Objective: To construct a ultraviolet-cross-linkable chitosan-carbon dots-morin (NMCM) hydrogel, observe whether it can repair cartilage injury by in vivo and in vitro experiments, and explore the related mechanism.
Methods: The chitosan was taken to prepare the ultraviolet (UV)-cross-linkable chitosan by combining methacrylic anhydride, and the carbon dots by combining acrylamide. The two solutions were mixed and added morin solution. After UV irradiation, the NMCM hydrogel was obtained, and its sustained release performance was tested. Chondrocytes were separated from normal and knee osteoarticular (KOA) cartilage tissue donated by patients with joint replacement and identified by toluidine blue staining. The 3rd generation KOA chondrocytes were co-cultured with the morin solutions with concentrations of 12.5, 25.0, 50.0 µmol/L and NMCM hydrogel loaded with morin of the same concentrations, respectively. The effects of morin and NMCM hydrogel on the proliferation of chondrocytes were detected by cell counting kit 8 (CCK-8). After co-cultured with NMCM hydrogel loaded with 50 µmol/L morin, the level of collagen type Ⅱ (COL-Ⅱ) of KOA chondrocytes was detected by immunofluorescence staining, and the level of reactive oxygen species (ROS) was detected by 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe. Twenty 4-week old Sprague Dawley rats were selected to construct a articular cartilage injury of right hind limb model, and were randomly divided into two groups ( n=10). The cartilage injury of the experimental group was repaired with NMCM hydrogel loaded with 25 µmol/L morin, and the control group was not treated. At 4 weeks after operation, the repair of cartilage injury was observed by micro-CT and gross observation and scored by the International Cartilage Repair Association (ICRS) general scoring. The cartilage tissue and subchondral bone tissue were observed by Safranine-O-fast green staining and COL-Ⅱ immunohistochemistry staining and scored by ICRS histological scoring. The expressions of tumor necrosis factor α (TNF-α), nuclear factor κB (NK-κB), matrix metalloproteinase 13 (MMP-13), and COL-Ⅱ were detected by Western blot and real-time fluorescence quantitative PCR.
Results: NMCM hydrogels loaded with different concentrations of morin were successfully constructed. The drug release rate was fast in a short period of time, gradually slowed down after 24 hours, and the amount of drug release was close to 0 at 96 hours. At this time, the cumulative drug release rate reached 88%. Morin with a concentration ≤50 µmol/L had no toxic effect on chondrocytes, and the proliferation of chondrocytes improved under the intervention of NMCM hydrogel ( P<0.05). NMCM hydrogel loaded with morin could increase the level of COL-Ⅱ in KOA chondrocytes ( P<0.05) and reduce the level of ROS ( P<0.05), but it did not reach the normal level ( P<0.05). Animal experiments showed that in the experimental group, the articular surface was rough and the defects were visible at 4 weeks after operation, but the surrounding tissues were repaired and the joint space remained normal; in the control group, the articular surface was rougher, and no repair tissue was found for cartilage defects. Compared with the control group, the experimental group had more chondrocytes, increased COL-Ⅱ expression, and higher ICRS gross and histological scores ( P<0.05); the relative expressions of MMP-13, NF-κB, and TNF-α protein and mRNA significantly decreased ( P<0.05), and the relative expressions of COL-Ⅱ protein/COL-2a1 mRNA significantly increased ( P<0.05).
Conclusion: NMCM hydrogel can promote chondrocytes proliferation, down regulate chondrocyte catabolism, resist oxidative stress, protect chondrocytes from cartilage injury, and promote cartilage repair.
目的: 构建负载桑黄素的碳点紫外光交联壳聚糖（ultraviolet-cross-linkable chitosan-carbon dots-morin，NMCM）水凝胶，通过体内外实验观察其能否修复软骨损伤，并探讨相关机制。.
方法: 取壳聚糖联合甲基丙烯酰酐制备紫外光（ultraviolet，UV）交联壳聚糖、联合丙烯酰胺制备碳点，两者混合后加入桑黄素溶液，在UV下照射交联，获得NMCM水凝胶，检测其缓释性能。取关节置换患者自愿捐赠的正常及膝关节骨关节炎（knee osteoarthritis，KOA）软骨组织，分离培养软骨细胞并经甲苯胺蓝染色鉴定。取第3代KOA软骨细胞，首先分别与12.5、25.0、50.0 µmol/L桑黄素及负载该浓度桑黄素的NMCM水凝胶混合培养，细胞计数试剂盒 8（cell counting kit 8，CCK-8）法检测上述浓度桑黄素及水凝胶对软骨细胞增殖的影响；然后与负载50 µmol/L桑黄素的NMCM水凝胶共培养，免疫荧光染色检测Ⅱ型胶原蛋白（collagen type Ⅱ，COL-Ⅱ）水平、2，7-二氯二氢荧光素二乙酸酯探针检测活性氧水平。取4周龄SD大鼠20只，构建右后肢膝关节软骨损伤模型，随机分为两组（ n=10）；其中实验组软骨缺损处注射负载25 µmol/L桑黄素的NMCM水凝胶，对照组不作处理。术后4周，取标本micro-CT观察股骨髁软骨缺损修复情况，大体观察缺损部位并行国际软骨修复协会（ICRS）大体评分，番红-O固绿染色及COL-Ⅱ免疫组织化学染色观察软骨和软骨下骨并行ICRS组织学评分，Western blot以及实时荧光定量PCR检测软骨组织中TNF-α、NF-κB、基质金属蛋白酶13（matrix metalloproteinase 13，MMP-13）、COL-Ⅱ蛋白及mRNA表达。.
结果: 实验成功构建负载不同浓度桑黄素的NMCM水凝胶，其短时间内药物释放速度较快，24 h后速度逐渐放缓，96 h时释放量接近于0，此时药物累积释放率达88%。浓度≤50 µmol/L的桑黄素对软骨细胞无毒性作用，且在NMCM水凝胶干预下，软骨细胞增殖能力提高（ P<0.05）。NMCM水凝胶可提高KOA软骨细胞中COL-Ⅱ水平（ P<0.05），降低活性氧水平（ P<0.05），但尚未达正常水平（ P<0.05）。动物实验显示，实验组术后4周关节面粗糙，软骨缺损可见，但其周围有组织修复，关节间隙仍保持正常；对照组关节面更粗糙，软骨缺损未见组织修复。与对照组相比，实验组软骨细胞较多，COL-Ⅱ表达增强，ICRS大体及组织学评分均较高（ P<0.05）；MMP-13、NF-κB、TNF-α蛋白及mRNA相对表达量降低，COL-Ⅱ蛋白及COL-2a1 mRNA升高，差异均有统计学意义（ P<0.05）。.
结论: NMCM水凝胶能促进大鼠膝关节软骨细胞增殖、抑制软骨细胞分解代谢，抵抗氧化应激反应，保护软骨细胞，促进软骨修复。.
Keywords: Knee; carbon dots; chitosan hydrogel; morin; osteoarthritis; rat.