Objective: To explore the effect of chitosan (CS) hydrogel loaded with tendon-derived stem cells (TDSCs; hereinafter referred to as TDSCs/CS hydrogel) on tendon-to-bone healing after rotator cuff repair in rabbits.
Methods: TDSCs were isolated from the rotator cuff tissue of 3 adult New Zealand white rabbits by Henderson step-by-step enzymatic digestion method and identified by multidirectional differentiation and flow cytometry. The 3rd generation TDSCs were encapsulated in CS to construct TDSCs/CS hydrogel. The cell counting kit 8 (CCK-8) assay was used to detect the proliferation of TDSCs in the hydrogel after 1-5 days of culture in vitro, and cell compatibility of TDSCs/CS hydrogel was evaluated by using TDSCs alone as control. Another 36 adult New Zealand white rabbits were randomly divided into 3 groups ( n=12): rotator cuff repair group (control group), rotator cuff repair+CS hydrogel injection group (CS group), and rotator cuff repair+TDSCs/CS hydrogel injection group (TDSCs/CS group). After establishing the rotator cuff repair models, the corresponding hydrogel was injected into the tendon-to-bone interface in the CS group and TDSCs/CS group, and no other treatment was performed in the control group. The general condition of the animals was observed after operation. At 4 and 8 weeks, real-time quantitative PCR (qPCR) was used to detect the relative expressions of tendon forming related genes (tenomodulin, scleraxis), chondrogenesis related genes (aggrecan, sex determining region Y-related high mobility group-box gene 9), and osteogenesis related genes (alkaline phosphatase, Runt-related transcription factor 2) at the tendon-to-bone interface. At 8 weeks, HE and Masson staining were used to observe the histological changes, and the biomechanical test was used to evaluate the ultimate load and the failure site of the repaired rotator cuff to evaluate the tendon-to-bone healing and biomechanical properties.
Results: CCK-8 assay showed that the CS hydrogel could promote the proliferation of TDSCs ( P<0.05). qPCR results showed that the expressions of tendon-to-bone interface related genes were significantly higher in the TDSCs/CS group than in the CS group and control group at 4 and 8 weeks after operation ( P<0.05). Moreover, the expressions of tendon-to-bone interface related genes at 8 weeks after operation were significantly higher than those at 4 weeks after operation in the TDSCs/CS group ( P<0.05). Histological staining showed the clear cartilage tissue and dense and orderly collagen formation at the tendon-to-bone interface in the TDSCs/CS group. The results of semi-quantitative analysis showed that compared with the control group, the number of cells, the proportion of collagen fiber orientation, and the histological score in the TDSCs/CS group increased, the vascularity decreased, showing significant differences ( P<0.05); compared with the CS group, the proportion of collagen fiber orientation and the histological score in the TDSCs/CS group significantly increased ( P<0.05), while there was no significant difference in the number of cells and vascularity ( P>0.05). All samples in biomechanical testing failed at the repair site during the testing process. The ultimate load of the TDSCs/CS group was significantly higher than that of the control group ( P<0.05), but there was no significant difference compared to the CS group ( P>0.05).
Conclusion: TDSCs/CS hydrogel can induce cartilage regeneration to promote rotator cuff tendon-to-bone healing.
目的: 探讨以壳聚糖（chitosan，CS）负载肌腱干细胞（tendon-derived stem cells，TDSCs）构建的可注射型水凝胶（以下简称TDSCs/CS水凝胶）促进兔肩袖腱-骨愈合的效果。.
方法: 取3只成年新西兰大白兔肩袖组织，采用Henderson分步酶消化法分离培养TDSCs，并经多向分化及流式细胞术鉴定。以CS包裹第3代TDSCs构建TDSCs/CS水凝胶，体外培养1～5 d以细胞计数试剂盒8（cell counting kit 8，CCK-8）法检测水凝胶中TDSCs增殖情况，并以单纯TDSCs作为对照，评估TDSCs/CS水凝胶细胞相容性。另取36只成年新西兰大白兔，随机分为3组（ n=12），分别为肩袖修复组（对照组）、肩袖修复+CS水凝胶注射组（CS组）、肩袖修复+DSCs/CS水凝胶注射组（TDSCs/CS组）。3组建立肩袖损伤+单排技术修复模型后，CS组及TDSCs/CS组将对应水凝胶注入腱-骨界面处修复肩袖，对照组不作其他处理。术后观察动物一般情况，于4、8周取材，实时定量PCR（real-time quantitative PCR，qPCR）检测腱-骨界面成肌腱相关基因（腱调蛋白、转录因子），成软骨相关基因（蛋白聚糖、性别决定区Y框蛋白9），成骨相关基因（ALP、人Runt相关转录因子2）表达；另于8周取材行HE、Masson染色观察并行组织学半定量评分，生物力学测试修复肩袖极限载荷和失效部位，评价组织愈合情况和生物力学特性改变。.
结果: CCK-8法检测示共培养后CS水凝胶可促进TDSCs增殖（ P<0.05）。术后各组动物均存活至实验完成。术后4、8周，TSDCs/CS组的成肌腱、成软骨、成骨相关基因相对表达量均高于CS组和对照组（ P<0.05），TSDCs/CS组组内术后8周上述基因相对表达量亦高于术后4周（ P<0.05）。组织学染色示TDSCs/CS组腱-骨界面处有清晰的软骨组织和致密有序胶原形成，半定量分析结果示TDSCs/CS组与对照组相比，细胞数量、胶原纤维分布以及组织学评分增高（ P<0.05），血管数量降低（ P<0.05）；TDSCs/CS组与CS组相比，胶原纤维分布以及组织学评分均增高（ P<0.05），而细胞数量、血管数量差异无统计学意义（ P>0.05）。生物力学检测示所有样本测试过程中均在修复部位失效，TDSCs/CS组极限载荷高于对照组（ P<0.05），但与CS组比较差异无统计学意义（ P>0.05）。.
结论: TDSCs/CS水凝胶可以诱导兔肩袖肌腱和骨之间的软骨再生，促进腱-骨愈合。.
Keywords: Rotator cuff repair; chitosan; hydrogel; rabbit; tendon-derived stem cells; tendon-to-bone healing.