透明质酸钠与软骨下钻孔对兔软骨损伤修复的对比试验
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摘要
试验通过建立兔部分软骨损伤模型,采用外源性注射透明质酸钠和软骨下钻孔两种治疗方法,比较两种方法的修复效果及早期修复机制,为临床应用提供试验依据。试验选用36只哈白兔建立膝关节软骨损伤模型后随机均分为3组:自愈组(阴性对照)、用药组(透明质酸钠注射)、钻孔组(软骨下钻孔)。术后1、4、6、8周,采用RTPCR方法检测修复物中Ⅱ型胶原(ColⅡ)、蛋白聚糖(Agg)、Ⅹ型胶原(ColⅩ)、Ⅰ型胶原(ColⅠ)及Runx2的mRNA表达量;术后8周,制作软骨切片进行HE染色、番红—固绿染色和甲苯胺蓝染色,分析3组的修复效果及修复机制。结果显示,两试验组软骨损伤都被明显修复,染色结果显示修复组织中软骨细胞增多,具有软骨典型的陷窝样结构,用药组阳性着色优于钻孔组。用药组软骨的特异性基因Agg和ColⅡ的mRNA表达量显著高于钻孔组(P<0.05),软骨细胞肥大相关基因ColⅩ和Runx2的表达量显著低于钻孔组(P<0.05)。自愈组ColⅠ的mRNA表达量最高,钻孔组次之,用药组最低。试验结果表明,用药及钻孔对关节软骨损伤都有不同程度的治疗修复作用,自愈组修复物成分主要为纤维组织,透明质酸钠治疗后产生了较多的透明软骨成分,而钻孔修复后的表层覆盖薄层纤维软骨成分,在骨缺损短期修复中,透明质酸钠的修复效果优于钻孔疗法。
The rabbit models of osteochondral defects were established and two methods of treatment with external injection of sodium hyaluronate and subchondral bone drilling were adopted,the repair effect and early repair mechanism of these two methods were compared to provide experimental basis for clinical application.The models of articular cartilage injury in 36 Harbin White rabbits were set up and randomly allocated to three groups:Self-healing group(negative control),medication group(sodium hyaluronate injection)and drilling group(subchondral bone drilling).Rabbits from each group were sacrificed 1,4,6 and 8 weeks after operation,RT-PCR method was adopted to detect ColⅠ,ColⅡ,Col Ⅹ,Agg,and Runx2 genes expression of repaired articular cartilage tissues;Postoperative 8 weeks,cartilage specimen were observed with HE staining,safranin green staining and toluidine blue staining to analyze the repair effect and repair mechanism of the three groups.The results showed that the cartilage damage of the two experimental groups were significantly repaired,staining showed that the chondrocytes in repaired tissues increased with typical cartilage lacuna like structure,the positive staining of medi-cation group was better than drilling group.The expression of cartilage specificity genes Aggand ColⅡ mRNA in medication group was significatly higher than that in drilling group(P<0.05).The expression of ColⅩ and Runx2 of drilling group were more active than medication group(P<0.05).The expression of major components of fibrous tissue,ColⅠ,self-healing group was the highest,then in drilling group,medication group was the lowest.The results showed that medication and drilling could repair the defects of the whole layer cartilage defects in various degrees.The main component of the restorations was fibrous tissue in self-healing group,more hyaline cartilage were produced by treated with hyaluronic acid and a thin layer of fibrous cartilage covering the surface of repair tissues in drilling group.In conclusion,in the short-term repair of bone defect,the effect of sodium hyaluronate was better than that of drilling.
The rabbit models of osteochondral defects were established and two methods of treatment with external injection of sodium hyaluronate and subchondral bone drilling were adopted,the repair effect and early repair mechanism of these two methods were compared to provide experimental basis for clinical application.The models of articular cartilage injury in 36 Harbin White rabbits were set up and randomly allocated to three groups:Self-healing group(negative control),medication group(sodium hyaluronate injection)and drilling group(subchondral bone drilling).Rabbits from each group were sacrificed 1,4,6 and 8 weeks after operation,RT-PCR method was adopted to detect ColⅠ,ColⅡ,Col Ⅹ,Agg,and Runx2 genes expression of repaired articular cartilage tissues;Postoperative 8 weeks,cartilage specimen were observed with HE staining,safranin green staining and toluidine blue staining to analyze the repair effect and repair mechanism of the three groups.The results showed that the cartilage damage of the two experimental groups were significantly repaired,staining showed that the chondrocytes in repaired tissues increased with typical cartilage lacuna like structure,the positive staining of medi-cation group was better than drilling group.The expression of cartilage specificity genes Aggand ColⅡ mRNA in medication group was significatly higher than that in drilling group(P<0.05).The expression of ColⅩ and Runx2 of drilling group were more active than medication group(P<0.05).The expression of major components of fibrous tissue,ColⅠ,self-healing group was the highest,then in drilling group,medication group was the lowest.The results showed that medication and drilling could repair the defects of the whole layer cartilage defects in various degrees.The main component of the restorations was fibrous tissue in self-healing group,more hyaline cartilage were produced by treated with hyaluronic acid and a thin layer of fibrous cartilage covering the surface of repair tissues in drilling group.In conclusion,in the short-term repair of bone defect,the effect of sodium hyaluronate was better than that of drilling.
引文
[1]李元城,张卫国.关节软骨损伤治疗进展[J].解放军医学杂志,2013,38(5):423-427.LI Y C,ZHANG W G.Advances in the treatment of articular cartilage injuries[J].Medical Journal of Chinese People’s Liberation Army,2013,38(5):423-427.(in Chinese)
[2]宋伟.膝关节骨性关节炎骨软骨结构病理学观察和形态计量学研究[D].重庆:第三军医大学,2012.SONG W.Structural pathological observation and morphometry of osteochondral structures in knee osteoarthritis[D].Chongqing:Third Military Medical University,2012.(in Chinese)
[3]梁茂华,张辉,孙磊,等.透明质酸钠预防兔膝关节固定后关节软骨退行性改变的作用[J].中国组织工程研究,2007,11(45):9043-9046.LIANG M H,ZHANG H,SUN L,et al.Preventive effect of sodium hyaluronate on degeneration of articular cartilage after knee joint fixation in rabbits[J].Chinese Journal of Tissue Engineering Research,2007,11(45):9043-9046.(in Chinese)
[4]吴海山,李晓华.关节内注射透明质酸钠对兔膝关节退变软骨的影响[J].第二军医大学学报,2000,21(7):680-682.WU H S,LI X H.Effects of intra-articular injection of sodium hyaluronate on degenerative cartilage of knee joint in rabbits[J].Academic Journal of Second Military Medical University,2000,21(7):680-682.(in Chinese)
[5]肖德明,李伟,王巨.SH关节腔内注射治疗兔膝关节炎的试验研究[J].中国矫形外科杂志,2006,14(9):690-692.XIAO D M,LI W,WANG J.Experimental study of intraarticular injection of SH for treatment of knee osteoarthritis in rabbits[J].Orthopedic Journal of China,2006,14(9):690-692.(in Chinese)
[6]张令涛,刘如明,肖建辉.透明质酸在骨组织工程中的应用[J].实用医学杂志,2016,32(8):1364-1366.ZHANG L T,LING R M,XIAO J H.The application of hyaluronic acid in bone tissue engineering[J].The Journal of Practical Medicine,2016,32(8):1364-1366.(in Chinese)
[7]TOSUN H B,GURGER M,GUMUSTAS S A,et al.The effect of sodium hyaluronate-chondroitin sulfate combined solution on cartilage formation in osteochondral defects of the rabbit knee:An experimental study[J].Therapeutics&Clinical Risk Management,2017,13:523-532.
[8]刘科,李靖年,杨冰,等.兔膝关节内注射透明质酸钠后胶原纤维及关节外相关韧带组织的变化[J].中国组织工程研究,2007,11(19):3734-3737.LIU K,LI J N,YANG B,et al.Changes of collagen fibers and extra articular ligament tissues after injection of sodium hyaluronate into rabbit knee joint[J].Chinese Journal of Tissue Engineering Research,2007,11(19):3734-3737.(in Chinese)
[9]倪磊,吕厚山.修复膝关节软骨缺损的关节镜技术——微骨折技术[J].中国微创外科杂志,2005,5(5):350-351.NI L,LIU H S.Arthroscopic technique for repair of cartilage defect of knee joint——Microfracture technique[J].Chinese Journal of Minimally Invasive Surgery,2005,5(5):350-351.(in Chinese)
[10]王浩,王伟.软骨下钻孔联合软骨源性形态发生蛋白缓释系统修复膝关节软骨缺损[J].中国组织工程研究,2014,18(21):3334-3340.WANG H,WANG W.Subchondral drilling combined with cartilage derived morphogenetic protein delivery system for repair of articular cartilage defects in the knee[J].Chinese Journal of Tissue Engineering Research,2014,18(21):3334-3340.(in Chinese)
[11]CHOI J I,LEE K B.Comparison of clinical outcomes between arthroscopic subchondral drilling and microfracture for osteochondral lesions of the talus[J].Knee Surgery Sports Traumatology Arthroscopy,2016,24(7):2140-2147.
[12]CALDWELL K L,WANG J.Cell-based articular cartilage repair:The link between development and regeneration[J].Osteoarthritis&Cartilage,2015,23(3):351.
[13]PROVOT S,SCHIPANI E.Molecular mechanisms of endochondral bone development[J].Biochemical&Biophysical Research Communications,2005,328(3):658-665.
[14]SOLHEIM E,HEGNA J,Oyen J,et al.Results at10to 14years after osteochondral autografting(mosaicplasty)in articular cartilage defects in the knee[J].The Knee,2013,20(4):287-290.
[15]蔺辉星,周振雷.Runx2研究进展[J].中国畜牧兽医,2009,36(2):53-57.LIN H X,ZHOU Z L.Advances in Runx2research[J].China Animal Husbandry&Veterinary Medicine,2009,36(2):53-57.(in Chinese)
[16]TUAN R S,CHEN A F,KLATT B A.Cartilage regeneration[J].Indian Journal of Orthopaedics,2016,21(5):303.
[17]吴昊,罗辉耀,朱冬承,等.微骨折+原位钻孔缝合法治疗股骨髁骨软骨骨折[J].临床骨科杂志,2016,19(4):466-468.WU H,LUO H Y,ZHU D C,Treatment of osteochondral fracture of femoral condyle by micro fracture plus in situ drilling and suture[J].Journal of Clinical Orthopaedics,2016,19(4):466-468.(in Chinese)
[18]张海宁,李汉秀,张洁,等.全层关节软骨缺损三种修复方法的比较试验研究[J].中华骨科杂志,2003,23(5):289-294.ZHANG H N,LI H X,ZHANG J,et al.Comparative experimental study of three methods for repair of full-thickness articular cartilage defect[J].Chinese Journal of Orthopaedics,2003,23(5):289-294.(in Chinese)
[19]葛志强,雷学锋.软骨下骨钻孔修复关节软骨缺损的研究进展[J].国际骨科学杂志,1997,1:34-37.GE Z Q,LEI X F.Research progress of subchondral bone drilling for repair of articular cartilage defects[J].International Journal of Orthopaedics,1997,1:34-37.(in Chinese)
[20]邵加华,陈松,谢金硕,等.Kartogenin与软骨修复的研究进展[J].第二军医大学学报,2016,37(4):471-476.SHAO J H,CHEN S,XIE J S,et al.Advances in the study of Kartogenin and cartilage repair[J].Academic Journal of Second Military Medical University,2016,37(4):471-476.(in Chinese)
[21]刘乐,王彦妮,庞玉娟,等.马骨髓间充质干细胞向软骨细胞的诱导分化[J].中国畜牧兽医,2017,44(2):417-424.LIU L,WANG Y N,PANG Y,et al.Differentiation of human bone marrow mesenchymal stem cells into chondrocytes[J].China Animal Husbandry&Veterinary Medicine,2017,44(2):417-424.(in Chinese)
[22]MARMOTTI A,BRUZZONE M,BONASIA D E,et al.One-step osteochondral repair with cartilage fragments in a composite scaffold[J].Knee Surgery Sports Traumatology Arthroscopy,2012,20(12):2590.
[23]冀晓明.透明质酸钠注射液对兔膝创伤性关节炎软骨修复试验研究[D].石家庄:河北医科大学,2011.YI X M.Experimental study of sodium hyaluronate injection on cartilage repair of traumatic knee osteoarthritis in rabbits[D].Shijiazhuang:Hebei Medical University,2011.(in Chinese)
[24]ABATE M,PULCINI D,DI I A,et al.Viscosupplementation with intra-articular hyaluronic acid for treatment of osteoarthritis in the elderly[J].Current Pharmaceutical Design,2010,16(6):631-640.
[25]庞金辉,刘明轩,石文俊,等.Ⅹ型胶原在膝骨关节炎软骨中的表达[J].实用临床医药杂志,2012,16(15):44-46.PANG J H,LIU M X,SHI W J,et al.Expression of typeⅩcollagen in cartilage of knee osteoarthritis[J].Journal of Clinical Medicine in Practice,2012,16(15):44-46.(in Chinese)
[26]刘菲,李学智,付妮妮,等.短刺法对兔膝骨关节炎软骨细胞Sox9、VEGF和ColⅩ表达的影响[J].南方医科大学学报,2016,36(7):997-1003.LIU F,LI X Z,FU N N,et al.Effect of short needling on the expression of Sox9,VEGF and ColⅩin chondrocytes of knee osteoarthritis in rabbits[J].Journal of Southern Medical University,2016,36(7):997-1003.(in Chinese)
[2]宋伟.膝关节骨性关节炎骨软骨结构病理学观察和形态计量学研究[D].重庆:第三军医大学,2012.SONG W.Structural pathological observation and morphometry of osteochondral structures in knee osteoarthritis[D].Chongqing:Third Military Medical University,2012.(in Chinese)
[3]梁茂华,张辉,孙磊,等.透明质酸钠预防兔膝关节固定后关节软骨退行性改变的作用[J].中国组织工程研究,2007,11(45):9043-9046.LIANG M H,ZHANG H,SUN L,et al.Preventive effect of sodium hyaluronate on degeneration of articular cartilage after knee joint fixation in rabbits[J].Chinese Journal of Tissue Engineering Research,2007,11(45):9043-9046.(in Chinese)
[4]吴海山,李晓华.关节内注射透明质酸钠对兔膝关节退变软骨的影响[J].第二军医大学学报,2000,21(7):680-682.WU H S,LI X H.Effects of intra-articular injection of sodium hyaluronate on degenerative cartilage of knee joint in rabbits[J].Academic Journal of Second Military Medical University,2000,21(7):680-682.(in Chinese)
[5]肖德明,李伟,王巨.SH关节腔内注射治疗兔膝关节炎的试验研究[J].中国矫形外科杂志,2006,14(9):690-692.XIAO D M,LI W,WANG J.Experimental study of intraarticular injection of SH for treatment of knee osteoarthritis in rabbits[J].Orthopedic Journal of China,2006,14(9):690-692.(in Chinese)
[6]张令涛,刘如明,肖建辉.透明质酸在骨组织工程中的应用[J].实用医学杂志,2016,32(8):1364-1366.ZHANG L T,LING R M,XIAO J H.The application of hyaluronic acid in bone tissue engineering[J].The Journal of Practical Medicine,2016,32(8):1364-1366.(in Chinese)
[7]TOSUN H B,GURGER M,GUMUSTAS S A,et al.The effect of sodium hyaluronate-chondroitin sulfate combined solution on cartilage formation in osteochondral defects of the rabbit knee:An experimental study[J].Therapeutics&Clinical Risk Management,2017,13:523-532.
[8]刘科,李靖年,杨冰,等.兔膝关节内注射透明质酸钠后胶原纤维及关节外相关韧带组织的变化[J].中国组织工程研究,2007,11(19):3734-3737.LIU K,LI J N,YANG B,et al.Changes of collagen fibers and extra articular ligament tissues after injection of sodium hyaluronate into rabbit knee joint[J].Chinese Journal of Tissue Engineering Research,2007,11(19):3734-3737.(in Chinese)
[9]倪磊,吕厚山.修复膝关节软骨缺损的关节镜技术——微骨折技术[J].中国微创外科杂志,2005,5(5):350-351.NI L,LIU H S.Arthroscopic technique for repair of cartilage defect of knee joint——Microfracture technique[J].Chinese Journal of Minimally Invasive Surgery,2005,5(5):350-351.(in Chinese)
[10]王浩,王伟.软骨下钻孔联合软骨源性形态发生蛋白缓释系统修复膝关节软骨缺损[J].中国组织工程研究,2014,18(21):3334-3340.WANG H,WANG W.Subchondral drilling combined with cartilage derived morphogenetic protein delivery system for repair of articular cartilage defects in the knee[J].Chinese Journal of Tissue Engineering Research,2014,18(21):3334-3340.(in Chinese)
[11]CHOI J I,LEE K B.Comparison of clinical outcomes between arthroscopic subchondral drilling and microfracture for osteochondral lesions of the talus[J].Knee Surgery Sports Traumatology Arthroscopy,2016,24(7):2140-2147.
[12]CALDWELL K L,WANG J.Cell-based articular cartilage repair:The link between development and regeneration[J].Osteoarthritis&Cartilage,2015,23(3):351.
[13]PROVOT S,SCHIPANI E.Molecular mechanisms of endochondral bone development[J].Biochemical&Biophysical Research Communications,2005,328(3):658-665.
[14]SOLHEIM E,HEGNA J,Oyen J,et al.Results at10to 14years after osteochondral autografting(mosaicplasty)in articular cartilage defects in the knee[J].The Knee,2013,20(4):287-290.
[15]蔺辉星,周振雷.Runx2研究进展[J].中国畜牧兽医,2009,36(2):53-57.LIN H X,ZHOU Z L.Advances in Runx2research[J].China Animal Husbandry&Veterinary Medicine,2009,36(2):53-57.(in Chinese)
[16]TUAN R S,CHEN A F,KLATT B A.Cartilage regeneration[J].Indian Journal of Orthopaedics,2016,21(5):303.
[17]吴昊,罗辉耀,朱冬承,等.微骨折+原位钻孔缝合法治疗股骨髁骨软骨骨折[J].临床骨科杂志,2016,19(4):466-468.WU H,LUO H Y,ZHU D C,Treatment of osteochondral fracture of femoral condyle by micro fracture plus in situ drilling and suture[J].Journal of Clinical Orthopaedics,2016,19(4):466-468.(in Chinese)
[18]张海宁,李汉秀,张洁,等.全层关节软骨缺损三种修复方法的比较试验研究[J].中华骨科杂志,2003,23(5):289-294.ZHANG H N,LI H X,ZHANG J,et al.Comparative experimental study of three methods for repair of full-thickness articular cartilage defect[J].Chinese Journal of Orthopaedics,2003,23(5):289-294.(in Chinese)
[19]葛志强,雷学锋.软骨下骨钻孔修复关节软骨缺损的研究进展[J].国际骨科学杂志,1997,1:34-37.GE Z Q,LEI X F.Research progress of subchondral bone drilling for repair of articular cartilage defects[J].International Journal of Orthopaedics,1997,1:34-37.(in Chinese)
[20]邵加华,陈松,谢金硕,等.Kartogenin与软骨修复的研究进展[J].第二军医大学学报,2016,37(4):471-476.SHAO J H,CHEN S,XIE J S,et al.Advances in the study of Kartogenin and cartilage repair[J].Academic Journal of Second Military Medical University,2016,37(4):471-476.(in Chinese)
[21]刘乐,王彦妮,庞玉娟,等.马骨髓间充质干细胞向软骨细胞的诱导分化[J].中国畜牧兽医,2017,44(2):417-424.LIU L,WANG Y N,PANG Y,et al.Differentiation of human bone marrow mesenchymal stem cells into chondrocytes[J].China Animal Husbandry&Veterinary Medicine,2017,44(2):417-424.(in Chinese)
[22]MARMOTTI A,BRUZZONE M,BONASIA D E,et al.One-step osteochondral repair with cartilage fragments in a composite scaffold[J].Knee Surgery Sports Traumatology Arthroscopy,2012,20(12):2590.
[23]冀晓明.透明质酸钠注射液对兔膝创伤性关节炎软骨修复试验研究[D].石家庄:河北医科大学,2011.YI X M.Experimental study of sodium hyaluronate injection on cartilage repair of traumatic knee osteoarthritis in rabbits[D].Shijiazhuang:Hebei Medical University,2011.(in Chinese)
[24]ABATE M,PULCINI D,DI I A,et al.Viscosupplementation with intra-articular hyaluronic acid for treatment of osteoarthritis in the elderly[J].Current Pharmaceutical Design,2010,16(6):631-640.
[25]庞金辉,刘明轩,石文俊,等.Ⅹ型胶原在膝骨关节炎软骨中的表达[J].实用临床医药杂志,2012,16(15):44-46.PANG J H,LIU M X,SHI W J,et al.Expression of typeⅩcollagen in cartilage of knee osteoarthritis[J].Journal of Clinical Medicine in Practice,2012,16(15):44-46.(in Chinese)
[26]刘菲,李学智,付妮妮,等.短刺法对兔膝骨关节炎软骨细胞Sox9、VEGF和ColⅩ表达的影响[J].南方医科大学学报,2016,36(7):997-1003.LIU F,LI X Z,FU N N,et al.Effect of short needling on the expression of Sox9,VEGF and ColⅩin chondrocytes of knee osteoarthritis in rabbits[J].Journal of Southern Medical University,2016,36(7):997-1003.(in Chinese)