| [Background]Currently,about 500 million people have diabetes.Most of them are type 2 diabetes mellitus(T2DM).They are at high risk of osteoporosis,fractures and poor bone healing.Even in patients with stable glycemic control,alveolar socket healing is worse than in non-diabetic patients.This may be related to abnormal bone metabolism,bone marrow microangiopathy,and vascular dysfunction.Bone tissue engineering technology has great advantages and prospects in the treatment of bone defects in diabetic patients.Mesenchymal stem cells(MSCs)with self-renewal ability and multi-differentiation ability as seed cells provide good osteogenic and osteoinductive properties for tissue engineered bone.At the same time,they have a wide range of sources and are easy to expand in vitro.It compensates for the shortcomings of autogenous bone that the source and quantity are very limited.When tissues are damaged or hypoxia,endothelial cells(ECs)can activate the signal cascade of cytoskeletal reorganization and cell budding to quickly form new blood vessels.The co-culture of ECs and MSCs is currently a popular solution to promote bone and angiogenesis in the bone defect area at the same time.Many previous studies have combined the two co-cultured cells with rigid scaffolds or gel materials for bone repair.Cell sheet technology can make cells highly aggregated and preserve abundant extracellular matrix,which enables co-cultured cells to be independently used without relying on scaffold materials,and has a good application prospect in bone tissue engineering.At present,there are a variety of methods to cultivate MSCs and ECs composite cell sheets to promote bone healing and angiogenesis,but there is no systematic comparison of these methods.Adipose-derived stem cells(ASCs)are rarely used in composite sheets for such studies.In addition,the effect of ASCs and ECs composite cell sheets on osteogenesis and angiogenesis in diabetic patients is still unclear.Compared with bone marrow mesenchymal stem cells and adult mesenchymal stem cells from other sources,ASCs can be isolated and extracted from adipose tissue of the whole body,which are more abundant and easier to obtain,and have less risk of trauma and complications in donor site.Therefore,this project studied the crosstalk between ASCs and ECs,used different methods to construct composite cell sheets of ASCs and ECs,and studied the osteogenic and angiogenic capabilities of each group of composite cell sheets in vitro and in T2DM rats.The study is to find out new ideas and directions for the clinical treatment of bone defects in patients with T2DM.[Objectives]To explore the effects of ASCs and ASCs sheets on the biological characteristics and vascular ability of ECs;to figure out the effects of ECs on the biological characteristics and osteogenesis ability of ASCs;to compare the osteogenesis ability of ASCs and ECs composite cell sheets constructed by different methods in vitro;to figure out the effects of composite cell sheets of ASCs and ECs on bone healing in T2DM rats.The purpose of this study was to explore a bone graft material with good osteogenic and angiogenic properties for the clinical treatment of T2DM bone defects.[Methods]Part Ⅰ: Studies on the crosstalk between ASCs and ECs in vitro.1.Primary ASCs and ECs of rats were isolated,cultured and identified.2.Conditioned media of ASCs and ASC sheets(ASC-CM and ASC sheet-CM)were prepared,and their effects on ECs proliferation were examined by CCK-8 and Edu(TMB)assays.Their effects on migration were explored by scratch tests and transwell migration assays.On the first 1,4,and 7 days when ECs were cultured in ASC-CM and ASC sheetCM,VEGF gene and protein expression of the ECs were detected by real-time quantitative PCR and western blot.The pro-angiogenic potential of ASC-CM and ASC sheet-CM was compared using a tube formation assay in vitro and matrigel plug assay in vivo.3.ASCs and ASC sheet exosomes(ASC-exo and ASC sheet-exo)were extracted based on the principle of polymer coprecipitation and then identified.Particle size and concentration of ASC-exo and ASC sheet-exo were compared.The effects of ASC-exo and ASC sheet-exo on migration ability of ECs were detected by scratch tests and transwell migration assays.The pro-angiogenic potential of ASC-exo and ASC sheet-exo was compared using a tube formation assay in vitro and matrigel plug assay in vivo.4.The effect of EC-CM on ASCs proliferation was examined by CCK-8 and Edu(TMB)assays.After cultured in EC-CM for 7 days,the apoptosis of ASCs was detected by FITC-Annexin V and PI apoptosis kit.Scratch tests and transwell migration assays were used to detect the effect of EC-CM on migration ability of ASCs.The expression of Runx2,OCN,ALP,BMP2 and VEGF were detected on the 0th,1st,3rd,5th and 7th day of EC-CM culture by RT-q PCR.Osteogenesis-related staining were performed to explore the effect of EC-CM on osteogenic ability of ASCs.Part Ⅱ: Studies on the effect of the cell sheets of co-cultured ASCs and ECs on bone regeneration and vascularization in T2DM rats5.Studies on osteogenesis ability of ASCs and ECs composite cell sheet in vitro.(1)ASCs and ECs were mixed at the ratio of 1:0,10:1,1:1,1:10 and 0:1,and then induced in cell-sheet induction medium for 7 days to prepare the co-culture sheets of ASCs and ECs;The expression of ALP,BMP2,Col-1,Runx2 and VEGF were detected by real-time PCR and Western Blot.Osteogenesis induction medium was used to induce osteogenesis of the co-cultured cell sheets in each group.Then the osteogenesis-related staining was performed.(2)ECs was inoculated on ASC sheet in the number of 2×10~6,2×10~5 and 2×10~4 cells/well,respectively,to prepare composite cell sheets;The expression of ALP,BMP2,Col-1,Runx2 and VEGF were detected by real-time PCR and Western Blot.Co-cultured cell sheets were cultured in osteogenesis induction medium for 7 days.And then sirius red staining and ALP staining were performed.(3)Five different kinds of membranes were prepared respectively: ASC sheet,EC sheet,1A1 E sheet in which ASCs and ECs were directly co-cultured at the ratio of 1:1,ASC sheet inoculated with ECs,ASC sheet +EC sheet,etc.The gene expression of ALP,BMP2,Col-1,Runx2 and VEGF was detected by real-time PCR.6.Studies on the effect of ASCs and ECs composite sheets on bone repair in T2DM ratsA T2DM rat model was established.The 48 T2DM rats were randomly divided into 6groups.The critical-sized defects(CSD)with a diameter of 5mm was prepared.The rats were divided into blank control group,ASC sheet group,EC sheet group,mixed group(the ASC sheet and EC sheet were mixed and overlapped),1A1 E sheet group(ASCs and ECs were cocultured at a ratio of 1:1 and then induced to cell sheet),and an ASC sheet+EC group(ECs were inoculated onto ASC sheets).The samples were taken 8 weeks after surgery and the healing of the bone defect was analyzed by Micro-CT and HE staining.[Results]Part Ⅰ: Studies on the crosstalk between ASCs and ECs in vitro.1.Rat ASCs and ECs were successfully extracted.2.Both ASC-CM and ASC sheet-CM promoted the proliferation of ECs,but there was no statistical difference between the two.Both of them could promote migration ability of ECs,and ASC sheet-CM was best.The expressions of VEGF in ASC sheet-CM group were significantly higher than those in ASC-CM group on the first 1,4,and 7 days after cultured in different media.More tubules of ECs were formed after exposure to ASC sheet-CM and ASC-CM.The number of branch points and the total tube length in the ASC sheet-CM group was highest.ASC-CM and ASC sheet-CM accelerated tissue vascularization more efficiently in vivo.However,no statistical difference was observed in ASC sheet-CM group compared with ASC-CM group.3.ASC-exo and ASC sheet-exo were successfully extracted,both of which exhibited bilayer membranes and cup-shaped or round-shaped structures and expressed exosome specific markers CD63,Alix and CD81.Nanoparticle tracking analysis showed that the concentration of ASC-exo was greater than that of ASC sheet-exo,but the average particle size of ASC-exo was smaller.The modal peak of ASC-exo and ASC sheet-exo had no significance.Both ASC-exo and ASC sheet-exo promoted the migration ability of ECs,and ASC sheet-exo group was the best.More tubules of ECs were formed after exposure to ASC-exo and ASC sheet-exo with different concentrations.When the concentrations were 10,100 and 200 g/L,the ASC sheet-exo group formed more tubules ASC-exo group.Both ASC-exo and ASC sheet-exo could promote angiogenesis in vivo,and ASC sheet-exo exhibited significantly highest potential to stimulate vascular network formation ASCs exhibited significantly higher potential to stimulate vascular network formation.4.EC-CM had no significant effect on the proliferation,appotosis and migration of ASCs as well as the expression of RUN2,OCN and BMP2,but it could promote the expression of ALP and VEGF.The results of the osteogenesis-related staining showed that the osteogenic ability of ASCs was enhanced after cultured in EC-CM for 7 days.Part Ⅱ: Studies on the effect of the cell sheets of co-cultured ASCs and ECs on bone regeneration and vascularization in T2DM rats5.Studies on osteogenesis ability of ASCs and ECs composite cell sheet in vitro.(1)The ASCs and ECs co-culture sheets were constructed at the ratio of 1:0,10:1,1:1,1:10 and 0:1.With the increase of ECs,the thickness of the co-culture sheets gradually decreased.The gene expression of ALP and BMP2 in 1:1 group was significantly higher than that in other 4 groups.Col-1 and Runx2 were highly expressed in 10:1 and 1:1 groups.The expression of VEGF in 1:1 group was at the middle level among the five groups.(2)The expressions of Runx2 and VEGF in the group with less ECs inoculation on ASC sheet were higher than other groups with more ECs.(3)The results of real-time quantitative PCR showed that the expressions of ALP,Runx2 and VEGF in ASC sheet + ECs group and ASC sheet + EC sheet group were significantly higher than those in other three groups.The ALP expression in ASC sheet +ECs group was the highest.The expression of Col-1 and VEGF in ASC sheet + EC sheet group was the highest.The expression of BMP2 in a1 esheet group was the highest.6.Studies on the effect of ASCs and ECs composite sheets on bone repair in T2DM rats(1)48 out of 50 rats successfully established a T2DM model with a success rate of96%.(2)The results of Micro-CT showed that EC sheet group could not promote bone formation;The effects of bone repair in ASC sheet group were superior to those of the blank control group and the EC sheet group.1A1 E sheet group,ASC sheet + ECs group and ASC sheet group had similar bone formation capacity,and there was no statistical difference between the three groups.The osteogenic capacity of ASC sheet + ECs group was the best in T2DM rats,followed by 1A1 E sheet group.(3)The results of HE staining showed that only a small amount of new bone was formed at the edge of bone defect in the blank control group and EC sheet group while a lot of new bone could be seen in the other groups.The number of blood vessels in ASC sheet group and EC sheet group were superior to that in control group.The numbers of blood vessels in the overlaid sheet group,the 1A1 E sheet group,and the ASC sheet + ECs group were similar and superior to those in the blank group,the ASC sheet group,and the EC sheet group.[Conclusion]1.We prepared ASC sheet-CM,and confirmed that it can significantly promote the proliferation ability of ECs like ASC-CM.In terms of promoting the migration of ECs,expression of VEGF and tube formation ability in vitro,ASC sheet-CM has significant advantages over ASC-CM.2.We extracted ASC sheet-exo,which is different from ASC-exo in particle size and concentration,and confirmed that ASC Sheet-exo has obvious advantages over ASC-exo in promoting migration and tube forming of ECs in vivo and in vitro.3.EC-CM had no significant effect on the proliferation,appotosis and migration of ASCs,but it could promote osteogenesis ability of ASCs and the gene expression of VEGF.4.When the ratio of ASCS to ECs is 1:1,the osteogenic ability of the composite sheets was the strongest.The composite sheets with a small amount of ECs inoculated on ASC sheet had the best osteogenesis and angiogenesis potential,which is superior to other groups.Three kinds of composite sheets had better osteogenesis and angiogenesis potential,which was superior to ASC sheet group and EC sheet group.5.EC sheet can promote angiogenesis but not bone healing in T2DM rats.Both composite sheets and ASC sheet can promote bone healing and neovascularization in T2DM rats.The effect of osteogenesis and angiogenesis in T2DM rats in ASC sheet + EC sheet group was the best.In summary,the composite sheet of ASCs and ECs,especially the mixture of ASC sheet and EC sheet,could promote T2DM bone healing and angiogenesis,and is expected to become a potential bone graft material for clinical treatment of T2DM bone defect. |
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