阿仑膦酸钠体外对软骨细胞的影响及对兔前交叉韧带切断后关节软骨和软骨下骨的作用

IN VITRO EFFECT OF ALENDRONATE ON CHONDROCYTES AND ARTICULAR CARTILAGE AND SUBCHONDRAL BONE IN RABBIT ANTERIOR CRUCIATE LIGAMENT TRANSECTION MODEL

目的探讨阿仑膦酸钠(alendronate,ALN)对受IL-1β干预后软骨细胞的影响,以及对前交叉韧带切断术(anterior cruciate ligament transection,ACLT)后兔骨性关节炎模型关节软骨和软骨下骨的保护作用。方法取3月龄雄性日本大耳白兔关节软骨采用酶消化法分离软骨细胞,并进行体外培养。取第3代软骨细胞随机分为3组:ALN组(A1组)及IL-1β组(B1组),采用含10ng/mL人重组IL-1β的DMEM完全培养液培养2d后,分别更换含或不含1×10-6mol/L ALN的DMEM完全培养液培养3d;空白组(C1组)细胞以DMEM完全培养液培养5d。取各组细胞行ColⅡ及MMP-13免疫细胞化学染色观察及实时RT-PCR检测。另取24只3月龄雄性日本大耳白兔,随机分为3组(n=8)。ACLT+ALN组(A2组)及ACLT组(B2组)制备ACLT模型,假手术组(C2组)以同法显露前交叉韧带后缝合切口。术后4d A2组以10μg/(kg·d)皮下注射浓度为25μg/mL的ALN,B2组和C2组予等量生理盐水。8周后处死动物行膝关节大体观察,取股骨内侧髁行组织学观察及ColⅡ及MMP-13免疫组织化学染色观察,对胫骨进行软骨下骨组织形态学分析。结果C1组软骨细胞胞浆中见ColⅡ免疫细胞化学染色表达呈强阳性,A1组呈阳性表达,B1组少见阳性显色。A1组积分吸光度(IA)值高于B1组(P<0.05),与C1组差异无统计学(P>0.05)。C1组软骨细胞胞浆中未见MMP-13免疫细胞化学染色阳性表达,A1组呈阳性表达,B1组呈强阳性表达。A1组IA值低于B1组(P<0.05),但高于C1组(P<0.05)。实时RT-PCR检测示A1组ColⅡmRNA表达高于B1组(P<0.05),与C1组差异无统计学意义(P>0.05);MMP-13mRNA表达低于B1组(P<0.05),但高于C1组(P<0.05)。体内实验8周后C2组膝关节面无溃疡,A2组膝关节面有少量溃疡,B2组关节面溃疡较多并有全层溃疡。A2组Mankin评分低于B2组(P<0.05),但高于C2组(P<0.05)。免疫组织化学染色见C2组关节软骨中ColⅡ蛋白表达呈强阳性,A2组呈阳性表达,B2组少见阳性显色;A2组IA值高于B2组(P<0.05),与C2组差异无统计学意义(P>0.05)。C2组关节软骨中未见MMP-13阳性表达,A2组呈弱阳性表达,B2组呈强阳性表达;A2组IA值低于B2组(P<0.05),但高于C2组(P<0.05)。骨形态计量学结果示:B2组软骨下骨骨小梁体积比、骨小梁厚度低于A2、C2组(P<0.05),骨小梁分离度、荧光百分率和骨形成率高于A2、C2组(P<0.05)。以上指数A2组与C2组差异均无统计学意义(P>0.05)。各组间骨小梁数目、矿化沉积率差异均无统计学意义(P>0.05)。结论ALN能抑制ACLT兔关节软骨降解,保护软骨下骨骨量和微观结构。在体内、体外ALN均能抑制MMP-13在软骨细胞中的表达,具有一定的软骨细胞保护功能。

Objective To examine the effects of alendronate （ALN） on IL-1β-stimulated chondrocyte of rabbit in vitro and on cartilage and subchondral bone in rabbit osteoarthritis （OA） induced by anterior cruciate ligament transection （ACLT）. Methods The chondrocytes from articular surface of healthy 3-month-old Japanese White rabbits were obtained by the method of enzyme digestion and cultured in vitro. The third generation chondrocytes were assigned into three groups：the chondrocytes were cultured in DMEM medium with 10 ng/mL IL-1β for 2 days,subsequently with （ALN group,group A1） or without （IL-1β group,group B1） 1 × 10-6 mol/L ALN for 3 days; the chondrocytes in vacant group （group C1） were cultured in DMEM medium for 5 days. The expression of Col II and MMP-13 were analyzed by immunocytochemical staining observation and real time RT-PCR test. Another twenty-four 3-month-old male Japanese White rabbits were randomized into three groups （n=8 per group）. The OA model was made by ACLT in ACLT＋ALN group （group A2） and ACLT group （group B2）; the joint cave was sutured after exposure of ACL in sham group （group C2）. After 4 days,the rabbits of group A2 received the subcutaneous injection of ALN at a dosage of 10 μg/（kg·d） for 8 weeks. Rabbits of group B2 and C2 received equal normal saline treatment. After 8 weeks,the rabbits were executed. The macro-pathologic changes of right knee joints were observed,so were the histological changes of femoral condyles. Expression levels of Col II and MMP-13 were detected by immunohistochemical staining. The bone histomorphometry analysis was applied to subchondral bone of proximal tibia. Results In vitro,the Col II immunocytochemical staining showed intensely positive staining in group C1,and the intensity of staining was slightly decreased in group A1,but the intensity of Col II immunocytochemical staining was extremely lower in the group B1. The integrated absorbance （IA） value for Col II in group A1 was significantly higher than that of group B1 （P 〈 0.05）,but there was no significant difference between group A1 and group C1 （P 〉 0.05）. Immunocytochemical detection of MMP-13 showed intense staining in group B1,and the intensity of staining was slightly decreased in group A1,but no MMP-13 expression was detected in the group C1. The IA value for MMP-13 in group A1 was significantly lower than that of group B1 （P 〈 0.05）,but significantly higher than that of group C1 （P 〈 0.05）. The real time RT-PCR analysis showed significantly higher mRNA levels of Col II in group A1 than in group B1 （P 〈 0.05）,but there was no significant difference between group A1 and group C1 （P 〉 0.05）. The MMP-13 mRNA level of the chondrocytes in group A1 was significantly lower than that of group B1 （P 〈 0.05）,but significantly higher than that of group C1 （P 〈 0.05）. In vivo,the gross appearance of surface of knee joint showed that there was no ulcer in group C2,and there was some ulcers in group A2,but many and all layers ulcers in group B2. Mankin score of group A2 was significantly lower than that of group B2 （P 〈 0.05）,but significantly higher than that of group C2 （P 〈 0.05）. Immunohistochemical staining showed that Col II in articular cartilage was intensely staining in group C2,the intensity of staining was slightly decreased in group A2,and the intensity of Col II immunohistochemical staining was extremely low in group B2,but there was no significant difference between group A2 and group C2 （P 〉 0.05）. The immunohistochemical staining for MMP-13 significantly increased in group B2,the intensity of staining was slightly decreased in group A2,and no MMP-13 expression was detectable in the group C2. The IA value for MMP-13 in group A2 was significantly lower than that of group B2 （P 〈 0.05）,but significant higher than that of group C2 （P 〈 0.05）. Bone histomorphometry showed that in group B2 percent trabecular area and trabecular thickness markedly decreased compared with those in group A2 and group C2 （P 〈 0.05）,but there was no significant difference between group A2 and group C2 （P 〉 0.05）. The trabecular separation,percent labeled perimeter and bone formation rate were significantly elevated in group B2 compared with those in group A1 and group C2 （P 〈 0.05）,but there was no significant difference between group A2 and group C2 （P 〉 0.05）. No significant difference was evident on trabecular number and mineral apposition rate values among groups A2,B2,and C2 （P 〉 0.05）. Conclusion For rabbits OA induced by ACLT,the subcutaneou injections of alendronate can inhibit cartilage degradation,prevent bone loss,and improve microarchitechture of subchondral bone. ALN can partially protect chondrocytes by inhibiting the expression of MMP-13 both in vitro and in vivo.