威灵仙干预可注射性C/β-GP凝胶复合同种异体软骨细胞体外三维培养及修复软骨缺损的研究
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摘要
背景及目的
关节软骨缺损修复困难,历来是医学界的难点及研究热点。目前,组织工程技术已经成为其主要的研究方向,并且取得了一定的进展,组织工程技术包括种子细胞、支架材料、生长因子三部分。另外,中医药在该领域中,已经被证实具有良好的临床疗效。我们筛选单味中药—威灵仙作为研究对象,有研究证明威灵仙能够维持和促进软骨细胞合成蛋白多糖与Ⅱ型胶原,并可能通过抑制白细胞介素1水平的增高保护关节软骨。本课题将威灵仙与组织工程技术结合,观察威灵仙促进软骨细胞增殖、干预C/β-GP凝胶复合同种异体软骨细胞体外三维培养以及修复软骨缺损的作用,探讨中医药与现代医学结合的方式与价值。
方法
1.体外分离培养兔膝关节软骨细胞,甲苯胺蓝染色鉴定后,取处于对数生长期第3代细胞随机分组进行培养,倒置显微镜观察软骨细胞形态变化,MTT法检测不同浓度威灵仙对软骨细胞增殖的影响,RT-PCR法检测TGF-β1mRNA表达。
2.按7：1比例制备C/β-GP复合软骨细胞凝胶,随机分为实验组和对照组,分别给予相应处理,MTT法检测不同浓度威灵仙对凝胶中软骨细胞增殖的影响,HE、TB和Ⅱ型胶原免疫组织化学染色观察1、4周凝胶中软骨细胞的组织学变化。
3.复制关节软骨缺损模型,24只新西兰白兔随机分为3组,即实验组(C/β-GP+软骨细胞+威灵仙培养基关节腔注射组)、对照组(C/β-GP+软骨细胞+普通培养基关节腔注射组)、模型组(模型组),分别给予相应处理,术后6、12周后分别行大体观察及HE、TB、Ⅱ型胶原免疫组织化学染色,并进行Wakitani评分。
结果
1.不同浓度威灵仙均能促进软骨细胞增殖,尤以0.5mg/ml增殖效果明显,且增殖高峰出现在威灵仙干预后的第3天。0.05、0.1、0.5、1.0mg/ml威灵仙均能促进软骨细胞TGF-β1 mRNA表达,4组组间差异无显著性意义,但作用的高峰为0.5mg/ml组。
2.软骨细胞能良好地在C/β-GP凝胶内伸展、粘附、生长和增殖。各浓度组威灵仙均能促进软骨细胞在凝胶中的增殖,其中0.5mg/ml威灵仙的效果最明显,且能促进软骨细胞Ⅱ型胶原、细胞外基质的分泌。
3.实验组、对照组可见缺损关节面被较好地填充,并形成透明软骨样结构,实验组表面平整度及与周围组织整合程度较对照组好,组织学切片上可见类软骨形成并分泌软骨基质和特异性Ⅱ型胶原,模型组未见修复,可见组织增生退变。实验组修复组织与周围组织整合程度、组织学及Ⅱ型胶原分泌量均好于对照组。Wakitani评分：实验、对照两组分值显著低于模型组分值(P<0.01),且实验组分值明显低于对照组分值(P<0.05)。
结论
1.威灵仙能促进软骨细胞增殖及TGF-β1 mRNA的表达,这种类细胞生长因子作用可能是其促进软骨修复的机制之一；
2.C／β-GP凝胶与软骨细胞具有良好的相容性,威灵仙能够促进软骨细胞在C／β-GP凝胶中的增殖、Ⅱ型胶原、蛋白多糖的分泌及成软骨化发展。
3.威灵仙能够促进可注射性C／β-GP凝胶复合同种异体软骨细胞修复关节软骨缺损,这为中医药与组织工程技术的结合提供了新思路、新方法。
Background and objective
Once the articular cartilage defect,the repair is quite difficult,and it has always been the difficulty and research focus of the medical profession. At present,tissue engineering technique has been main research direction and has got advancement.The technique includes of seeds cell, frame and growth factor.Chinese medicine has been manifested satisfactory clinical therapeutic effect on repairing cartilage defect. We filt Weilingxian as study object. Studies have shown that Weilingxian can maintain and promote the synthesis of proteoglycan and collagen II of chondrocyte, and protect articular cartilage by inhibiting the level of interleukin-1 (IL-1) possibly.The topic combine Weilingxian with tissue engineering technique,and observe effect of Weilingxian on developing proliferation of chondrocytes and interfereing in injectable chitosan/β-glycerol PHosPHate(C/β-GP)encapsulating allograft chondrocytes there-dimension cultured in vitro and repairing articular cartilage defects. Our study try to explore methode and value of combineding Chinese medicine with modern medicine.
Methods
1.chondrocytes were isolated and cultured by the way of enzymatic digestion in vitro,after the PHenotype was identified by toluidine blue staining,the third-passage cells in the logarithmic growth PHase were randomly divided into groups and given the treatment respectively.MorPHology of chondrocytes was observed under inverted PHase contrast microscope,Methyl Thiazolyl Tetrazolium (MTT) assay method was adopted to observe the influence of Weilingxian with different concentrations on the proliferation of chondrocytes, and anti-transcription-polymerase chain-type reaction (RT-PCR) was used to assay the expression changes of transforming growth factor-β1 mRNA.
2.Chitosan/β-glyceroPHosPHate gel encapsulating allograft chondrocytes was prepared at ratio of 7:1,then was randomly divided into experimental and control groups divided into groups and given corresponding treatment respectively.MTT assay method was used to detect the influence of Weilingxian with different concentrations on the proliferation of chondrocytes three-dimensional cultured in gel;HE, TB and type II collagen immunohistochemical staining was used to detect histological changes of chondrocytes at 1、4 week.
3.After the model of knee articular cartilage defects was established,24 New Zealand rabbits were randomly divided into three groups,that is experimental group (C/β-GP+chondrocytes+Weilingxian medium intra-articular injection group), control group (C/β-GP+chondrocytes+normal medium intra-articular injection group), model group (model group),and given corresponding treatment respectively.The specimens were excised at the 6th and 12th week,examined grossly and analyzed by haematoxylin eosine,toluidine blues staining and typeⅡcollagen immunohistochemistry reaction.Wakitani score method was counted to evaluate the effect of repair.
Result
1.All groups of Weilingxian can promote proliferation of chondrocytes,effect of 0.5mg/ml group was especially obvious,and the peak of proliferation was at the third day.Group of 0.05、0.1、0.5 and 1.0 mg/ml could promote the expression of TGF-β1 mRNA, and there was no significant difference between the four groups (P>0.05), but the peak occurred at 0.5 mg/ml group.
2.Chondrocytes behave good state of stretch、adhesion、proliferation in C/β-GP gel.All groups of Weilingxian can promote chondrocyte proliferation in the gel,and the effect of 0.5mg/ml group was especially obvious.Weilingxian can also enhance the secretion of collagen II and extracellular matrix.
3.Defects of articular surface were well filled in experimental group and control group,and hyaline cartilage-like structure were formed,the surface flatness and degree of integration with surrounding tissue of experimental group was better than control group,and model group was not repaired.Formation of cartilage-like and secretion of cartilage matrix and specificity of collagen type II were found in histological slices.Integration of repair tissue with surrounding tissue,histology and amount of type II collagen secretion in experimental group were better than control group. Wakitani scores:scores of experimental group and control group were significantly lower than model group (P<0.01),and scores of experimental group was significantly lower than group control group (P<0.05).
Conclusions
1.Weilingxian can promote chondrocyte proliferation and TGF-β1mRNA expression,these role like cell growth factor may be one of the possible mechanisms to promote the repair of cartilage defect.
2.It has good compatibility of chondrocytes in the co-culture with C/-GP gel,Weilingxian can promote chondrocytes proliferation and secretion of collagenⅡ、proteoglycan cultured in the C/β-GP gel, and also can induce the development to cartilage.
3.Weilingxian can promote repair of cartilage defect by the injectable C/β-GP gel composite allogenic chondrocytes,this offer a new idea and method for the combination of Chinese medicine and tissue engineering technologies.
关节软骨缺损修复困难,历来是医学界的难点及研究热点。目前,组织工程技术已经成为其主要的研究方向,并且取得了一定的进展,组织工程技术包括种子细胞、支架材料、生长因子三部分。另外,中医药在该领域中,已经被证实具有良好的临床疗效。我们筛选单味中药—威灵仙作为研究对象,有研究证明威灵仙能够维持和促进软骨细胞合成蛋白多糖与Ⅱ型胶原,并可能通过抑制白细胞介素1水平的增高保护关节软骨。本课题将威灵仙与组织工程技术结合,观察威灵仙促进软骨细胞增殖、干预C/β-GP凝胶复合同种异体软骨细胞体外三维培养以及修复软骨缺损的作用,探讨中医药与现代医学结合的方式与价值。
方法
1.体外分离培养兔膝关节软骨细胞,甲苯胺蓝染色鉴定后,取处于对数生长期第3代细胞随机分组进行培养,倒置显微镜观察软骨细胞形态变化,MTT法检测不同浓度威灵仙对软骨细胞增殖的影响,RT-PCR法检测TGF-β1mRNA表达。
2.按7：1比例制备C/β-GP复合软骨细胞凝胶,随机分为实验组和对照组,分别给予相应处理,MTT法检测不同浓度威灵仙对凝胶中软骨细胞增殖的影响,HE、TB和Ⅱ型胶原免疫组织化学染色观察1、4周凝胶中软骨细胞的组织学变化。
3.复制关节软骨缺损模型,24只新西兰白兔随机分为3组,即实验组(C/β-GP+软骨细胞+威灵仙培养基关节腔注射组)、对照组(C/β-GP+软骨细胞+普通培养基关节腔注射组)、模型组(模型组),分别给予相应处理,术后6、12周后分别行大体观察及HE、TB、Ⅱ型胶原免疫组织化学染色,并进行Wakitani评分。
结果
1.不同浓度威灵仙均能促进软骨细胞增殖,尤以0.5mg/ml增殖效果明显,且增殖高峰出现在威灵仙干预后的第3天。0.05、0.1、0.5、1.0mg/ml威灵仙均能促进软骨细胞TGF-β1 mRNA表达,4组组间差异无显著性意义,但作用的高峰为0.5mg/ml组。
2.软骨细胞能良好地在C/β-GP凝胶内伸展、粘附、生长和增殖。各浓度组威灵仙均能促进软骨细胞在凝胶中的增殖,其中0.5mg/ml威灵仙的效果最明显,且能促进软骨细胞Ⅱ型胶原、细胞外基质的分泌。
3.实验组、对照组可见缺损关节面被较好地填充,并形成透明软骨样结构,实验组表面平整度及与周围组织整合程度较对照组好,组织学切片上可见类软骨形成并分泌软骨基质和特异性Ⅱ型胶原,模型组未见修复,可见组织增生退变。实验组修复组织与周围组织整合程度、组织学及Ⅱ型胶原分泌量均好于对照组。Wakitani评分：实验、对照两组分值显著低于模型组分值(P<0.01),且实验组分值明显低于对照组分值(P<0.05)。
结论
1.威灵仙能促进软骨细胞增殖及TGF-β1 mRNA的表达,这种类细胞生长因子作用可能是其促进软骨修复的机制之一；
2.C／β-GP凝胶与软骨细胞具有良好的相容性,威灵仙能够促进软骨细胞在C／β-GP凝胶中的增殖、Ⅱ型胶原、蛋白多糖的分泌及成软骨化发展。
3.威灵仙能够促进可注射性C／β-GP凝胶复合同种异体软骨细胞修复关节软骨缺损,这为中医药与组织工程技术的结合提供了新思路、新方法。
Background and objective
Once the articular cartilage defect,the repair is quite difficult,and it has always been the difficulty and research focus of the medical profession. At present,tissue engineering technique has been main research direction and has got advancement.The technique includes of seeds cell, frame and growth factor.Chinese medicine has been manifested satisfactory clinical therapeutic effect on repairing cartilage defect. We filt Weilingxian as study object. Studies have shown that Weilingxian can maintain and promote the synthesis of proteoglycan and collagen II of chondrocyte, and protect articular cartilage by inhibiting the level of interleukin-1 (IL-1) possibly.The topic combine Weilingxian with tissue engineering technique,and observe effect of Weilingxian on developing proliferation of chondrocytes and interfereing in injectable chitosan/β-glycerol PHosPHate(C/β-GP)encapsulating allograft chondrocytes there-dimension cultured in vitro and repairing articular cartilage defects. Our study try to explore methode and value of combineding Chinese medicine with modern medicine.
Methods
1.chondrocytes were isolated and cultured by the way of enzymatic digestion in vitro,after the PHenotype was identified by toluidine blue staining,the third-passage cells in the logarithmic growth PHase were randomly divided into groups and given the treatment respectively.MorPHology of chondrocytes was observed under inverted PHase contrast microscope,Methyl Thiazolyl Tetrazolium (MTT) assay method was adopted to observe the influence of Weilingxian with different concentrations on the proliferation of chondrocytes, and anti-transcription-polymerase chain-type reaction (RT-PCR) was used to assay the expression changes of transforming growth factor-β1 mRNA.
2.Chitosan/β-glyceroPHosPHate gel encapsulating allograft chondrocytes was prepared at ratio of 7:1,then was randomly divided into experimental and control groups divided into groups and given corresponding treatment respectively.MTT assay method was used to detect the influence of Weilingxian with different concentrations on the proliferation of chondrocytes three-dimensional cultured in gel;HE, TB and type II collagen immunohistochemical staining was used to detect histological changes of chondrocytes at 1、4 week.
3.After the model of knee articular cartilage defects was established,24 New Zealand rabbits were randomly divided into three groups,that is experimental group (C/β-GP+chondrocytes+Weilingxian medium intra-articular injection group), control group (C/β-GP+chondrocytes+normal medium intra-articular injection group), model group (model group),and given corresponding treatment respectively.The specimens were excised at the 6th and 12th week,examined grossly and analyzed by haematoxylin eosine,toluidine blues staining and typeⅡcollagen immunohistochemistry reaction.Wakitani score method was counted to evaluate the effect of repair.
Result
1.All groups of Weilingxian can promote proliferation of chondrocytes,effect of 0.5mg/ml group was especially obvious,and the peak of proliferation was at the third day.Group of 0.05、0.1、0.5 and 1.0 mg/ml could promote the expression of TGF-β1 mRNA, and there was no significant difference between the four groups (P>0.05), but the peak occurred at 0.5 mg/ml group.
2.Chondrocytes behave good state of stretch、adhesion、proliferation in C/β-GP gel.All groups of Weilingxian can promote chondrocyte proliferation in the gel,and the effect of 0.5mg/ml group was especially obvious.Weilingxian can also enhance the secretion of collagen II and extracellular matrix.
3.Defects of articular surface were well filled in experimental group and control group,and hyaline cartilage-like structure were formed,the surface flatness and degree of integration with surrounding tissue of experimental group was better than control group,and model group was not repaired.Formation of cartilage-like and secretion of cartilage matrix and specificity of collagen type II were found in histological slices.Integration of repair tissue with surrounding tissue,histology and amount of type II collagen secretion in experimental group were better than control group. Wakitani scores:scores of experimental group and control group were significantly lower than model group (P<0.01),and scores of experimental group was significantly lower than group control group (P<0.05).
Conclusions
1.Weilingxian can promote chondrocyte proliferation and TGF-β1mRNA expression,these role like cell growth factor may be one of the possible mechanisms to promote the repair of cartilage defect.
2.It has good compatibility of chondrocytes in the co-culture with C/-GP gel,Weilingxian can promote chondrocytes proliferation and secretion of collagenⅡ、proteoglycan cultured in the C/β-GP gel, and also can induce the development to cartilage.
3.Weilingxian can promote repair of cartilage defect by the injectable C/β-GP gel composite allogenic chondrocytes,this offer a new idea and method for the combination of Chinese medicine and tissue engineering technologies.
引文
[1]O'Driscoll SW.The healing and regeneration of articular cartilage[J].Bone Joint Surg Am,1998,80:17 95-1812.
[2]Temenoff JS,Mikos AG.Injectable biodegradable materials for orthopedic tissue engineering[J].Biomat erials,2000,21 (23):2405-2412.
[3]Lee CR,Breinan HA,Nehrer S,et al.Articular cartilage chondrocytes in type Ⅰ and type Ⅱ collagen-G AG matrices exhibit contractile behavior in vitro[J].Tissue Eng,2000,6(5):555-565.
[4]杨贵勇,卢世璧,王继芳,等.自体骨膜游离移植修复髋膝关节软骨大面积缺损的远期观察[J].中国修复重建外科杂志,2004,18(1)：8-11.
[5]Czitrom AA,Keating S,Gross AE,et al.The viability of artic-ular cartilage in fresh osteochondral allo grafts after clinical transplantation[J].J Bone Joint Surg(Am),1990,72(4):574-581.
[6]皇甫小桥,赵金忠,杨星光.关节镜下自体骨软骨移植修复软骨缺损[J].中国修复重建外科杂志,2008,22(7)：891-893.
[7]谭琪,田清业,刘光军,等.同种异体软骨移植修复关节软骨缺损的实验研究[J].国际骨科学杂志,2008,29(5)：336-339.
[8]孙涛,王英振.同种异体软骨细胞移植术后关节软骨蛋白多糖的测定[J].中国骨与关节损伤杂志,2008,23(3)：214-217.
[9]孙骏,侯筱魁,李旭,等.人转化生长因子β1基因转染骨髓基质干细胞复合藻酸钙修复骨软骨缺损[J].中国组织工程研究与临床康复,2008,12(29)：5636-5638.
[10]Langer R,Vacanti JP.Tissue engineering [J].Science,1993,260(5110):920-926.
[11]霍建忠,陈峥嵘,蒋淳,等.转化生长因子TGF-β 1基因修饰骨髓间充质干细胞复合壳聚糖修复兔关节软骨缺损[J].中华风湿病学杂志,2005,9(7)：393-396.
[12]王会才,张震宇,辛伟光.微重力三维动态诱导骨髓间充质干细胞复合可注射型支架材料Pluronic F-127修复关节软骨缺损[J].中国组织工程研究与临床康复,2007,11(11)：2609-2611.
[13]陈飞雁,顾湘杰,钟明康,等.威灵仙注射液对骨关节炎关节液与软骨白介素-1水平的影响[J].中国矫形外科杂志,2004,12(7)：524-526.
[14]陈飞雁,王旭,黄加张,等.威灵仙注射液对骨关节炎模型动物软骨组织形态和胶原表达的影响[J].中国矫形外科杂志,2006,14(5)：360-363.
[1]Manning WK,Bonner WM.Isolation and culture ofchondrocytes from human articular cartilage[J].Arthritis Rheum,1967,10(1):235-239.
[2]宁旭,尚显文,尹培荣,等.胶原酶一次消化法培养兔软骨细胞[J].贵州医药,2004,28(6)：490.
[3]Hu DN,Yang PY,KuMC,et al.Isolation and cultivation of human articular chondrocytes[J].Kaohsiung J MedSci,2002,18(3)：113.
[4]周强,李起鸿,戴刚,等.三步酶消化法高效分离兔原代关节软骨细胞及体外培养观察[J].中华外科杂志,2005,43(8)：522.
[5]张志光,郑卫平,苏凯,等.兔关节软骨细胞的分离培养和形态学特征[J].中山大学学报,2004,25(1)：63-66.
[6]周新社,戴尅戎,汤亭亭,等.人负重关节软骨细胞的体外分离与培养[J].临床骨科杂志,2004,7(2)：206.
[7]李文辉,侯筱魁.软骨组织工程种子细胞及其培养方法[J].中华创伤骨科杂志,2003,5(3)：244-246.
[8]Rodrigues A,Pap S,Vacanti CA.The effect of long-term culture on the ability of human auricular chondrocytes to generate tissue engineered cartilage.In:Stard GB,Horch R,Tanczos E,eds[M].Biological matrices and tissue reconstruction.New York:Springer,1998:163-168.
[9]何清义,李起鸿,许建中,等.人关节软骨细胞的永生化及其表型诱导[J].中华创伤杂志,2002,18(12)：727-731.
[10]邓欢,黄年根,张吉翔.DNA甲基化与细胞周期[J].国际遗传学杂志,2006,9(2)：106-109.[11]赵瑜,冯齐凤,朱祝生,等.中药补肾方对体外培养软骨细胞影响[J].时珍国医国药杂志,2008,19(4)：960-961.
[12]冯坤,谢文,陈宝龙,等.5种淫羊藿对软骨组织生长和软骨细胞增殖影响的对比研究[J].中国中药杂志,2006,34(24)：2065-2067.
[13]叶蕻芝,李西海,梁文娜,等.透骨消痛颗粒含药血清对兔软骨细胞增殖影响的研究[J].中国临床药理学 与治疗学,2009,14(6)：624-628.
[14]朱文菁.MTT法分析培养成骨细胞的存活和增殖能力[J].上海医科大学学报,1995,22(33)：254.
[15]孙卫民,王慧琴.细胞因子研究方法学[M].北京：人民卫生出版社,1999,65-67.
[16]朱萱萱,支月芳,邱召娟,等.痛痹颗粒及拆方的含药血清对家兔软骨细胞增殖与凋亡的影响[J].实用中医内科学杂志,2006,20(6)：603-604.
[17]王新东,张前德,纪伟.痛痹方血清对体外培养乳兔关节软骨细胞增殖和MMP-3与TIMP-1的影响[J].上海中医药杂志,2006,40(6)：64-65.
[18]章敏,王勇,李媛媛,等.痹痛灵颗粒含药血清对家兔关节软骨细胞体外培养的影响[J].中华中医药杂志,2009,24(1)：75-77.
[19]欧阳冰,林绵辉,左文建,等.结缔组织生长因子对体外培养的兔关节软骨细胞增殖和表型的影响[J].中国组织工程研究与临床康复,2009,13(20)：3816-3820.
[20]Lieberman JR,Dalusikl A,Einhorn TA.The role of growth factors in the repair of bnoe、biology and clinical applicatinos[J].J bone Joint Sugr Am JT,2002,84A(6):1032-1044.
[21]Frenkel SR,Saadeh PB,Mehrara BJ,et al.Transforming growth factor beta superfamily members:role in cartilage modeling.Plast Reconstr Surg.2000,5(3):980-990.
[22]王传家,李宇,纪影畅,等.TGF-β1对猪关节软骨细胞合成糖胺多糖的作用[J].汕头大学医学院学报,2003,16(3)：159-161.
[23]Blumenfeld I,Gaspar R,Laufer D,et al.Enhancement of toluidine blue staining by transf-orming growth factor-beta、insulinlike growth factor and growth hormone in the temporo-mandibular joint of aged mice.Cell Tissue Organ.2000;167:121-129.
[24]亓建洪,赵庆华,朱宝林,等.TGF-β1对人软骨细胞基质金属蛋白酶-1mRNA及其阻滞剂-1mRNA表达的影响[J].中华创伤骨科杂志,2005,7(8)：753-757.
[25]Sporn MB,Roberts AB,Wabefield,et al.Some recent advances in thechemistry and biologyof transforming growth factor-betaCell Biol.1987;5(3):1039.
[26]Ferguson CM,Schwarz EM,Reynolds PR,et al.Smad 2 and 3 mediate transforming gro-wth factor-betal-induced inhibition of chondrocyte maturation.Endocrinology.2000;141(12):4728-4735.
[27]王东武,杨柳,段小军,等.可注射温固化支架复合异体软骨细胞修复关节软骨缺损的初步研究[J].重庆医学,2006,35(12)：1082-1087.
[28]Yang K-C,Wu C-C,Lin F-H,et al.Chitosan/gelatin hydrogel as immunoisolative matrix for injectable bioartificial pancreas[J].Xenotransplantation,2008,15:407-416.
[29]Bryan J.Studies on donal cartilage strains II selective effects of diffeveuf growth conditions.Exp Cell' Res.1988;52:327-337.
[30]Cruz M. Structural lesion and disability-not only rheumatoid arthritis but also osteoart-hritis.Acta Reumatol Port,2008,33(4):475-476.
[31]彭程,林绵辉,肖涛.细胞因子与软骨损伤及修复研究进展[J].国际骨科学杂志,2006,27(4)：239-243.
[32]Chetina EV, Pul AR.The role of growth factors in inhibition of collagen degeneration and chondrocyte differentiation in patients with osteoarthrosis.Vestn Ross Akad Med Nauk,2008,(9):40-45.
[33]Veilleux N,Spector M. Effects of FGF-2 and IGF-1 on adult canine articular chondrocytes in type Ⅱ collagen-glycosaminoglycan scaffolds in vitro. Osteoarthritis Cartilage.2005,13(4):278-286.
[34]Hicks DL,Sage AB,Shelton E,et al. Effect of bone morPHogenetic proteins 2 and 7 on septal chondrocytes in alginate. Otolaryngol Head Neck Surg,2007,136(3):373-379.
[35]王勇,陆长青,王凡.丹参诱导大鼠骨髓间充质干细胞向神经样细胞分化[J].解剖学杂志,2007,30(2)：207-210.
[36]李楠,王和鸣,李文顺,等.龟鹿二仙胶汤含药血清对兔体外BMSCs向软骨细胞分化的干预作用.上海中医药大学学报,2007,2 1(6)：58-61.
[37]刘维统,王跃,郝鹏,等.黄芪甲苷对关节软骨细胞功能影响的实验研究.实用医院临床杂志,2009,6(4)：44-47.
[38]吴涛,徐俊昌,南开辉,等.淫羊藿苷促进羊骨髓间充质干细胞的增殖和成骨分化.中国组织工程研究与临床康复,2009,13(19)：3725-3729.
[39]杜晓岩,毛艳,李慕勤,等.羟基磷灰石-磷酸三钙／壳聚糖骨碎补复合材料修复骨缺损的实验研究.中国美容医学,2009,18(、8)：1125-1127.
[40]彭锐,李胜利,邹阳.明胶白芨胶／血竭多孔材料的生物相容性评价.中国中医骨伤科杂志,2007,15(1)：38-40.
[41]张杨,李秀兰,张文海,等.含有生肌液的组织工程皮肤修复兔皮肤缺损.中国组织工程研究与临床康复,2008,12(46)：9001-9005.
[42]Jeong B,Kim SW,Bae YH.Thermosensitive sol-gel reversible hydrogels.Adv Drug Deliv Rev.2002,54:37-51.
[43]Risbud M, Ringe J, Bhonde R, et al. In vitro expression of cartilage specific markers by chondrocytes on a biocompatible hydrogel:implications for engineering cartilage tissue[J]. Cell Transplant,2001,10(8):755。
[44]Chenite A,Chaput C,Wang D,et al.Novel injectable neutral solutions of chitosan form biodegradable gels in situ.Biomaterials,2000,21(21):2155-2161.
[45]高学敏.中药学[M].北京：人民卫生出版社,2000：645-655.
[46]陈飞雁,顾湘杰,钟明康,等.威灵仙注射液对骨关节炎关节液与软骨白介素-1水平的影响[J].中国矫形外科杂志,2004,12(7)：524-526.
[47]陈飞雁,王旭,黄加张,等.威灵仙注射液对骨关节炎模型动物软骨组织形态和胶原表达的影响[J].中国矫形外科杂志,2006,14(5)：360-363.
[48]Hua Wu,Ying Wan,Xiaoying Cao,et,al.Proliferation of chondrocytes on porous poly(DL-lactide)/chitosan scaffolds[J]. Acta Biomaterialia,2008,4:76-87.
[49]Yang KQSaris DB,Geuze RE,et al.Impact of expansion and redifferentitation conditions on chondrogenic capacity of cultured chondrocytes.Tissue Eng,2006,12(9):2435-2447.
[50]ATSUSHI FUNAYAMA,YASUO NIKI,HIDEO MATSUMOTO,et al.Repair of full-thickness articular cartilage defects using injectable type Ⅱ collagen gel embedded with cultured chondrocytes in a rabbit model[J].J Orthop Sci,2008,13:225-232.
[51]Atsushi Funayama,Uuayama,Yasuo Niki,Hideo Matsumoto,et al.Repair of full-thickness articular cartilage defects using injectable type Ⅱ collagen gel embedded with cultured chondrocytes in a rabbit model[J].J Orthop Sci 2008,13(3):225-232.
[52]Wakitani S,Nawata M,Tensho K.Repair of articular cartilage defects in the patello-femoral joint with autologous bone marrow mesenchymal cell transplantation:three case reports involving nine defects in five knees[J].Tissue Eng Regen Med,2007,1(1):74-79.
[53]Jubeck B,Muth E,Gohr CM.Type Ⅱ collagen levels correlate with mineralization by articular cartilage vesicles[J].Arthritis Rheum,2009,60(9):2741-2746.
[1]O'Driscoll SW. The healing and regeneration of articular cartilage[J]. Bone Joint Surg Am,1998,80: 1795-1812.
[2]王传家等,转化生长因子-β1对猪关节软骨细胞合成糖胺多糖的作用[J].汕头大学医学院学报,2003,16(3)：159-161.
[3]Lee KH,Song SU,Hwang TS,et al.Regeneration of hyaline cartilage by cell-mediated gene therapy using transforming growth factor beta 1 producing fibroblasts[J]. Hum Gene Ther,2001,12:1805-181.
[4]Madry H,Emkey G,Zurakowski D,et al. Overexpression of human fibroblast growth factor 2 stimulates cell proliferation in an ex vivo model of articular chondrocyte transplantation[J].Gene Med,2004,6(2):238-45.
[5]Mizuta H, Kudo S, Nakmura E,et al. Active proliferation of mesenchymal cells prior to the chond rogenic repair response in rabbit full-thickness defects of articular cartilage.Osteoarthritis cartilage,2004, 12(7):586-596.
[6]Schmidt MB, chen EH, Lynch SE A review of the effects of insulin-like growth factor and platelet derived growth factor on in vivo cartilage healing and repair[J].Osteoarthritis cartilage.2006,14(5):403-412.
[7]Tian H, Stogiannidis I Up-regulation of cartilage digomeric matrix protein gene expression by insulin-like growth factor-1 revealed by real-time reverse transcription-polymerase chain reaction[J].Acta Biochim BioPHy Sin(shanghai),2006,38(10):677-682.
[8]Brower-Toland BD,Saxer RA,Goodrich LR, et al.Direct adenovirus mediated insulin-like growth factor I gene transfer enhances transplant chondrocyte function[J].Hum Gene Ther,2001,12:117-129.
[9]Fukuoka H,Shibata S,Suda N,et al,Bone morPHogenetic protein rescues the lack of secondary cartilage in Runx2-deficient mice [J]J Anat,2007,211(1):8-15.
[10]倪滨,李明.BMP生物活性的研究现状[J].重庆医学,2007,36(10)：970-973.
[11]Grimsrud CD,Romano PR,D'Souza M,et al.BMP-6 is an autocrine stimulator of chondrocyte differentiation[J].J Bone Min Res 1999,14(4):475-482.
[12]刘凌,张洪涛,王现彬等.腺病毒介导骨形态发生蛋白2基因对兔软骨细胞的影响[J].中国组织工程研究与临床康复.2008,12(50)：9811-9813.
[13]Mason JM,Breitbart AS,Barcia M,et al.Cartilage and bone regeneration using gene enhanced tissue engineering[J].Clin Orthop 2000,(379Suppl):S171-S178.
[14]Madry H,Zurakowski D,Trippel SB.Overexpression of human insulin-like growth factor-I promotes new tissue formation in anexvivomodel of articular chondrocyte transplantation[J]. Gene Ther,2001,8:1443-1449.
[15]Nixon AJ,Brower-Toland BD,Bent SJ,et al,Insulinlike growth factor-1 gene therapy applications for cartilage repair[J].Clin Orthop 2000,390:201-13.
[16]Oligino T,Ghivizzani S, Wolfe D,et al.Intra-articular delivery of a herpes simplex virus IL-1Ra gene vector reduces inflammation in a rabbit model of arthritis[J].Gene Ther,1999,6:1713-1720.
[17]Kwon GY, Jeong J,Woo JK,et al. Co-expression of bfl-1 enhanceshost response in the herpes simplex virus-thymidine kinase/ganciclovir gene therapy system [J]. Biochem BioPHys Res Commun,2003,303(3): 756-759.
[18]曲福军,侯宣,周余来等.重组pcDNA3.1-BMP7转染兔膝关节软骨细胞及其表达[J].中国临床康复.2006,10(17)：79-82.
[19]Goomer RS,Deftos LI,Terkeltaub R,et al.High-efficiency non-viral transfection of primary chondrocytes and perichondrial cells for ex vivo gene therapy to repair articular cartilage defects[J]. Osteoarthritis Cartilage,2001,9:248-256.
[20]Brunner S,Saneri,Cerottas,et al.Cell cycle dependence of gene transfer by lipoplex,polyplex and recombinant adenovirus [J].Gene The 2000,7:401-7.
[21]Evans CH,Ghivizzani SC,Kang R et al. Gene therapy for rheumatic diseases[J].Arthritis Rheum,1999, 42(1):1-16.
[22]Grande DA,Breitbart AS,Mason J,et al.Cartilage tissue engineering:current limitations and solution s[J].Clin Orthop Relat Res,1999,367(suppl):s176-s186.
[23]Mason JM, Grande DA,Barcia M, et al. Expression of human bone morPHogenic protein 7 in primary rabbit periosteal cells:potential utility in gene therapy for osteochondral repair[J].Gene Ther,1998, 5(8)：1098-1104.
[24]秦宏敏,沙广钊,佟向阳等.兔骨髓间充质干细胞经Ad-TGF β1重组腺病毒载体转染定向分化为软骨细胞[J].中国组织工程研究与临床康复.2007,11(42)：8444-8448.
[25]Gelse K,Jiang QJ,Aigner T,etal.Fibroblast-mediated delivery of growth factor complementary DNA into mouse joints induces chondrogenesis but avoids the disadvantages of direct viral gene transfer[J]. Arthritis Rheum,2001; 44(8):1943-1953.
[1]Langer R,Vacanti JP.Tissue engineering [J].Science.1993,260(5110):920-926.
[2]靳小兵,组织工程方法修复关节软骨缺损[J].中国微创外科杂志.2005,5(8)：685-687.
[3]Friendenstein A J,Gorskaja J F,Kulagina N N.Fibroblast Precursors in Normal and Irradi-ated Mouse Hema-topoietic Organs[J].Exp Hematol.1976,4(5):267-274.
[4]Gerstenfeld LC,Ctuceta J,Shea C M,et al.Chondro-cytes provide morPHogenic signals that selectivel y induce os-teogenic differentiation of mesenchymal stem cell[J].J BoneMiner Re-s.2002,17(2):221.
[5]Pereira R F,Halford K W,O'hara M D,et al.Cul-tured Adherent Cells from Marrow can Serve as Pr ecursorCells for Bone,Cartilage,and Lung in Irradiated Mice[J].ProcNatl Acad S-ci USA.1995,92:4857-4 861.
[6]马力,刘大军,李德天,等.不同分离方法及培养条件对兔骨髓间充质干细胞生长增殖及生物学特性的影响[J].中国组织工程研究与临床康复.2008,12(38)：7401-7406.
[7]蒋文慧,马爱群,董安平,等.骨髓间质干细胞的分离培养与鉴定[J].西安交通大学学报(医学版).2005,26(3)：224-227.
[8]赵瑛.SD大鼠骨髓间充质干细胞的分离培养和鉴定[J].重庆师范大学学报(自然科学版).2008,25(1)：75-77.
[9]王超,徐蕴,宋文刚,等.大鼠骨髓间充质干细胞分离培养方法的建立及其表型分析[J].ISSN 1007-8738细胞与分子免疫杂志.2007,23(5)：466-468.
[10]苏秋香,王忠华,王淑华,等.不同分离液和培养基对骨髓间充质干细胞原代培养的影响研究[J],沈阳医学院学报.2005,7(4)：252-254.
[11]张永先,蔡锦方,汤继文,等.兔骨髓基质细胞的分离培养[J].中华显微外科杂志.2004,27(2)：120-123.
[12]马力,刘大军,李德天,等.不同分离方法及培养条件对兔骨髓间充质干细胞生长增殖及生物学特性的 影响[J].中国组织工程研究与临床康复.2008,12(38)：7401-7406.
[13]王超,徐蕴,宋文刚,等.大鼠骨髓间充质干细胞分离培养方法的建立及其表型分析[J].ISSN 1007-8738细胞与分子免疫杂志.2007,23(5)：466-468.
[14]霍建忠,陈峥嵘,蒋淳,等.转化生长因子TGF-β1基因修饰骨髓间充质干细胞复合壳聚糖修复兔关节软骨缺损[J].中华风湿病学杂志.2005,9(7)：393-396.
[15]杨物鹏,许建中.BMP-2和TGF β1对培养兔关节软骨细胞代谢的影响[J].第三军医大学学报.2000,22(12)：1139-1141.
[16]Madry H,Kaul QCucchiarini M,et al.Enhanced repair of articular cartilage defects in vivo by trans planted chondrocytes over expressing insulin-like growth factor Ⅰ (IGF-Ⅰ)[J].Gene Ther.2005,12(5):117 1-1179.
[17]Kotev-EmethS,PitaruS,Pri-ChenS,etal.Establishmentofaratlong-termcultureexpressingtheosteogenicPHeno type:dependenceon dexamethasone and FGF-2[J].Connect Tissue Res.2002,43:606-612.
[18]俞猛,夏仁云,高飙,等.bFGF和地塞米松对骨髓基质干细胞增殖与分化的影响[J].中国康复.2006,21(1)：12-15.
[19]Hughes S E, Hall P A.The fibroblast growth factor and receptor moltigene families [J].J Patho.19 93,170(3):219-21.
[20]Gelse K,von der Mark K.Articular cartilage repair by gene therapy using growth factor-producing mesenchymal cells[J].Arthritis Rheum.2003,48(2):430-441.
[21]丁幸坡,金先庆,刘伟.HIGF-1基因修饰骨髓基质干细胞复合PLGA移植修复关节软骨缺损[J].重庆医科大学学报.2006,31(3)：354-357.
[22]孙骏,侯筱魁,李旭,等.人转化生长因子β1基因转染骨髓基质干细胞复合藻酸钙修复骨软骨缺损[J].中国组织工程研究与临床康复.2008,12(29)：5636-5638.
[23]许建中,范东伟,张文霞.经Ⅱ型胶原修饰后的PLGA对诱导后的骨髓基质干细胞粘附及分化的影响[J].郑州大学学报.2005,40(3)：424-426.
[24]龙华,袁华,马保安,等.腺病毒介导TGF-β 1及BMP-7基因共表达感染骨髓基质干细胞修复兔关节软骨缺损[J].中国骨与关节损伤杂志.2008,23(6)：471-474.
[25]Corsi K, Chellat F, YahiaL, et al. Mesenchymal stemcells, MG63 and HEK293 transfe-ction using chitosan-DNA nanoparticles[J]. Biomaterials.2003,24(7):1255.
[26]Ponticiello MS,Schinagl RM,Kadiyala S,et al.Gelatin-based resorbable sponge as a carri-er matrix f or human mesenchymal stem cells in cartilage regeneration therapy[J].J Biomed Mater Res.2000,52(2): 246-255
[27]郝俊龙,汪玉良，孙正义,等.新型支架材料羟基磷灰石／聚磷酸钙纤维／左旋聚乳酸复合自体骨髓间充质干细胞修复关节软骨全层缺损[J].中国临床康复.2006,10(37)：64-67.
[28]丁幸坡,金先庆,刘伟HIGF-1基因修饰骨髓基质干细胞复合PLGA移植修复关节软骨缺损[J].重庆医科大学学报.2006,31(3)：354-357.
[29]王觅格,夏亚一,王栓科,等.骨髓间充质干细胞复合消旋聚乳酸／明胶修复兔关节软骨缺损的实验研究[J].中国修复重建外科杂志.2007,21(7)：753-758.
[30]王会才,张震宇,辛伟光.微重力三维动态诱导骨髓间充质干细胞复合可注射型支架材料Pluronic F-127修复关节软骨缺损[J].中国组织工程研究与临床康复.2007,11(14)：2609-2612.
[2]Temenoff JS,Mikos AG.Injectable biodegradable materials for orthopedic tissue engineering[J].Biomat erials,2000,21 (23):2405-2412.
[3]Lee CR,Breinan HA,Nehrer S,et al.Articular cartilage chondrocytes in type Ⅰ and type Ⅱ collagen-G AG matrices exhibit contractile behavior in vitro[J].Tissue Eng,2000,6(5):555-565.
[4]杨贵勇,卢世璧,王继芳,等.自体骨膜游离移植修复髋膝关节软骨大面积缺损的远期观察[J].中国修复重建外科杂志,2004,18(1)：8-11.
[5]Czitrom AA,Keating S,Gross AE,et al.The viability of artic-ular cartilage in fresh osteochondral allo grafts after clinical transplantation[J].J Bone Joint Surg(Am),1990,72(4):574-581.
[6]皇甫小桥,赵金忠,杨星光.关节镜下自体骨软骨移植修复软骨缺损[J].中国修复重建外科杂志,2008,22(7)：891-893.
[7]谭琪,田清业,刘光军,等.同种异体软骨移植修复关节软骨缺损的实验研究[J].国际骨科学杂志,2008,29(5)：336-339.
[8]孙涛,王英振.同种异体软骨细胞移植术后关节软骨蛋白多糖的测定[J].中国骨与关节损伤杂志,2008,23(3)：214-217.
[9]孙骏,侯筱魁,李旭,等.人转化生长因子β1基因转染骨髓基质干细胞复合藻酸钙修复骨软骨缺损[J].中国组织工程研究与临床康复,2008,12(29)：5636-5638.
[10]Langer R,Vacanti JP.Tissue engineering [J].Science,1993,260(5110):920-926.
[11]霍建忠,陈峥嵘,蒋淳,等.转化生长因子TGF-β 1基因修饰骨髓间充质干细胞复合壳聚糖修复兔关节软骨缺损[J].中华风湿病学杂志,2005,9(7)：393-396.
[12]王会才,张震宇,辛伟光.微重力三维动态诱导骨髓间充质干细胞复合可注射型支架材料Pluronic F-127修复关节软骨缺损[J].中国组织工程研究与临床康复,2007,11(11)：2609-2611.
[13]陈飞雁,顾湘杰,钟明康,等.威灵仙注射液对骨关节炎关节液与软骨白介素-1水平的影响[J].中国矫形外科杂志,2004,12(7)：524-526.
[14]陈飞雁,王旭,黄加张,等.威灵仙注射液对骨关节炎模型动物软骨组织形态和胶原表达的影响[J].中国矫形外科杂志,2006,14(5)：360-363.
[1]Manning WK,Bonner WM.Isolation and culture ofchondrocytes from human articular cartilage[J].Arthritis Rheum,1967,10(1):235-239.
[2]宁旭,尚显文,尹培荣,等.胶原酶一次消化法培养兔软骨细胞[J].贵州医药,2004,28(6)：490.
[3]Hu DN,Yang PY,KuMC,et al.Isolation and cultivation of human articular chondrocytes[J].Kaohsiung J MedSci,2002,18(3)：113.
[4]周强,李起鸿,戴刚,等.三步酶消化法高效分离兔原代关节软骨细胞及体外培养观察[J].中华外科杂志,2005,43(8)：522.
[5]张志光,郑卫平,苏凯,等.兔关节软骨细胞的分离培养和形态学特征[J].中山大学学报,2004,25(1)：63-66.
[6]周新社,戴尅戎,汤亭亭,等.人负重关节软骨细胞的体外分离与培养[J].临床骨科杂志,2004,7(2)：206.
[7]李文辉,侯筱魁.软骨组织工程种子细胞及其培养方法[J].中华创伤骨科杂志,2003,5(3)：244-246.
[8]Rodrigues A,Pap S,Vacanti CA.The effect of long-term culture on the ability of human auricular chondrocytes to generate tissue engineered cartilage.In:Stard GB,Horch R,Tanczos E,eds[M].Biological matrices and tissue reconstruction.New York:Springer,1998:163-168.
[9]何清义,李起鸿,许建中,等.人关节软骨细胞的永生化及其表型诱导[J].中华创伤杂志,2002,18(12)：727-731.
[10]邓欢,黄年根,张吉翔.DNA甲基化与细胞周期[J].国际遗传学杂志,2006,9(2)：106-109.[11]赵瑜,冯齐凤,朱祝生,等.中药补肾方对体外培养软骨细胞影响[J].时珍国医国药杂志,2008,19(4)：960-961.
[12]冯坤,谢文,陈宝龙,等.5种淫羊藿对软骨组织生长和软骨细胞增殖影响的对比研究[J].中国中药杂志,2006,34(24)：2065-2067.
[13]叶蕻芝,李西海,梁文娜,等.透骨消痛颗粒含药血清对兔软骨细胞增殖影响的研究[J].中国临床药理学 与治疗学,2009,14(6)：624-628.
[14]朱文菁.MTT法分析培养成骨细胞的存活和增殖能力[J].上海医科大学学报,1995,22(33)：254.
[15]孙卫民,王慧琴.细胞因子研究方法学[M].北京：人民卫生出版社,1999,65-67.
[16]朱萱萱,支月芳,邱召娟,等.痛痹颗粒及拆方的含药血清对家兔软骨细胞增殖与凋亡的影响[J].实用中医内科学杂志,2006,20(6)：603-604.
[17]王新东,张前德,纪伟.痛痹方血清对体外培养乳兔关节软骨细胞增殖和MMP-3与TIMP-1的影响[J].上海中医药杂志,2006,40(6)：64-65.
[18]章敏,王勇,李媛媛,等.痹痛灵颗粒含药血清对家兔关节软骨细胞体外培养的影响[J].中华中医药杂志,2009,24(1)：75-77.
[19]欧阳冰,林绵辉,左文建,等.结缔组织生长因子对体外培养的兔关节软骨细胞增殖和表型的影响[J].中国组织工程研究与临床康复,2009,13(20)：3816-3820.
[20]Lieberman JR,Dalusikl A,Einhorn TA.The role of growth factors in the repair of bnoe、biology and clinical applicatinos[J].J bone Joint Sugr Am JT,2002,84A(6):1032-1044.
[21]Frenkel SR,Saadeh PB,Mehrara BJ,et al.Transforming growth factor beta superfamily members:role in cartilage modeling.Plast Reconstr Surg.2000,5(3):980-990.
[22]王传家,李宇,纪影畅,等.TGF-β1对猪关节软骨细胞合成糖胺多糖的作用[J].汕头大学医学院学报,2003,16(3)：159-161.
[23]Blumenfeld I,Gaspar R,Laufer D,et al.Enhancement of toluidine blue staining by transf-orming growth factor-beta、insulinlike growth factor and growth hormone in the temporo-mandibular joint of aged mice.Cell Tissue Organ.2000;167:121-129.
[24]亓建洪,赵庆华,朱宝林,等.TGF-β1对人软骨细胞基质金属蛋白酶-1mRNA及其阻滞剂-1mRNA表达的影响[J].中华创伤骨科杂志,2005,7(8)：753-757.
[25]Sporn MB,Roberts AB,Wabefield,et al.Some recent advances in thechemistry and biologyof transforming growth factor-betaCell Biol.1987;5(3):1039.
[26]Ferguson CM,Schwarz EM,Reynolds PR,et al.Smad 2 and 3 mediate transforming gro-wth factor-betal-induced inhibition of chondrocyte maturation.Endocrinology.2000;141(12):4728-4735.
[27]王东武,杨柳,段小军,等.可注射温固化支架复合异体软骨细胞修复关节软骨缺损的初步研究[J].重庆医学,2006,35(12)：1082-1087.
[28]Yang K-C,Wu C-C,Lin F-H,et al.Chitosan/gelatin hydrogel as immunoisolative matrix for injectable bioartificial pancreas[J].Xenotransplantation,2008,15:407-416.
[29]Bryan J.Studies on donal cartilage strains II selective effects of diffeveuf growth conditions.Exp Cell' Res.1988;52:327-337.
[30]Cruz M. Structural lesion and disability-not only rheumatoid arthritis but also osteoart-hritis.Acta Reumatol Port,2008,33(4):475-476.
[31]彭程,林绵辉,肖涛.细胞因子与软骨损伤及修复研究进展[J].国际骨科学杂志,2006,27(4)：239-243.
[32]Chetina EV, Pul AR.The role of growth factors in inhibition of collagen degeneration and chondrocyte differentiation in patients with osteoarthrosis.Vestn Ross Akad Med Nauk,2008,(9):40-45.
[33]Veilleux N,Spector M. Effects of FGF-2 and IGF-1 on adult canine articular chondrocytes in type Ⅱ collagen-glycosaminoglycan scaffolds in vitro. Osteoarthritis Cartilage.2005,13(4):278-286.
[34]Hicks DL,Sage AB,Shelton E,et al. Effect of bone morPHogenetic proteins 2 and 7 on septal chondrocytes in alginate. Otolaryngol Head Neck Surg,2007,136(3):373-379.
[35]王勇,陆长青,王凡.丹参诱导大鼠骨髓间充质干细胞向神经样细胞分化[J].解剖学杂志,2007,30(2)：207-210.
[36]李楠,王和鸣,李文顺,等.龟鹿二仙胶汤含药血清对兔体外BMSCs向软骨细胞分化的干预作用.上海中医药大学学报,2007,2 1(6)：58-61.
[37]刘维统,王跃,郝鹏,等.黄芪甲苷对关节软骨细胞功能影响的实验研究.实用医院临床杂志,2009,6(4)：44-47.
[38]吴涛,徐俊昌,南开辉,等.淫羊藿苷促进羊骨髓间充质干细胞的增殖和成骨分化.中国组织工程研究与临床康复,2009,13(19)：3725-3729.
[39]杜晓岩,毛艳,李慕勤,等.羟基磷灰石-磷酸三钙／壳聚糖骨碎补复合材料修复骨缺损的实验研究.中国美容医学,2009,18(、8)：1125-1127.
[40]彭锐,李胜利,邹阳.明胶白芨胶／血竭多孔材料的生物相容性评价.中国中医骨伤科杂志,2007,15(1)：38-40.
[41]张杨,李秀兰,张文海,等.含有生肌液的组织工程皮肤修复兔皮肤缺损.中国组织工程研究与临床康复,2008,12(46)：9001-9005.
[42]Jeong B,Kim SW,Bae YH.Thermosensitive sol-gel reversible hydrogels.Adv Drug Deliv Rev.2002,54:37-51.
[43]Risbud M, Ringe J, Bhonde R, et al. In vitro expression of cartilage specific markers by chondrocytes on a biocompatible hydrogel:implications for engineering cartilage tissue[J]. Cell Transplant,2001,10(8):755。
[44]Chenite A,Chaput C,Wang D,et al.Novel injectable neutral solutions of chitosan form biodegradable gels in situ.Biomaterials,2000,21(21):2155-2161.
[45]高学敏.中药学[M].北京：人民卫生出版社,2000：645-655.
[46]陈飞雁,顾湘杰,钟明康,等.威灵仙注射液对骨关节炎关节液与软骨白介素-1水平的影响[J].中国矫形外科杂志,2004,12(7)：524-526.
[47]陈飞雁,王旭,黄加张,等.威灵仙注射液对骨关节炎模型动物软骨组织形态和胶原表达的影响[J].中国矫形外科杂志,2006,14(5)：360-363.
[48]Hua Wu,Ying Wan,Xiaoying Cao,et,al.Proliferation of chondrocytes on porous poly(DL-lactide)/chitosan scaffolds[J]. Acta Biomaterialia,2008,4:76-87.
[49]Yang KQSaris DB,Geuze RE,et al.Impact of expansion and redifferentitation conditions on chondrogenic capacity of cultured chondrocytes.Tissue Eng,2006,12(9):2435-2447.
[50]ATSUSHI FUNAYAMA,YASUO NIKI,HIDEO MATSUMOTO,et al.Repair of full-thickness articular cartilage defects using injectable type Ⅱ collagen gel embedded with cultured chondrocytes in a rabbit model[J].J Orthop Sci,2008,13:225-232.
[51]Atsushi Funayama,Uuayama,Yasuo Niki,Hideo Matsumoto,et al.Repair of full-thickness articular cartilage defects using injectable type Ⅱ collagen gel embedded with cultured chondrocytes in a rabbit model[J].J Orthop Sci 2008,13(3):225-232.
[52]Wakitani S,Nawata M,Tensho K.Repair of articular cartilage defects in the patello-femoral joint with autologous bone marrow mesenchymal cell transplantation:three case reports involving nine defects in five knees[J].Tissue Eng Regen Med,2007,1(1):74-79.
[53]Jubeck B,Muth E,Gohr CM.Type Ⅱ collagen levels correlate with mineralization by articular cartilage vesicles[J].Arthritis Rheum,2009,60(9):2741-2746.
[1]O'Driscoll SW. The healing and regeneration of articular cartilage[J]. Bone Joint Surg Am,1998,80: 1795-1812.
[2]王传家等,转化生长因子-β1对猪关节软骨细胞合成糖胺多糖的作用[J].汕头大学医学院学报,2003,16(3)：159-161.
[3]Lee KH,Song SU,Hwang TS,et al.Regeneration of hyaline cartilage by cell-mediated gene therapy using transforming growth factor beta 1 producing fibroblasts[J]. Hum Gene Ther,2001,12:1805-181.
[4]Madry H,Emkey G,Zurakowski D,et al. Overexpression of human fibroblast growth factor 2 stimulates cell proliferation in an ex vivo model of articular chondrocyte transplantation[J].Gene Med,2004,6(2):238-45.
[5]Mizuta H, Kudo S, Nakmura E,et al. Active proliferation of mesenchymal cells prior to the chond rogenic repair response in rabbit full-thickness defects of articular cartilage.Osteoarthritis cartilage,2004, 12(7):586-596.
[6]Schmidt MB, chen EH, Lynch SE A review of the effects of insulin-like growth factor and platelet derived growth factor on in vivo cartilage healing and repair[J].Osteoarthritis cartilage.2006,14(5):403-412.
[7]Tian H, Stogiannidis I Up-regulation of cartilage digomeric matrix protein gene expression by insulin-like growth factor-1 revealed by real-time reverse transcription-polymerase chain reaction[J].Acta Biochim BioPHy Sin(shanghai),2006,38(10):677-682.
[8]Brower-Toland BD,Saxer RA,Goodrich LR, et al.Direct adenovirus mediated insulin-like growth factor I gene transfer enhances transplant chondrocyte function[J].Hum Gene Ther,2001,12:117-129.
[9]Fukuoka H,Shibata S,Suda N,et al,Bone morPHogenetic protein rescues the lack of secondary cartilage in Runx2-deficient mice [J]J Anat,2007,211(1):8-15.
[10]倪滨,李明.BMP生物活性的研究现状[J].重庆医学,2007,36(10)：970-973.
[11]Grimsrud CD,Romano PR,D'Souza M,et al.BMP-6 is an autocrine stimulator of chondrocyte differentiation[J].J Bone Min Res 1999,14(4):475-482.
[12]刘凌,张洪涛,王现彬等.腺病毒介导骨形态发生蛋白2基因对兔软骨细胞的影响[J].中国组织工程研究与临床康复.2008,12(50)：9811-9813.
[13]Mason JM,Breitbart AS,Barcia M,et al.Cartilage and bone regeneration using gene enhanced tissue engineering[J].Clin Orthop 2000,(379Suppl):S171-S178.
[14]Madry H,Zurakowski D,Trippel SB.Overexpression of human insulin-like growth factor-I promotes new tissue formation in anexvivomodel of articular chondrocyte transplantation[J]. Gene Ther,2001,8:1443-1449.
[15]Nixon AJ,Brower-Toland BD,Bent SJ,et al,Insulinlike growth factor-1 gene therapy applications for cartilage repair[J].Clin Orthop 2000,390:201-13.
[16]Oligino T,Ghivizzani S, Wolfe D,et al.Intra-articular delivery of a herpes simplex virus IL-1Ra gene vector reduces inflammation in a rabbit model of arthritis[J].Gene Ther,1999,6:1713-1720.
[17]Kwon GY, Jeong J,Woo JK,et al. Co-expression of bfl-1 enhanceshost response in the herpes simplex virus-thymidine kinase/ganciclovir gene therapy system [J]. Biochem BioPHys Res Commun,2003,303(3): 756-759.
[18]曲福军,侯宣,周余来等.重组pcDNA3.1-BMP7转染兔膝关节软骨细胞及其表达[J].中国临床康复.2006,10(17)：79-82.
[19]Goomer RS,Deftos LI,Terkeltaub R,et al.High-efficiency non-viral transfection of primary chondrocytes and perichondrial cells for ex vivo gene therapy to repair articular cartilage defects[J]. Osteoarthritis Cartilage,2001,9:248-256.
[20]Brunner S,Saneri,Cerottas,et al.Cell cycle dependence of gene transfer by lipoplex,polyplex and recombinant adenovirus [J].Gene The 2000,7:401-7.
[21]Evans CH,Ghivizzani SC,Kang R et al. Gene therapy for rheumatic diseases[J].Arthritis Rheum,1999, 42(1):1-16.
[22]Grande DA,Breitbart AS,Mason J,et al.Cartilage tissue engineering:current limitations and solution s[J].Clin Orthop Relat Res,1999,367(suppl):s176-s186.
[23]Mason JM, Grande DA,Barcia M, et al. Expression of human bone morPHogenic protein 7 in primary rabbit periosteal cells:potential utility in gene therapy for osteochondral repair[J].Gene Ther,1998, 5(8)：1098-1104.
[24]秦宏敏,沙广钊,佟向阳等.兔骨髓间充质干细胞经Ad-TGF β1重组腺病毒载体转染定向分化为软骨细胞[J].中国组织工程研究与临床康复.2007,11(42)：8444-8448.
[25]Gelse K,Jiang QJ,Aigner T,etal.Fibroblast-mediated delivery of growth factor complementary DNA into mouse joints induces chondrogenesis but avoids the disadvantages of direct viral gene transfer[J]. Arthritis Rheum,2001; 44(8):1943-1953.
[1]Langer R,Vacanti JP.Tissue engineering [J].Science.1993,260(5110):920-926.
[2]靳小兵,组织工程方法修复关节软骨缺损[J].中国微创外科杂志.2005,5(8)：685-687.
[3]Friendenstein A J,Gorskaja J F,Kulagina N N.Fibroblast Precursors in Normal and Irradi-ated Mouse Hema-topoietic Organs[J].Exp Hematol.1976,4(5):267-274.
[4]Gerstenfeld LC,Ctuceta J,Shea C M,et al.Chondro-cytes provide morPHogenic signals that selectivel y induce os-teogenic differentiation of mesenchymal stem cell[J].J BoneMiner Re-s.2002,17(2):221.
[5]Pereira R F,Halford K W,O'hara M D,et al.Cul-tured Adherent Cells from Marrow can Serve as Pr ecursorCells for Bone,Cartilage,and Lung in Irradiated Mice[J].ProcNatl Acad S-ci USA.1995,92:4857-4 861.
[6]马力,刘大军,李德天,等.不同分离方法及培养条件对兔骨髓间充质干细胞生长增殖及生物学特性的影响[J].中国组织工程研究与临床康复.2008,12(38)：7401-7406.
[7]蒋文慧,马爱群,董安平,等.骨髓间质干细胞的分离培养与鉴定[J].西安交通大学学报(医学版).2005,26(3)：224-227.
[8]赵瑛.SD大鼠骨髓间充质干细胞的分离培养和鉴定[J].重庆师范大学学报(自然科学版).2008,25(1)：75-77.
[9]王超,徐蕴,宋文刚,等.大鼠骨髓间充质干细胞分离培养方法的建立及其表型分析[J].ISSN 1007-8738细胞与分子免疫杂志.2007,23(5)：466-468.
[10]苏秋香,王忠华,王淑华,等.不同分离液和培养基对骨髓间充质干细胞原代培养的影响研究[J],沈阳医学院学报.2005,7(4)：252-254.
[11]张永先,蔡锦方,汤继文,等.兔骨髓基质细胞的分离培养[J].中华显微外科杂志.2004,27(2)：120-123.
[12]马力,刘大军,李德天,等.不同分离方法及培养条件对兔骨髓间充质干细胞生长增殖及生物学特性的 影响[J].中国组织工程研究与临床康复.2008,12(38)：7401-7406.
[13]王超,徐蕴,宋文刚,等.大鼠骨髓间充质干细胞分离培养方法的建立及其表型分析[J].ISSN 1007-8738细胞与分子免疫杂志.2007,23(5)：466-468.
[14]霍建忠,陈峥嵘,蒋淳,等.转化生长因子TGF-β1基因修饰骨髓间充质干细胞复合壳聚糖修复兔关节软骨缺损[J].中华风湿病学杂志.2005,9(7)：393-396.
[15]杨物鹏,许建中.BMP-2和TGF β1对培养兔关节软骨细胞代谢的影响[J].第三军医大学学报.2000,22(12)：1139-1141.
[16]Madry H,Kaul QCucchiarini M,et al.Enhanced repair of articular cartilage defects in vivo by trans planted chondrocytes over expressing insulin-like growth factor Ⅰ (IGF-Ⅰ)[J].Gene Ther.2005,12(5):117 1-1179.
[17]Kotev-EmethS,PitaruS,Pri-ChenS,etal.Establishmentofaratlong-termcultureexpressingtheosteogenicPHeno type:dependenceon dexamethasone and FGF-2[J].Connect Tissue Res.2002,43:606-612.
[18]俞猛,夏仁云,高飙,等.bFGF和地塞米松对骨髓基质干细胞增殖与分化的影响[J].中国康复.2006,21(1)：12-15.
[19]Hughes S E, Hall P A.The fibroblast growth factor and receptor moltigene families [J].J Patho.19 93,170(3):219-21.
[20]Gelse K,von der Mark K.Articular cartilage repair by gene therapy using growth factor-producing mesenchymal cells[J].Arthritis Rheum.2003,48(2):430-441.
[21]丁幸坡,金先庆,刘伟.HIGF-1基因修饰骨髓基质干细胞复合PLGA移植修复关节软骨缺损[J].重庆医科大学学报.2006,31(3)：354-357.
[22]孙骏,侯筱魁,李旭,等.人转化生长因子β1基因转染骨髓基质干细胞复合藻酸钙修复骨软骨缺损[J].中国组织工程研究与临床康复.2008,12(29)：5636-5638.
[23]许建中,范东伟,张文霞.经Ⅱ型胶原修饰后的PLGA对诱导后的骨髓基质干细胞粘附及分化的影响[J].郑州大学学报.2005,40(3)：424-426.
[24]龙华,袁华,马保安,等.腺病毒介导TGF-β 1及BMP-7基因共表达感染骨髓基质干细胞修复兔关节软骨缺损[J].中国骨与关节损伤杂志.2008,23(6)：471-474.
[25]Corsi K, Chellat F, YahiaL, et al. Mesenchymal stemcells, MG63 and HEK293 transfe-ction using chitosan-DNA nanoparticles[J]. Biomaterials.2003,24(7):1255.
[26]Ponticiello MS,Schinagl RM,Kadiyala S,et al.Gelatin-based resorbable sponge as a carri-er matrix f or human mesenchymal stem cells in cartilage regeneration therapy[J].J Biomed Mater Res.2000,52(2): 246-255
[27]郝俊龙,汪玉良，孙正义,等.新型支架材料羟基磷灰石／聚磷酸钙纤维／左旋聚乳酸复合自体骨髓间充质干细胞修复关节软骨全层缺损[J].中国临床康复.2006,10(37)：64-67.
[28]丁幸坡,金先庆,刘伟HIGF-1基因修饰骨髓基质干细胞复合PLGA移植修复关节软骨缺损[J].重庆医科大学学报.2006,31(3)：354-357.
[29]王觅格,夏亚一,王栓科,等.骨髓间充质干细胞复合消旋聚乳酸／明胶修复兔关节软骨缺损的实验研究[J].中国修复重建外科杂志.2007,21(7)：753-758.
[30]王会才,张震宇,辛伟光.微重力三维动态诱导骨髓间充质干细胞复合可注射型支架材料Pluronic F-127修复关节软骨缺损[J].中国组织工程研究与临床康复.2007,11(14)：2609-2612.
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