Cell origin was identified by immunofluorescence.Then the composites were placed in α-MEM medium to obtain the extracts of each composite for in vitro cell experiments.CCK-8 was used to verify the effect of the extract on BMMSCs proliferation.Quantitative real time polymerase(qRT-PCR),Western Blot(WB)validation of BMMSCs osteogenesis leaching solution to differentiation.The third part:The composite material was transplanted into the SD rat skull critical bone defect model,and the bone repair was detected by micro-CT.Then the area of new bone formation and the percentage of total area of defect in each group were calculated to observe the repair effect of the composite material for bone tissue defect in vivo.The osteogenic properties of the materials were tested by HE and Masson.ResμltsIn the first part,mechanical tests show that the mechanical strength of TDM/α-CSH composites decreases with the increase of TDM content.The more TDM content,the lower the compressive strength of the composites.SEM showed that the TDM films obtained after gradient demineralization by EDTA showed that the dentin tubules were fully opened and the collagen fibers in part of the matrix were exposed,which made the collagen fiber bundles in the matrix of the dentin relaxed.It is proved that EDTA can open dentin tubules after gradient demineralization.The TDM particles were ground into TDM particles.Scanning electron microscopy showed that the TDM particles were quasi-round particles,and many kinds of round particles formed loose sheet structure with each other.α-CSH is a loose prismatic structure.When TDM and α-CSH are uniformly mixed,the α-CSH structure can be seen in granular TDM.With the increase of TDM content,the content of prismaticα-CSH decreases,and the material becomes more loose.FT-IR and XRD analysis showed that TDM/α-CSH composite was a physical mixture of TDM and α-CSH,and no new material was produced after the mixture.The hemolysis test showed that the hemolysis rate of all the composite materials was lower than 5%,which proved that our composite materials would not cause hemolysis reaction of red blood cells and had good compatibility with blood.Degradation experiments showed that the composite had degradation effect,and the degradation rate increased with the increase of TDM content.In the second part,we obtained BMMSCs that showed elongated polygonal morphology under light microscopy.After 14 days of adipogenic induction and 21 days of osteogenic induction,BMMSCs presented lipid droplets and mineralized junctions.Flow cytometry showed that the expression rates of CD29,CD90 and CD73 were higher than 95%and were positive.CD31 is less than 5%,which is negative.Immunofluorescence assay showed positive results for Vimentin on mesenchymal surface and negative results for CK14 on epithelial cells.This proved that the cells we extracted were indeed BMMSCs with multidirectional differentiation potential.The results of CCK-8 showed that the composite material had a certain influence on cell proliferation.With the increase of TDM content,the promotion effect of composite material on cell proliferation was significantly enhanced.QRT-PCR and WB results showed that each group could significantly promote the expression of OPN,RUNX2 and ALP in BMMSCs.In the third part,when the composite material was transplanted into the rat skull critical bone defect model,the results of micro-CT three-dimensional reconstruction showed that the blank control group only had a small amount of new bone formation at the defect edge,and the amount of new bone formation was significantly lower than that of the other materials groups.Comparison of new bone formation area in each group:blank control group<α-CSH group<10%TDM/α-CSH group<30%TDM/α-CSH group<50%TDM/α-CSH group,and there were statistical differences among all groups(P<0.05).In addition,HE and Masson results showed that the composite material could significantly induce bone formation.The results showed that the composite material has a certain ability to promote the formation of new bone,and with the increase of the amount of TDM composite material,the promotion of osteogenic repair will be enhanced after implantation of bone tissue defects.ConclusionThe combination of TDM/α-CSH osteogenic materials did not produce new materials,and the osteoinduction performance was stronger with the increase of TDM mass ratio.TDM/α-CSH is a composite bone repair material with certain plasticity,strength,degradability,good bone induction and bone conduction properties,and may be an ideal bone graft material in artificial bone transplantation.