| Background:Diabetes is the third largest non-communicable disease in the world today,and China is known as the world’s "largest country of diabetes." With blood glucose increasing for a long time,large blood vessels and microvascular will be damaged,high blood glucose can endanger the heart,brain,kidney,peripheral nerves,eyes,feet and other organs at the same time.Reconstruction of large tissue defects caused by trauma or cancer remains a challenge for surgeons.The current gold standard for the treatment of large tissue defects is autologous tissue transplantation.Meanwhile,autologous tissue transplantation is limited by defect size and donor site complications.Therefore,how to reconstruct large bone defects in diabetic patients is becoming more urgent and difficult.A new field—bone tissue engineering(BTE)has begun to rise.Insufficient vascularization is one of the challenges in repairing massive bone defects in tissue engineering.Oxygen and other nutrients play a very important role in the process of osteogenesis,and blood vessels can only supply oxygen to tissues in the range of 100-200 um at most.Tissue necrosis will occur if oxygen supply is not available in a timely and effective manner,especially in the center of tissue engineered bone.Bone marrow mesenchymal cells(BMSCs)are ideal seed cells for bone tissue engineering because of their self-replication ability,multiplex differentiation potential and low immunogenicity.However,it can not effectively solve the problem of vascularization.The discovery of endothelial progenitor cells(EPCs)brings a glimmer of hope for the vascularization of tissue engineered bone,because it can proliferate and differentiate into vascular endothelial cells,and can also participate in vascular repair and regeneration,and has low immunogenicity.Studies have shown that direct co-culture of BMSCs and EPCs not only promotes the osteogenic differentiation of BMSCs,but also promotes the angiogenesis of EPCs.The cell sheet technology,which provides a high density of transplanted cells containing extracellular matrix(ECM)with intact cell-cell junctions,is an effective method in tissue engineering.Purpose:The objective of this study was to explore the effects of high glucose environment on the proliferation and osteogenic differentiation of BMSCs,and on the proliferation and angiogenesis of EPCs.To explore the effects of cell sheets,which containing BMSCs and EPCs,on calvarial critical size defects in T2 DM rats.Methods:1.BMSCs and EPCs were separated by density gradient centrifugation combined with differential attachment method.BMSCs were identified by induction of adipogenesis,osteogenesis and chondrogenesis.EPCs were identified by double fluorescence staining and Matrigel tubule formation experiments.BMSCs,EPCs,BMSCs and EPCs double-cell sheets were continuously induced and cultured by cell-sheet inducing culture medium.The structure of cell sheets were observed by scanning electron microscopy.2.Firstly,we screened the glucose concentration.BMSCs and EPCs were cultured in culture media containing different glucose concentration and their growth curves were drawn.The optimum glucose concentration was selected and used in subsequent experiments.High glucose culture medium was applied to BMSCs and EPCs,and ALP activity and PCR were used to detect its effect on osteogenic differentiation of BMSCs,and Matrigel tube formation experiment,RT-PCR and WB were used to detect the effect of EPCs on angiogenesis.3.In vivo: The model of type 2 diabetes mellitus rats was established by high fat and high sugar diet combined with STZ injection,and then the model of calvarial bilateral critical-sized defects in type 2 diabetic rats were constructed.The defect was round in diameter of 4 mm.Then these rats were randomly divided into four groups:(A)blank control group,(B)EPCs cell sheet group,(C)BMSCs cell sheet group,(D)BMSCs/EPCs cell sheet group.Eight weeks after the operation,the calvaria was taken out and the number of trabeculae(Tb.N),bone volume fraction(BV/TV),trabecular space(Tb.Sp)and trabecular thickness(Tb.Th)of the new bone were analyzed by micro-CT.4.Statistical analysis: All results were expressed by mean(±standard deviation).The experimental data were analyzed by SPSS17.0 statistical software.The mean of two independent samples was compared by t test.The mean of multiple samples was compared by one way of Variance,if the variance is homogeneous.If heterogeneity of variance,the square root of the data is transformed and then the variance analysis is used.When compare the mean of multiple samples between two group,we use LSD post hoc test if the variance is homogeneous.If the variance is heterogeneous,Games-Howell test is used.P < 0.05 indicates the difference is significant.Results:1.BMSCs and EPCs were successfully isolated by density gradient centrifugation combined with differential attachment method.BMSCs were identified to have multipotent differentiation potential and EPCs were identified to have angiogenesis potential.The cell sheets were translucent and milky white film-like material curled to the middle,and could easily be separated from the bottom of the dish completely,and had certain elasticity.Scanning electron microscopy showed that a large number of matrix secretion and layered deposition were observed on the surface of the cell sheet in each group,and the cells were wrapped in it.2.Increasing the glucose concentration in culture medium inhibited the proliferation of EPCs.However,appropriately increasing glucose concentration was beneficial to cell growth,but if glucose concentration went higher,cell proliferation would be inhibited.(P < 0.05).So 30mmol/L glucose concentration was selected for subsequent experiment.In high glucose environment,the ALP activity of BMSCs decreased(P < 0.05),and the expression of osteogenesis related genes,such as Runx2 and Osterix,also decreased(P < 0.05).In high glucose environment,EPCs Matrigel tubule formation decreased significantly,and the results of RTPCR and WB also showed that the expression of vascular endothelial growth factor decreased at both gene and protein levels(P < 0.05).3.Using high-fat and high-sugar diet combined with STZ injection successfully constructed the model of T2 DM rats.After 8 weeks of cell sheets implantation,rats were sacrificed.BMSCs/EPCs group had the best ability to repair calvarial defects.Micro-CT results showed that the new bone of the double cell sheet group had the highest bone volume fraction,the largest number of trabeculae,the thickest trabecular thickness and the smallest trabecular space,compared with the other three groups,and there were statistical differences.Conclusion:1.BMSCs and EPCs could be successfully isolated and cultured by density gradient centrifugation combined with differential attachment method.Cell membranes could be induced by cell culture medium containing ascorbic acid.2.In vitro,increasing the glucose concentration in culture medium inhibited the proliferation of EPCs.However,appropriately increasing glucose concentration was beneficial to BMSCs growth,but if glucose concentration went higher,cell proliferation would be inhibited.High glucose inhibited osteogenic differentiation of BMSCs and inhibited the angiogenesis of EPCs.3.The application of BMSCs and EPCs co-cultured cell sheet in the calvarial bilateral defect of type 2 diabetic rats can significantly promote bone regeneration.This study provided experimental basis for further research and clinical application of tissue engineering bone. |
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