PRP联合ADSCs负载藻酸钙凝胶修复软骨缺损的效果研究OACSTPCD

Objective To investigate the effect of platelet-rich plasma(PRP)combined with adipose-derived stem cells(ADSCs)loaded calcium alginate gel on repairing cartilage defects.Methods ADSCs were isolated and cultured from subcutaneous fat of New Zealand white rabbits.The rabbit articular cartilage defect model was created,and then ADSCs were implanted onto PRP loaded calcium alginate gel to construct tissue engineered cartilage and transplant to the defect.The experiment was divided into three groups:the control group,the PRP group and the PRP ADSCs calcium alginate gel group,with 10 rabbits in each group.The morphology and growth of cells were observed after operation.Toluidine blue staining and type Ⅱ collagen immunohistochemical staining were used to detect the chondrogenic characteristics of ADSCs.CCK-8 method was used to detect the proliferation activity of ADSCs.RT-PCR was used to detect the expression of type Ⅱcollagen mRNA and proteoglycan mRNA.Results Under the inverted phase contrast microscope,ADSCs showed typical morphology of mesenchymal stem cells,which was spindle or oval,with abundant cytoplasm and regular nuclear shape.With the extension of culture time,ADSCs gradually showed a good growth state,the number of cells increased,the connection between cells was tight,and the morphology was regular.In the control group,there was no obvious repair in the cartilage defect area,with light blue staining and a large number of ADSCs.The cartilage defect area in the PRP group was partially repaired and stained blue,and the number of ADSCs was more than that in the control group.The repair effect of cartilage defect area in the PRP ADSCs calcium alginate gel group was the best,the staining was dark blue,and the number of ADSCs was more than that in the PRP group.After 14 days of induction,the ADSCs were positive for toluidine blue metachromostaining,and purple red metachromostaining appeared in the cytoplasm and the surrounding cells.Type Ⅱ collagen immunohis-tochemical staining showed that the average gray value of ADSCs cells in the PRP ADSCs calcium alginate gel group was significantly higher than that in other groups(P<0.05);and the positive cells showed brownish yellow granules in the cytoplasm and yellow staining around the cells.CCK-8 test showed that the ADSCs in the PRP ADSCs calcium alginate gel group entered exponential growth on the second day,and that in the control group began to enter exponential growth on the third day.During the observation period,the OD value of ADSCs in the PRP ADSCs calcium alginate gel group was significantly higher than that in other groups(P<0.05).The expression of type II collagen mRNA in the PRP ADSCs calcium alginate gel group was significantly higher than that in other groups(P<0.05).The proteoglycan mRNA expression of ADSCs in the three groups increased with time.On the 14th and 21st day,the proteoglycan mRNA expression of ADSCs in the PRP ADSCs calcium alginate gel group was significantly higher than that in other groups(P<0.05).Conclusion The tissue engineered cartilage constructed by the complex formed by PRP,ADSCs and calcium alginate gel can improve the proliferation activity of ADSCs and the mRNA expression levels of collagen type II and proteoglycan,and the defective articular cartilage can be repaired significantly.