| Objective:To study the effect of adipose-derived stem cells?Adipose-Derived Stem Cells,ADSCs?in combination with sustained-release transforming growth factor-beta1,TGF-?1)-based hydroxyapatite?Hydroxyapatite,HA?nanospheres scaffolds for constructing ADSCs-HA nano-microsphere complex to repair traumatic cartilage defects in rabbit knee joints.Methods:?1?HA nanospheres containing cytokine TGF-?1were prepared and their morphology was analyzed by electron microscope.The slow release concentration and duration of the TGF-?1 were detected by immersion experiments in simulated body fluids.?2?Adipose tissue was extracted ADSCs,subcultured from three-month-old New Zealand to observe cell morphology and flow cytometry identification.?3?HA nanospheres were attached to absorbable gelatin sponge to construct HA nanospheres scaffolds.The third generation ADSCs were inoculated with 1×105 cells/mL concentrations to co-culture the nanospheres scaffolds to construct ADSCs-HA nanospheres complexes for MTT detection and electron microscopy observation.?4?The animal model of traumatic cartilage defect in rabbit knee joint was made by using 18 new zealand white female rabbits of 3months old.The diameter of the femoral intercondylar trochlear was 4.5 mm,and the depth3 mm cylindrical cartilage defects.A random average of 18 New Zealand rabbits were divided into 3 groups:A?ADSCs-HA nanospheres composite implanted at cartilage defect?;B?nanospheres scaffold implanted at cartilage defect?;and C?blank control,cartilage defect without any treatment?.?5?The repair effect of cartilage defect in rabbit knee joint was detected 12 weeks after operation.The texture of repair tissue and the binding degree of surrounding tissue and basal part were observed by gross observation.Tissue sections of repair area of knee cartilage defect were taken,collagen II was observed by immunohistochemical staining,glucosamine was observed by toluidine blue staining,the expression of collagen II was detected by Western Blot method,and the repair effect of cartilage defect was evaluated by Wakitani score.Results:?1?The HA nanospheres containing TGF-?1cytokines were successfully prepared.The microspheres showed a spherical shape with an average particle size of about8.85?m±3.48?m,and the microspheres were easily aggregated and had adhesion.The sustained slow release within TGF-?1 microspheres was obtained by ELISA detection for29 days to maintain effective concentrations for 27 days.?2?The adipose-derived stem cells were isolated from the subcutaneous fat of rabbit's inguinal sulcus and subcultured.the expression CD90,CD29 cell surface antigen was 59.74%,42.90%,and the CD45,CD14expression was 1.27%and 0.86%,respectively.?3?HA nano-microsphere scaffolds were cultured ADSCs 3 days.Electron microscopy showed that the HA microspheres scattered in the scaffold and the cells attached to grow.MTT detection indicated that HA growth and proliferation of ADSCs in the scaffold of nano-microspheres were good.?4?In 18 cases,there was no infection in the incision,no abscission of the graft,normal activity and diet.Twelve weeks later group C showed a small amount of fibrous tissue growth,no chondrocyte filling;group B repair tissue was loose,toluidine blue staining positive,type II collagen immunohistochemistry staining positive,margin and normal cartilage tissue boundary clear,very few chondrocyte structure;group A repair histogen scaffold has been absorbed,repair tissue and normal cartilage tissue boundary is clear,repair tissue methylaniline blue staining positive,type II collagen immunohistochemistry staining positive,can see chondrocyte arrangement disorder.Statistical analysis of Western Blot tests and Wakitani Scores:The relative gray values of II collagen were detected were0.437±0.103,0.325±0.076,0.175±0.045,respectively.The relative gray value difference of II collagen between the three groups was statistically significant?P<0.05?,among which the difference between group A and C,group B and C was more significant?P<0.01?.Wakitani score of these groups was 5.50±2.59,8.83±2.79,11.50±1.05,respectively.There was no significant difference between the Wakitani Score of group B and C?P>0.05?.There were significant differences between A and B,A and C groups?P<0.05?.Conclusion:The TGF-?1 in vitro sustained-release effect of HA nanospheres was good in this experiment.Construction of HA nano-microspheres/gelatin sponge composite scaffolds in the treatment of cartilage defects has a promising prospect.ADSCs-HA nano-microsphere complexes can differentiate into chondrocytes and provide theoretical basis for tissue engineering repair of cartilage defects. |
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