透明质酸促进撞击性膝关节软骨损伤修复的生物学机理研究

Biological Mechanism and Repair Effects of Hyaluronate on Impact Knee Articular Cartilage Injury

直接或间接撞击是引起膝关节软骨损伤的常见诱因,关节软骨本身没有血供,基质陷窝中的软骨细胞代谢活性低,损伤后难以自我修复.透明质酸是软骨基质和关节液的重要组成成分,是促进软骨修复的一个有希望的载体与支架.本实验通过手术建立兔膝关节股骨內髁撞击损伤模型,术后每周关节内注射透明质酸0.5ml,伤后8 周收集标本,关节液用ELISA 法做Ⅱ型胶原、粘多糖和肿瘤坏死因子TNF-α含量测定.用组织学和免疫组织化学观察股骨內髁软骨中细胞层次结构、形态和Ⅱ型胶原表达,并用Tunel 法观察了软骨及软骨下骨中凋亡细胞的发生率.结果直接撞击可引起股骨內髁软骨面的损伤,组织学上表现为从浅表层到软骨下骨深浅不一的微裂纹、塌陷或缺损.ELISA 分析显示撞击后8 周关节液中Ⅱ型胶原和粘多糖含量减少,肿瘤坏死因子TNF-α含量增加.透明质酸可增加关节液中Ⅱ型胶原和粘多糖含量并降低TNF-α含量.免疫组化显示透明质酸治疗组关节软骨中Ⅱ型胶原阳性细胞数量增加;软骨下骨层凋亡细胞数减少.透明质酸诱导软骨细胞集簇性增生,但增生的软骨细胞未能按正常组织结构排列,未能向损伤处趋化;从而影响软骨损伤的直接修复能力,裂纹和缺损的软骨层未见明显的修复.结论：关节内注射透明质酸可增加Ⅱ型胶原和粘多糖的分泌;降低凋亡细胞数量,显示有稳定软骨的作用.但增生的软骨细胞未能向损伤处趋化, 从而未能直接修复损伤的软骨;如何联合利用透明质酸,干细胞和骨髓刺激促进软骨的修复需进一步研究.

Direct or indirect impacts are the common cause of knees articular cartilage injury. Articular cartilage has only limited capacity for regeneration due to lack of bloodsupply and low metabolic activity of chondrocytes in cartilage matrix lacuna. Hyaluronate（HA） is one of the important compounds of extracellular matrix and joint fluid. It’s apromising scaffold to promote cartilage repair. In this study, we established a rabbit animal model of impact cartilage injury on femoral condyle. 0.5ml HA was intra-articular injectedimmediately and every week after surgery. Joint fluid and femoral condyle cartilage were harvested at 8 weeks after the surgery for Elisa, histology and immunohistochemistryexamination, respectively. COL-Ⅱ, GAG and TNF-α levels in joint fluid were determined by ELISA. The cellular organization in the zone of cartilage was histologically observedwith microscope. The expression of COL-Ⅱin femoral condyle were also analyzed by immunohistochemistry stain. In addition, cells underwent apoptosis in cartilage and subcondralbone were detected by TUNEL assay. Results direct impact can cause articular cartilage injury, including micro-cracks, collapses or defects from articular surface to subchondral bone.ELISA results indicated that COL-Ⅱ and GAG levels were decreased, and TNF-α level was increased at 8 weeks after impact injury. Intra-articular injection of HA significantlyincreased COL-Ⅱ and GAG levels and decreased TNF-α level in joint fluid. immunohistochemistry stain showed that the number of COL-Ⅱ positive cells was increased in HA treatedgroup, while apoptotic cells in subchondral bone were reduced. At the superficial and intermediate zone, increased cluster of cartilage cells founded in HA treated rabbits. However, thecracks and defects were not repaired. Conclusion： 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. However, HA failed to Induce direct cartilage regeneration and restoration probably due to lack of blood supply, limited chemotaxis and migration ofchondrocytes. Further studies are needed to clarify whether combination of HA with marrow stimulation or stem cells can improve its effeciency for cartilage repair.