几种构建组织工程关节软骨方法的比较研究
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摘要
软骨病变是骨科较为常见的疾患。创伤、骨软骨炎、骨性关节炎、髌骨软化等均可引起软骨的病损。但关节软骨的病变难以自身修复，自1743年Hunter提出软骨一旦破坏即不可自身修复，两个半世纪过去了，至今软骨病变仍无理想的治疗办法，关节软骨的病变最终导致骨性关节炎而不得不接受关节置换。
组织工程软骨的研究为修复关节软骨的病变开辟了新途径。1987年瑞典人Britterg进行了第一例临床应用，目前全世界已经有超过10，000例，透明质酸钠和胶原膜复合自体软骨细胞构建的组织工程软骨已经作为产品推广应用。大量文献证明，利用组织工程软骨，能产生类似正常关节软骨的透明软骨组织，且大宗病例长期随访资料证明临床效果优良。
影响组织工程软骨质量的因素很多，但主要可以概括为三个方面：种子细胞、支架和构建方法。本课题的目的主要是通过比较研究寻找优化的制备组织工程软骨的技术方法，并为临床应用搭建技术平台。
自体软骨细胞是目前唯一应用于临床的软骨组织工程种子细胞，但成熟软骨细胞扩增困难，自体软骨组织取材有限，如何从极少量关节软骨组织中分离、培养并大量扩增出足够数量和优质的软骨细胞，是组织工程软骨临床应用的瓶颈。取材组织消化获取的原代软骨细胞的数量和活性直接决定了细胞扩增的时间周期、最终获得的细胞的数量、可传代次数和细胞的质量，从而进一步影响组织工程软骨修复的质量。本实验从兔的关节软骨做起，观察组织块剪切的大小、消化收集步骤以及胰酶和胶原酶浓度等对获取软骨细胞的影响，证明对少量关节软骨组织，用培养基配制的0.2％的胶原酶直接消化一步收集细胞的方法是比较优化的消化分离细胞的方法。成熟软骨细胞传3～4代后增殖逐渐缓慢，趋向停滞，无法达到组织工程软骨所需要的细胞数量，通过对软骨细胞培养中培养基、添加成份、血清及生长因子等方面的优化组合研究，表明添加了维生素C、脯氨酸、胰岛素、非必须氨基酸等成分的软骨
博士论文:几种构建组织工程关节软骨方法的比较研究
中国人民解放军军医进修学院
细胞培养基对软骨细胞生长更有利。继之对8例成年人关节软骨细胞体外培
养条件进行研究，结果表明使用20%胎牛血清效果优于10cy0;人AB血清优于
胎牛血清;10%人血清的浓度是足够而且合适的;FGF一2、TGF一pl、PDGF一bb、
HGF四种因子单独使用最佳促增殖浓度分别为:SOng/m1、ing/m1、Ing/ml、
Zong/ml。sng/ml FGF一2与一ng/ml TGFpl组合是一种比较好的适于成年人关
节软骨细胞生长的因子组合，它在促进软骨细胞增殖的同时，也较好的保持
了软骨细胞的分化表型。
本实验通过在兔膝关节陈旧性软骨缺损模型上对三种自体软骨细胞移植
方法—自体软骨细胞骨膜下注射、骨膜下细胞团块移植和细胞复合胶原支
架的比较研究，我们发现骨膜下细胞团块移植是比较好的修复软骨缺损的手
段，操作简单，修复效果好。
本实验用目前研究较多的三种支架—胶原、脱钙骨基质(DBM)和PLGA
组成工程软骨，观察其对修复兔膝关节软骨陈旧性缺损的效果，结果表明胶
原蛋白海绵是比较好的组织工程软骨支架材料，DBM需要进一步研究改进材料
孔径，而PLGA由于其代谢产物的毒性作用，对关节软骨修复的效果较差。
由于自体关节软骨组织来源困难，造成关节再损伤，本实验利用藻酸凝
胶包埋同种异体软骨细胞修复兔膝关节软骨缺损，取得了很好的修复效果，
表明同种异体软骨细胞作为软骨组织工程的种子细胞是可行的，可以作为对
自体软骨组织取材困难、培养失败的替代方法;通过对脂肪组织来源干细胞
的分离、培养、鉴定、定向分化过程及修复兔关节软骨缺损的实验研究，表
明脂肪千细胞是较好的软骨组织工程干细胞来源。
Articular cartilage defects resulted from trauma, osteoarthritis and osteochondritis diseases is a major challenges for the orthopedic surgeons. Because of the unique feature of articular cartilage which is avascular and the differentiated chondrocytes trapped within compact matrix, mobilization of regenerative cells to areas of injury is insufficient; cartilage tissue has a limited self-regenerative capacity. None of the current surgical options for treating cartilage defects are satisfactory, including lavage and debridement, shaving the cartilage surface, perforating the underlying subchondral bone, periosteal or perichondrial or cartilage transplantation and osteotomy. These procedures are insufficient to resurfacing large articular cartilage defect with lasting hyaline-like cartilage. Cartilage damage may lead to gradual degeneration of joint and ultimately artifical prosthesis needed.
Engineered cartilage tissue offers a new strategy for the repair and regeneration of damage or diseased articular cartilage. The first clinical application of autologous chondrocyte implantation (ACI) was reported by Mats Brittberg in 1987. Since then, more than 10,000 cases have been performed worldwide. ACI-P is considered as the first generation tecqnic which is injected autologous chondrocytes beneath the periosteal flap. The second generation (ACI-M) is base on the type I /III collagen membrane or hyaluronate derived scaffold. Now all three procedures are used extensively worldwide. ACI or tissue engineered cartilage has shown very promising results, and high percentage of hyaline-like cartilage tissue would present in the cartilage defects. Retrospective studies of more than 1,000 cases over 10 years showed that 74% had good to excellent results
Many factors influence the quality of tissue engineered cartilage and the key
elements include cells, scaffolds and transplanting methods. The purpose of this study was to establish a simple, safe, effective and low-cost procedure to construct engineered articular cartilage, which can be used for clinical treatment.
Autologous chondrocyte is still the only one permitted in clinical usage. Even the limit cartilage tissue could be obtained from biopsies, and the limit capacity of proliferation of mature chondrocytes. The first technical challenge is in vitro generation of human chondrocytes from the little biopsies ranged from 200 to 300 milligrams. Isolating chondrocytes from the biopsy is the first key process and the quantity of living cells obtained will determine directly proliferating quantity and quality of the collection cells, So, size of cutting cartilage pieces, procedures for digesting and the methods of collecting cells, the proper concentration of trypsine and collagenase were studied, The resaults showed that for adult human articular cartilage biopsies, cutting the biopsies to sand-like, digesting with 0.2% collagenase in medium, and collecting cells by one step may be the optimum procedure. Different medium, different cytokines, and different serum were also compared, The results showed that: 1,Adding some supplement substances such as vitamin C, proline, insulin and none essential amino acids (NEAA) are necessary. 2, The optimal concentration of four cytokines,FGF-2, TGF-P1, PDGF-bb and HGF, when used separately, is 50ng/ml, 1ng/ml, 1ng/ml, 20ng/ml respectively. While 5ng/ml FGF-2 combined with Ing/ml TGFbeta1 could achieve the best effect, adding PDGF-bb, HGF or both could not enhance proliferation.3, Human AB serum is better than fetal bovine serum (FBS), the proper concentration of human AB serum is 10% .
Three transplanting materials of ACI were applied in the full-thickness defects in femoral condyles of adult rabbits, 1, chondrocytes, 2,conglomeration of chondrocyte, 3,chondrocyte-seeded collagen scaffold, all transplanted beneath the periosteal flap. The primary results showed that last two methods had better results
than that of chondrocytes injecting. Also we compared the methods for fixing periosteal flap, and
组织工程软骨的研究为修复关节软骨的病变开辟了新途径。1987年瑞典人Britterg进行了第一例临床应用，目前全世界已经有超过10，000例，透明质酸钠和胶原膜复合自体软骨细胞构建的组织工程软骨已经作为产品推广应用。大量文献证明，利用组织工程软骨，能产生类似正常关节软骨的透明软骨组织，且大宗病例长期随访资料证明临床效果优良。
影响组织工程软骨质量的因素很多，但主要可以概括为三个方面：种子细胞、支架和构建方法。本课题的目的主要是通过比较研究寻找优化的制备组织工程软骨的技术方法，并为临床应用搭建技术平台。
自体软骨细胞是目前唯一应用于临床的软骨组织工程种子细胞，但成熟软骨细胞扩增困难，自体软骨组织取材有限，如何从极少量关节软骨组织中分离、培养并大量扩增出足够数量和优质的软骨细胞，是组织工程软骨临床应用的瓶颈。取材组织消化获取的原代软骨细胞的数量和活性直接决定了细胞扩增的时间周期、最终获得的细胞的数量、可传代次数和细胞的质量，从而进一步影响组织工程软骨修复的质量。本实验从兔的关节软骨做起，观察组织块剪切的大小、消化收集步骤以及胰酶和胶原酶浓度等对获取软骨细胞的影响，证明对少量关节软骨组织，用培养基配制的0.2％的胶原酶直接消化一步收集细胞的方法是比较优化的消化分离细胞的方法。成熟软骨细胞传3～4代后增殖逐渐缓慢，趋向停滞，无法达到组织工程软骨所需要的细胞数量，通过对软骨细胞培养中培养基、添加成份、血清及生长因子等方面的优化组合研究，表明添加了维生素C、脯氨酸、胰岛素、非必须氨基酸等成分的软骨
博士论文:几种构建组织工程关节软骨方法的比较研究
中国人民解放军军医进修学院
细胞培养基对软骨细胞生长更有利。继之对8例成年人关节软骨细胞体外培
养条件进行研究，结果表明使用20%胎牛血清效果优于10cy0;人AB血清优于
胎牛血清;10%人血清的浓度是足够而且合适的;FGF一2、TGF一pl、PDGF一bb、
HGF四种因子单独使用最佳促增殖浓度分别为:SOng/m1、ing/m1、Ing/ml、
Zong/ml。sng/ml FGF一2与一ng/ml TGFpl组合是一种比较好的适于成年人关
节软骨细胞生长的因子组合，它在促进软骨细胞增殖的同时，也较好的保持
了软骨细胞的分化表型。
本实验通过在兔膝关节陈旧性软骨缺损模型上对三种自体软骨细胞移植
方法—自体软骨细胞骨膜下注射、骨膜下细胞团块移植和细胞复合胶原支
架的比较研究，我们发现骨膜下细胞团块移植是比较好的修复软骨缺损的手
段，操作简单，修复效果好。
本实验用目前研究较多的三种支架—胶原、脱钙骨基质(DBM)和PLGA
组成工程软骨，观察其对修复兔膝关节软骨陈旧性缺损的效果，结果表明胶
原蛋白海绵是比较好的组织工程软骨支架材料，DBM需要进一步研究改进材料
孔径，而PLGA由于其代谢产物的毒性作用，对关节软骨修复的效果较差。
由于自体关节软骨组织来源困难，造成关节再损伤，本实验利用藻酸凝
胶包埋同种异体软骨细胞修复兔膝关节软骨缺损，取得了很好的修复效果，
表明同种异体软骨细胞作为软骨组织工程的种子细胞是可行的，可以作为对
自体软骨组织取材困难、培养失败的替代方法;通过对脂肪组织来源干细胞
的分离、培养、鉴定、定向分化过程及修复兔关节软骨缺损的实验研究，表
明脂肪千细胞是较好的软骨组织工程干细胞来源。
Articular cartilage defects resulted from trauma, osteoarthritis and osteochondritis diseases is a major challenges for the orthopedic surgeons. Because of the unique feature of articular cartilage which is avascular and the differentiated chondrocytes trapped within compact matrix, mobilization of regenerative cells to areas of injury is insufficient; cartilage tissue has a limited self-regenerative capacity. None of the current surgical options for treating cartilage defects are satisfactory, including lavage and debridement, shaving the cartilage surface, perforating the underlying subchondral bone, periosteal or perichondrial or cartilage transplantation and osteotomy. These procedures are insufficient to resurfacing large articular cartilage defect with lasting hyaline-like cartilage. Cartilage damage may lead to gradual degeneration of joint and ultimately artifical prosthesis needed.
Engineered cartilage tissue offers a new strategy for the repair and regeneration of damage or diseased articular cartilage. The first clinical application of autologous chondrocyte implantation (ACI) was reported by Mats Brittberg in 1987. Since then, more than 10,000 cases have been performed worldwide. ACI-P is considered as the first generation tecqnic which is injected autologous chondrocytes beneath the periosteal flap. The second generation (ACI-M) is base on the type I /III collagen membrane or hyaluronate derived scaffold. Now all three procedures are used extensively worldwide. ACI or tissue engineered cartilage has shown very promising results, and high percentage of hyaline-like cartilage tissue would present in the cartilage defects. Retrospective studies of more than 1,000 cases over 10 years showed that 74% had good to excellent results
Many factors influence the quality of tissue engineered cartilage and the key
elements include cells, scaffolds and transplanting methods. The purpose of this study was to establish a simple, safe, effective and low-cost procedure to construct engineered articular cartilage, which can be used for clinical treatment.
Autologous chondrocyte is still the only one permitted in clinical usage. Even the limit cartilage tissue could be obtained from biopsies, and the limit capacity of proliferation of mature chondrocytes. The first technical challenge is in vitro generation of human chondrocytes from the little biopsies ranged from 200 to 300 milligrams. Isolating chondrocytes from the biopsy is the first key process and the quantity of living cells obtained will determine directly proliferating quantity and quality of the collection cells, So, size of cutting cartilage pieces, procedures for digesting and the methods of collecting cells, the proper concentration of trypsine and collagenase were studied, The resaults showed that for adult human articular cartilage biopsies, cutting the biopsies to sand-like, digesting with 0.2% collagenase in medium, and collecting cells by one step may be the optimum procedure. Different medium, different cytokines, and different serum were also compared, The results showed that: 1,Adding some supplement substances such as vitamin C, proline, insulin and none essential amino acids (NEAA) are necessary. 2, The optimal concentration of four cytokines,FGF-2, TGF-P1, PDGF-bb and HGF, when used separately, is 50ng/ml, 1ng/ml, 1ng/ml, 20ng/ml respectively. While 5ng/ml FGF-2 combined with Ing/ml TGFbeta1 could achieve the best effect, adding PDGF-bb, HGF or both could not enhance proliferation.3, Human AB serum is better than fetal bovine serum (FBS), the proper concentration of human AB serum is 10% .
Three transplanting materials of ACI were applied in the full-thickness defects in femoral condyles of adult rabbits, 1, chondrocytes, 2,conglomeration of chondrocyte, 3,chondrocyte-seeded collagen scaffold, all transplanted beneath the periosteal flap. The primary results showed that last two methods had better results
than that of chondrocytes injecting. Also we compared the methods for fixing periosteal flap, and
引文
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alginate/chondrocyte constructs: effects of guluronic acid content and matrix synthesis, Journal of Orthopaedic Research, 2001, 19: 493-499.
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[61] Mizuno H, Zuk PA, Zhu M, at al. Myogenic differentiation by human processed lipoaspirate cells. Plastic and Reconstructive Surgery, 2002, 109(1): 199-211
[62] Gronthos S, Franklin DM, Leddy HA, et al. Surface protein characterization of human adipose tissue-derived stromal cells. J Cellular Physiology, 2001, 189: 54-63.
[63] Winter A, Breit S, Parsch D, et al. Cartilage-like gene expression in differential human stem cell spheriods: a comparison of bone marrow-derived and adi[pose tissue-derived stromal cells. Arthritis Rheum 2003, 48: 418-29.
[64] Wickham MQ, Erickson GR, Gimble JM, et al. Multipotent stromal cells derived from the infrapatellar fat of the knee. Clin Orthop, 2003, Jul: 196-212.
[65] Ahdjoudi S, Lasmoles F, Oyajobi BO, et al. Reciprocal control of osteoblast/chondroblast and osteoblast/adipocyte differentiation of multipotential clonal human marrow stromal F/STRO-1(-) cells. J cell Biochem, 2001, 81: 23-38.
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