| Background and objectiveAs we all know,angiogenesis plays an important role in wound healing,bone formation and metabolism.The literature points out that copper is essential in the process of angiogenesis,and animal experiments verify that copper sulfate can promote corneal angiogenesis.At the same time,calcium sulfate has good biocompatibility and can promote the formation of blood vessels around tibial bone defect.Therefore,the purpose of this study is to verify the effect of new composite scaffolds mixed with copper sulfate and calcium sulfate,which own the properties of good compatibility,moderate degradation rate and mutual enhancement of angiogenesis/bone repair effect.Method(1)Preparation,physical characterization and in vitro degradation experiment of a novel copper doped calcium sulfate bone cementFirst,the ?-calcium sulfate hemihydrate powders and the different concentrations of copper sulfate pentahydrate solution were mixed according to the fixed solid-liquid ratio,and the cylindrical composite samples were obtained by cylindrical mold,dried in the drying oven for 24 hours.The materials were characterized by XRD,FT-IR,Mechanical pressure and SEM.Then the degradation characteristics of the composite were studied by immersing in simulated body fluid.(2)Effect of extracts of different concentrations of composite on the proliferation,differentiation,tube formation and migration of human umbilical vein endothelial cells(HUVECs)According to international standards,the extracts of different concentrations of composite material were prepared to incubate with human umbilical vein endothelial cells(HUVECs),and the effects of the extracts on the proliferation and migration of HUVECs were detected by CCK-8 experiment and scratch assay;the effects of the extracts on the angiogenesis of vein endothelial cells were detected by tube formation experiment;angiogenic-related genes expression(VEGF,HIF-1? and eNOS)were detected by QRT PCR and angiogenic-related protein(VEGF,HIF-1?)were detected by Immunofluorescence staining.(3)Effect of copper doped calcium sulfate composite on the proliferation and differentiation of bone marrow mesenchymal cells(BMSCs)According to international standards,different concentrations of composite extracts were prepared to incubate with BMSCs,and the proliferation of the extracts on BMSCs was detected by CCK-8 experiment;the expression of related osteogenic differentiation genes(OCN,ALP,Runx2 and BSP)was detected by qRT-PCR;the differentiative effect of the extracts on BMSCs was detected by alizarin red staining.(4)Experimental study on the effect of angiogenesis and bone repair around critical tibial defect in rats on the Cu(0.5)?Cu(0)and the blank group.A critical bone defect model was made in the upper tibia of SD rats.The model was filled with a Cu(0.5)and Cu(0)sample separately.After 6 weeks,the angiographic reconstruction and Immunohistochemistry method were used to evaluate the effect of Cu(0.5)and Cu(0)sample on the formation of blood vessels.the effect of the composite on bone defect was evaluated by means of Micro CT and Histomorphology.Result1.SEM shows that the specific surface of the material is calcium sulfate dihydrate in the form of plate-like shape,and the inner layer is ?-calcium sulfate hemihydrate in the form of rod shape.The compressive strength of the composite increased with the increase of copper concentration,and the degradation of the composite was the fastest in the first three weeks.2.CCK-8 showed that the extract with low concentration of copper could promote the proliferation of vein endothelial cells,while the extract with high concentration of copper had toxicity to cells.The results of scratch assay showed that the extracts of copper doped calcium sulfate and pure calcium sulfate had migration effect on endothelial cells,but the high concentration of copper appeared cytotoxicity after 24 hours later.At the same time,the tube formation study showed that the extracts had a tube-formation effect on endothelial cells;the expression of angiogenic-related gene expression(VEGF,HIF-la and eNOS)increased significantly with the increase of copper concentration,while the expression of eNOS gene was promoted slightly,and it was not significant between groups.At the same time,the cellular Immunofluorescence staining also proved.3.CCK-8 assay confirmed that the low concentration of copper doped calcium sulfate and pure calcium sulfate had significant proliferation effect on bone marrow mesenchymal cells,while the high concentration of copper doped composite[Cu(1)and Cu(2.5)]had slightly proliferation reaction;the expression of osteogenic-related genes decreased with the increasing concentration of copper ion;In addition,alizarin red experiment also proved that the BMSCs differentiated gradually inhibited with the increasing concentration of copper ions.3.Compared with pure calcium sulfate and the blank group,the quantity of blood vessels around tibial bone defect increased after 6 weeks in the Cu(0.5)group.Immunohistochemistry showed that the density of blood vessels in copper doped calcium sulfate group was higher than that in calcium sulfate group and the blank group.Micro CT and Histomorphology showed that compared with pure calcium sulfate,the ability of bone defect repair was weakened in Cu(0.5),but compared with the blank group,there was still a certain ability of bone repair in Cu(0.5).Conclusion1.Compared with pure calcium sulfate,the released copper ions and calcium ions have synergistic effect on promoting blood vessels.2.The low concentration of copper has a proliferational effect on HUVECs and BMSCs.The copper can promote the expression of angiogenic-related genes and inhibit the expression of osteogenic-related genes and the ability of BMSCs differentiation.3.The effect of copper doped calcium sulfate composite promote blood vessels around the upper tibial bone defect in SD rats.4.Compared with the pure calcium sulfate,the ability of defect repair in the Cu(0.5)were inhibited to some extent,and the Cu(0.5)group was better than the blank group. |
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