犬骨关节炎软骨下骨重塑的病理特点及机制

Pathological characteristics and mechanism of subchondral bone remodeling in canine osteoarthritis

目的:通过构建比格犬膝关节前交叉韧带横断(ACLT)骨关节炎模型探究关节软骨下骨重塑的病理特点及发病机制。方法:本研究选取8周岁,雄性,体重10~15 kg的比格犬,将18只犬按随机数字表分为两组,即对照组和骨关节炎(OA)模型组。通过离断犬膝关节前交叉韧带构建关节不稳的OA模型,术后16周后处以安乐死,取材。通过番红固绿染色和国际骨关节炎研究学会-改良Manking(OARSI-Modified Manking)评分评估关节软骨退变程度。采用微计算机断层扫描(Micro-CT)分析软骨下骨的骨体积分数(BV/TV)、骨小梁分离度(SP.Tb)、骨小梁模式因子(Tb.Pf),通过经免疫染色测定软骨下骨中pSmad2/3,Osterix和CD31的表达水平。采用独立样本t检验进行数据的统计学分析。结果:OA模型组肉眼可见多处不同程度的关节软骨退变,番红固绿染色显示关节软骨龟裂,但尚未侵犯深层结构,OARSI-Modified Manking评分显著升高(4.7±2.3),差异有统计学意义(t=-3.213,P<0.01)。Micro-CT结果显示,与对照组比较,OA模型组的软骨下骨BV/TV显著高于对照组(0.4±0.3),而SP.Tb和Tb.Pf均显著低于对照组(0.3±0.1、-2.0±3.5),且组间差异均有统计学意义(t=-6.124、2.519、3.393,P<0.05)。酶联免疫吸附试验(ELISA)结果表明,与对照组比较,OA模型组的外周血中含有高浓度的转化生长因子(TGF)-β1(81.7±5.1),且组间差异有统计学意义(t=-16.580,P<0.01)。免疫组织化学染色结果显示,与对照组比较,OA模型组的关节软骨下骨髓腔中聚集了大量的pSmad2/3+MSC、Osterix+成骨前体细胞和CD31+血管内皮细胞(109.9±3.9、70.5±1.8、26.8±1.8),且组间差异有统计学意义(t=-29.814、-42.475、-8.937,P<0.05)。结论:比格犬膝关节OA的软骨下骨中出现异常骨重塑,其中活跃的骨吸收和TGF-β1/Smad2/3通路是疾病发生与发展中的关键靶点。

Objective To investigate the pathological features and pathogenesis of articular subchondral bone remodeling through constructing an anterior cruciate ligament transection(ACLT)osteoarthritis(OA)model in Beagle dogs.Methods The total of 18 male beagles weighing 10-15 kg were selected,and divided into two groups according to a random number table:a control group and an OA model group.The OA model of joint instability was constructed by ACLT of the knee in dogs.All dogs were euthanized at 16 weeks postoperatively and materials were obtained.The degree of articular cartilage degeneration was evaluated by Safranin O-Fast Green staining and OARSI-Modified Manking score.The bone volume fraction(BV/TV),trabecular bone separation(SP.Tb),and trabecular pattern factor(Tb.Pf)of subchondral bone were analyzed by Micro-CT.The pSmad2/3,osterix and CD31 expression levels in subchondral bone were detected by immunostaining.Independent sample t test was used for statistical analysis of data.Results In the OA model group,multiple articular cartilage degenerations were visible.Safranin O-Fast Green staining showed articular cartilage cracking,but the deep structure was not violated.The OARSI-Modified Manking score was significantly increased(4.7±2.3,t=-3.213,P<0.01).Micro-CT results showed that as compared with the control group,the subchondral bone BV/TV in the OA model group was significantly increased(0.4±0.3),while the SP.Tb and Tb.Pf were significantly reduced(0.3±0.1,-2.0±3.5),and the differences between two groups were statistically significant(t=-6.124,2.519,3.393,P<0.05).ELISA results showed that as compared with the control group,concentrations of TGF-β1 in the peripheral blood in the OA model group were significantly increased(81.7±5.1,t=-16.580,P<0.01).Immunohistochemical staining showed that as compared with the control group,the number of pSmad2/3+MSC,osterix+osteoblast precursor cells and CD31+vascular endothelial cells(109.9±3.9,70.5±1.8,26.8±1.8)were significantly increased in the OA model group(t=-29.814,-42.475,-8.937,P<0.01).Conclusion Abnormal bone remodeling occurs in the knee subchondral bone of Beagle dogs with OA,in which active bone resorption and the TGF-β1/Smad2/3 pathway are key targets in the occurrence and development of the disease.