透明质酸促进撞击性膝关节软骨损伤修复的实验研究

Repairing effects of hyaluronate on cartilage impact injury of the knee

目的 探讨关节内注射透明质酸（HA）促进撞击性膝关节软骨损伤修复的作用和机理.方法 选取20只新西兰兔,通过手术建立双侧膝关节股骨内髁撞击损伤模型,左膝关节术后每周关节内注射HA 0.5 mL（治疗组）,右膝关节术后注射等量生理盐水（对照组）.伤后8周采用组织学和免疫组织化学染色方法观察软骨中细胞层次结构、形态和Ⅱ型胶原的表达,并用Tunel法观察软骨及软骨下骨中细胞凋亡的发生情况.应用酶联免疫吸附试验法测定关节液中Ⅱ型胶原、黏多糖（GAG）和肿瘤坏死因子-α的含量.结果 术后8周,光学显微镜下对照组浅层局部可见少量增殖成簇群状的软骨细胞;治疗组浅层簇群状增殖的软骨细胞数量增加,但增殖细胞未按正常浅层软骨细胞方向排列,没有向损伤处趋化、移动.术后8周,对照组和治疗组关节软骨中Ⅱ型胶原阳性细胞数平均分别为（26±8）、（183 ±37）个/mm^2,凋亡细胞数平均分别为（137 ±26）、（14±6）个/mm^2,两组比较差异均有统计学意义（P＜0.05）.术后8周,治疗组关节液中Ⅱ型胶原和GAG含量较对照组增加,肿瘤坏死因子-α含量较对照组减少,差异均有统计学意义（P＜0.05）.结论 关节内注射HA可以增加Ⅱ型胶原和GAG的分泌,减少细胞凋亡,具有稳定软骨的作用.增生的软骨细胞向损伤处趋化能力有限,影响软骨损伤的直接修复.

Objective To investigate the role and mechanism of hyaluronate （HA） repairing impact injury to the knee articular cartilage.Methods We established animal models of impact injury to the knee cartilage on both femoral condyles in a total of 20 rabbits.Immediately and every week after surgery,each left knee joint received an intra-articular injection of 0.5 mL HA （treatment group） while an equal volume of physiological saline was injected into each right knee joint （control group）.At 8 weeks after the surgery,type Ⅱ collagen （Col-Ⅱ）,glycosaminoglycan （GAG） and tumor necrosis factor-α （TNF-α） levels in the joint fluid were determined using enzyme linked immunosor bent assay.The cellular organization in the zone of cartilage was histologically observed with microscopy.The expression of Col-Ⅱ in femoral condyles was also analyzed by immunohistochemistry.In addition,cells that underwent apoptosis in cartilage and subchondral bone were detected by Tunel assay.Results Few proliferating cells were found in the superficial and intermediate zones of cartilage in the control group at 8 weeks.The number of proliferating cartilage cells was significantly increased in the treatment group.However,the growing cartilage cells did not organize normally and failed to migrate to the injured area.Immunohistochemistry staining showed that the control group had 26 ±8/mm^2 of Col-Ⅱ positive cells and 137 ± 26/mm^2 of the apoptotic cells in subchondral bone while the treatment group had 183 ±37/mm^2 and 14 ±6/mm^2 respectively,showing significant differences between groups （P 〈0.05）.On the other hand,Col-Ⅱ and GAG levels in the articular fluid were significantly increased and TNF-α level was decreased in the treatment group compared to the control group at 8 weeks （P 〈 0.05）.Conclusions Intra-articular injection of HA may stable the articular cartilage after impact injury by increasing the secretion of Col-Ⅱ and GAG and reducing apoptotic cells.HA treatment may increase the proliferative cells but fail to repair the damaged cartilage probably due to limited capacity of the proliferative cells migrating to the injured area.