| ObjectiveTo investigate whether transplantation of human adipose stromal cells(hASCs)can enhance tendon-bone healing.MethodsUnilateral ACL reconstruction using autologous semitendinosus tendons were conducted on 48 New Zealand rabbits(weighing 2.5kg-3.0 kg),which were randomly divided into 2 groups.The experimental group were injected hASCs in the tibial and femoral bone tunnels while the control group were injected with equal normal saline.All experimental animal were sacrificed at 2 weeks,4 weeks and 8 weeks after surgery.The specimens were harvested for histological analysis and biomechanical analysis.Statistical analysisThe results are expressed as the mean ± SD and Statistical analysis was done using Statistical Package for Social Science(SPSS)20.0.Comparisons among groups were performed by two-independent sample t-test.Differences were considered as statistically significant when the p value was less than 0.05.Results1.Identification of human adipose-derived stem cellsFlow cytometric results showed that hASCs were positive for CD90(99.5%),CD51(96.8%),CD105(91.1%),and negative for CD34(10.6%),CD45(10.7%),CD49e(12.2%).2.The results of ALP stainingThe results of alkaline phosphatase staining showed that the positive staining of the experimental group was more obvious than that of the control group.Semi-quantitative analysis of the stained images was performed by Image-pro plus 6.0.The semi-quantitative results showed that the IOD/Area in experimental group was significantly higher than that of the control group and there was significant difference between erperimental group and control group.(P = 0.0257).3.The results of qRT-PCR and related osteogenic gene expressionCompared with control group,the OCN expression was not significant.The expression of OPN gene was 6.8 fold higher than that on the control group(p=0.018).The expression of RUNX2 gene was 2.6 fold higher than that on the control group(p=0.015).ALP was 3.1 fold higher than that on the control group(P=0.019).Collagen-1 was 4.1 fold higher than that on the control group(P=0.043).4.Histological analysis4.1 The results of HE stainingThe results of HE staining showed that inflammatory cells were mainly infiltrated at 2 weeks postoperatively.Inflammatory cells decreased at 4 weeks postoperatively,and connective tissue mainly formed by fibrous connective tissue on the tendon bone interface at.8 weeks postoperatively.There were few fibrochondrocytes and osteocytes in the experimental group at 4 weeks postoperatively.New bone formation occurred at 8 weeks postoperatively,and a direct healing at the tendon bone interface was formed.4.2 Masson’s trichrome staining resultsMasson’s trichrome staining showed that the inflammatory cells infiltrated mainly in the experimental group and the control group at 2 weeks.In the experimental group,fibrous chondrocytes and fibrous connective tissue were formed at 4 weeks postoperatively.At 8 weeks postoperatively,new bone and dense connective tissue were formed at the tendon bone interface,while the control group had no new bone formation.5.Biomechanical analysisFrom the biomechanical data,it was found that the maximal failure load in the experimental group was higher than that of control group,but there was no significant difference at 2 weeks after operation(21.02±8.30 N vs 12.36±7.67N,p=0.09).At 4 weeks(87.52=10.29N vs 14.76±4.10N,P<0.01)and 8 weeks(126.31±12.85 N vs 47.0±12.81N,P<0.01)after operation,the maximal failure load in the experimental group were higher than that of control group and there were significant differences between erperimental group and control group.The stiffness of the graft tendon in the experimental group were higher than that of control group but there were no significant differences between experimental group and control group at 2 weeks(9.76±5.07N/mm vs 7.85±4.11N/mm,P=0.49)and 8 weeks(37.18±5.62N/mm vs 30.28±23.86 N/mm,P=0.51)after operation.The stiffness of the experimental group was significantly higher than that of the control group at 4 weeks after operation and there was significant differences between erperimental group and control group.(27.80±7.24N/mm vs 8.98±4.12,P<0.01)Conclusions:1.hASCs can differentiate into osteoblasts in vitro osteogenic induction environment,and have osteogenic ability.2.Histological staining showed that hASCs were able to promote ossification of the tendon-bone interface,which facilitated the direct migration of the tendon-bone interface.3.Transplantation of osteoblast-derived human adipose-derived stem cells at the tendon-bone interface enhances the biomechanical properties of the tendon-bone interface and has potential for clinical applications. |
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