| Objective:1)To explore the application of a simple method to construct human amniotic mesenchymal stem cells(hAMSCs)sheet and to study their potential to differentiate into chondrocyte.2)To explore the feasibility of articular cartilage defects repaired with particulated juvenile cartilage allograft.3)To explore the feasibility of repairing osteochondral defects with human Amniotic Mesenchymal Stem Cells(hAMSCs)sheet encapsulated cartilage particles.Methods:1)The hAMSCs were separated through mechanical-enzymatic digestion from the maternal placenta,and the obtained cells were subjected to phenotypic identification by flow cytometry and immunofluorescence.The third generation of hAMSCs were used for subsequent experiments.After 14 days of in vitro induced culture,the ability to differentiate into osteocytes and lipocytes was observed using different methods.CCK-8 was used to detect cell proliferation.hAMSCs sheet and chondrocyte –induced hAMSCs sheet were constructed by hAMSCs,respectly;Scanning electron microscope(SEM)were used to observe the cell phenotype,distribution and structural characteristics of hAMSCs sheet;Chondrogenic-induced hAMSCs sheet was detected to differentiate into chondrocytes by toluidine blue staining and immunohistochemistry(IHC),and m RNA expression(SOX9,COLⅡ,ACAN)and relative protein expression were also evaluated.2)The cartilage particles were obtained from juvenile minipig and cultured in vitro.Brdu immunofluorescence assay was performed at 1,3 and 7 days of culture.The cartilage particles/fibrin gel composites were subcutaneously transplanted into the nude mice.The specimens were taken for hematoxylin-eosin staining,safranin O staining and immunohistochemistry after 1 month.Full-thickness cartilage defects of 8mm in diameter were created in the left patellar grooves of 10 adult minipig and the defects were treated with cartilage particles/fibrin glue(n=5)and left untreated(n=5).The specimens were taken for histological score and histological evaluation at 3 months after transplantation.3)Osteochondral defects(3.5mm in diameter,3mm in depth)were created in the left patellar grooves of 20 New Zealand White rabbits and the defects were treated with hAMSCs sheet/cartilage particles(n=5),cartilage particles(n=5),hAMSCs sheet(n=5),fibrin glue(n=5).After 3 months,The results were evaluated by general observation,histological examination and histological scores.The survival time and differentiation of transplanted hAMSCs in cartilage defects were evaluated by immunofluorescence.Results:1)The hAMSCs on the third passage highly expressed the phenotype of MSCs,and hAMSCs sheet still highly expressed the phenotype of MSCs.Vimentin expression is positive,and cytokeratin19(CK19)is negative.Alizarin red staining and oil red staining is positive after 14 days,respectively;CCK-8 assay showed that the cell growth curve was Sshaped,cell proliferation rate was relatively slow on the first day,entered logarithmic growth phase on the third day,and reached the peak on the eighth day.Scanning electron microscope(SEM)results showed that hAMSCs sheet was multiple and cells were stacked layer-by-layer.Toluidine blue staining and immunohistochemistry were positive,and m RNA expression and relative protein expression were higher in chondrogenic-induced hAMSCs sheet than hAMSCs sheet(P<0.05).2)The results of immunofluorescence showed that the brdu signal gradually increased with the prolongation of culture time.Subcutaneous transplantation of cartilage particles: HE staining showed the chondrocytes is uniform;Safranin O staining and IHC was positive.The results of in vivo experiments showed that there was no obvious repaired tissue in the control group,and the defect boundary was obvious.A large number of new tissues were produced in the defects of the cartilage particles group.The color of the new tissue was close to the normal cartilage tissue and the defect boundary was not clear,however,the surface was not smooth.The results of HE staining showed that only a small amount of new tissue was produced in the defects of the control group.A large amount of new tissue was produced in the defects of the cartilage particles group,and the cells in the new tissue were evenly distributed.The results of toluidine blue staining and safranin O staining showed that the new tissue of the control group was not stained.The new tissue of the cartilage particles group was obviously stained.The results of immunohistochemistry showed that the new tissue in the control group was hardly stained,and the staining of the new tissue in the cartilage particles grouop was obvious.The histological score was 15.8±0.83 for the cartilage particles group and 4.8±0.83 for the control group,the difference was statistically significant(P<0.05).3)The defect of the hAMSCs sheet/cartilage particles group was almost completely filled by the new tissue,and the defect boundary was not clear.The cartilage particles group and the hAMSCs sheet group also had more new tissue,and the defect boundary was still observed;There was only a small amount of new tissue in the defects of the fibrin glue group,the defect boundary was obvious.The results of histological examination showed that the formation of new tissue,subchondral bone regeneration and osteochondral interface integration of the hAMSCs sheet/cartilage particles group were superior to the other groups.The histological scores of the hAMSCs sheet/cartilage particles group were significantly higher than the other groups,the difference was statistically significant(P<0.05).A large number of human nuclear specific antigen positive cells were observed in the defect area of the hAMSCs sheet/cartilage particles group and the hAMSCs sheet group;and some of the positive cells expressed SOX9.Conclusions:1)A simple method was successfully used to construct a hAMSCs sheet,and the sheet have a good chondrogenic differentiation potential in vitro research.2)Particulated juvenile cartilage allograft can achieve good results in repairing full-thickness cartilage defects.3)HAMSCs sheet encapsulated cartilage particles can enhance osteochondral defects repair,... |
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