| Objective:The experiment aims to study the effect of SVF on angiogenesis of granular cartilage and its possible mechanism by using rabbit fat-derived SVF mixed with rabbit ear granule cartilage autotopic transplantation to observe the tissue structure,blood vessel number and growth factor expression of grafts.Methods: Firstly,24 New Zealand rabbits were surgically cut to obtain bilateral inguinal adipose tissue,and the vascular matrix components(SVF)were isolated by enzymatic digestion.reserve part of fresh extracted SVF spare.Adipose stem cells and endothelial cells in vascular matrix components were identified by flow cytometry;then,the unilateral ear cartilage of New Zealand rabbit was cut to make about 1.0 mm3 granular cartilage.Design control group(granular cartilage complete medium)and experimental group(granular cartilage SVF).The rabbits were randomly sacrificed and sampled at 4 weeks,12 weeks and 24 weeks after operation to observe the gross specimen of granular cartilage regeneration and compare with wet weight.Histological staining was used to observe chondrocyte activity and microvessel growth. Immunohistochemistry was used to observe level of vascular endothelial cell markers CD31;fluorescence imprinting PCR detection in the transplanted body.Results:(1)Taking the fresh SVFs for flow cytometry detection.CD45CD31 was added to the CD90C73 as a positive marker.CD90C73 expression was high and CD45CD31 expression was low.Added CD34CD31 positive markers,CD34CD31 high expression;(2)Weight of the plant at 4,12,and 24 weeks after operation.:The weight of the 4weeks,12 weeks and 24 weeks of the control group was(2.080±0.224)g?(1.990± 0.205)g?(1.995± 0.192)g.The weight of the plant was(2.141±0.257)g?(2.215±0.203)g?(2.210±0.205).At 4 weeks,there was no significant difference in wet weight between the two groups(P>0.05).and the weight of the experimental group at 12 and 24 weeks was significantly higher than that of the control group(P<0.05).weight of each control group was not significantly changed than before operation(P>0.05).There was no significant difference in weight between the four-week experimental group and the preoperative weight(P>0.05).HE staining showed that the control group: a small number of new chondrocytes were seen in the granular cartilage graft at 4 weeks under inverted microscope,and a small number of cartilage apoptosis and inflammatory reaction were seen in the central area,and no obvious vascular growth was seen.At 12 weeks and 24 weeks,a small amount of cartilage proliferation,fewer layers of chondrocytes than the experimental group,less vascular distribution;experimental group :4 weeks under the inverted microscope chondrocyte proliferation is more active,more layers of chondrocytes than the control group,a small number of inflammatory cells can be seen,a small number of vascular formation around the cartilage.At 12 weeks and 24 weeks,a large number of new chondrocytes were observed,the volume gradually increased,the inflammatory cells decreased,and more new blood vessels were seen.The number of CD31 blood vessels in the control group was significantly lower than that in the experimental group at 4,12±24 weeks after operation :[(35.27)/(55.42± 2.15),(41.80)/(67.32±2.21),(57.55)/(1.89)/(71.12± 1.87)](all P<0.05).last,according to the detection RT-PCR and WB of the HIF-1? ? VEGFm RNA level and product expression level: according to the detection RT-PCR,it can be seen that the expression of m RNA in the experimental group and the experimental group is improved.the m RNA level was still higher than that of the control group at the beginning of 12 weeks after operation(P <0.05);the relative expression level of the experimental group was higher at 4,12 and 24 weeks after operation than that of the control group(P<0.05).Conclusion:Autologous adipose tissue SVF combined with granular cartilage injection transplantation can promote the angiogenesis of granular cartilage.SVF can improve the VEGF and HIF-1? expression level of the composite graft,which may be part of the mechanism of vascular matrix components promoting granular cartilage angiogenesis;... |
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