摘要
【目的】：β-TCP是一种较理想的骨组织工程无机支架材料，传统的β-TCP生物陶瓷制作方法制作的生物陶瓷，其微结构难以控制，应用于软骨组织工程的较少。近年来法国地中海大学成功地研制了微孔结构可控的β-磷酸三钙多孔陶瓷材料，该材料不仅具有良好的生物相容性和较高的机械强度，而且可调控微结构以适合软骨组织工程构建的需要。我们和上海贝奥路公司合作，通过研究不同微结构的生物陶瓷支架复合软骨细胞体外培养的结果，发现适合软骨组织工程构建的生物陶瓷支架材料微结构。通过对兔解剖结构的测量，建立兔关节软骨缺损模型，进行体内生物陶瓷支架软骨修复动物实验，观察并评估软骨修复效果，从而找到适合体内软骨组织构建的生物陶瓷支架。为β-TCP材料进一步临床应用于关节软骨缺损的修复提供实验依据。
【方法】：(一)选取二月龄健康新西兰大白兔，耳缘静脉注射空气栓塞处死，取双侧股骨髁、肱骨及胫骨，剔除周围软组织，分别使用游标卡尺及64排CT重建进行测量，获取各项解剖数据。(二)消化分离成年兔肋软骨细胞及关节软骨细胞进行体外培养，观察两种软骨细胞的细胞获得率、存活率、细胞贴壁率、形态学变化、MTT法测定细胞增殖速度，并且进行甲苯胺兰染色、亮绿-番红花“0”染色、二甲基亚甲基蓝法测定硫酸糖胺多糖。从而优选出适合软骨组织工程研究的合适的软骨细胞及其传代数。(三)通过对肋软骨细胞复合不同微结构的生物陶瓷材料进行体外培养，计算软骨细胞在支架内增殖生长的不同趋势，优选出适合软骨细胞生长的β-TCP生物陶瓷支架的合适微结构。(四)利用专利技术，在前期研究的基础上，烧结出适合软骨组织工程构建的具有合适微结构的β-TCP生物陶瓷支架材料。(五)将体外实验优选的适合软骨细胞生长的微结构的β-TCP生物陶瓷支架材料复合肋软骨细胞植入兔关节软骨缺损模型中，以验证该微结构修复动物体内关节软骨缺损的可行性。
【结果】：(一)解剖测量的结果得到了兔股骨髁、肱骨及胫骨的各项数据，并且进行了CT三维重建，测量结果显示肱骨头软骨面至骺线距离4．5±0．2mm。胫骨内侧平台宽度为6．1±0．4 mm，外侧平台宽度为6．4±0．3mm，但胫骨平台表面覆盖有半月板组织，无法进行手术显露，这两个部位都无法满足体内试验的需要。股骨内侧髁宽度为5．0±0．2mm，外侧髁宽度为4．0±0．1mm，两侧髁的宽度都较小，并且表面形态较不规整，同样不满足体内试验的需要。股骨滑车宽度为6．5±0．5mm，滑车近髁间窝表面软骨至髓腔距离为9．1±0．6mm，并且表面形态较规整，可以适合体内试验的需要。(二)利用专利技术进行适合软骨组织工程研究的合适微结构β-TCP生物陶瓷支架材料的烧制，并且进行相关理化性能的表征检测。(三)两种软骨细胞体外培养的结果显示关节软骨：消化时间为6．49±1．87h，细胞获得率为5．79±1．7×10~5／100mg。肋软骨：消化时间为4．14±1．45h，细胞获得率为4．76±1．2×10~5／100mg，肋软骨细胞和关节软骨细胞在细胞获得率、存活率、贴壁率上都不具有统计学意义，而肋软骨细胞的消化时间却低于关节软骨细胞，P值为0．0032。二甲基亚甲基蓝法测定细胞传代培养液中GAG的含量，显示1代软骨细胞及2代软骨细胞分泌GAG的量和原代细胞相似无统计学差异，但从第3代软骨细胞开始则开始具有统计学差异。(四)对软骨细胞复合不同微结构的支架材料培养的结果显示，培养第一周，不同内连接径及孔径的支架材料中种植的肋软骨细胞的生长速度无明显差异，但是从第二周开始，在孔径相同的条件下内连接径120μm的支架材料生长速度明显快于其他内连接径的支架材料，这种情况一直延续到第四周。(五)进行体内动物实验认证，结果证明肋软骨细胞复合β-TCP生物陶瓷材料可以修复兔关节软骨缺损，4月后的2代肋软骨细胞组组织学评分为20．76±2．13，甲苯氨兰、番红及Ⅱ型胶原染色均显示软骨组织修复较为完全。
【结论】：
兔股骨滑车近髁间窝软骨缺损的动物模型是有效的，适合软骨细胞组织工程研究。
软骨种子细胞优选的结果发现肋软骨细胞在体外生长速度较快，分泌糖胺聚糖的量更多，从体外扩增数量的角度出发，2代软骨细胞较适合软骨组织工程构建的需要。
微结构不同的生物陶瓷支架材料对肋软骨细胞体外培养的结果是有差异的，孔径为500-630μm，内连接径120 m的生物陶瓷材料体外培养较适合软骨细胞体外生长的需要。
体内动物实验的结果发现，不同的复合方式软骨修复结果是有差异的。肋软骨细胞经体外培养至2代后复合生物陶瓷支架材料修复动物关节软骨缺损的结果较原代消化细胞直接复合支架材料修复动物关节软骨缺损效果为好。
硬质生物陶瓷材料完全可以作为软骨工程的支架材料。
硬质支架材料上层软骨细胞成软骨，下层软骨细胞则未成软骨组织，趋向转化成骨。无编改内容
【Objective】:
β-TCP is an ideal scaffold for bone tissue engineering of inorganicmaterials with attractive applications. However, the traditionalmaking-method ofβ-TCP bioceramics is hard to control the microstructureof the scaffold, soβ-TCP bioceramics were seldom applied to thecartilage tissue engineering.Recently,French Mediterranean Universityhas successfully developed a controllable porous structure ofβ-tricalcium phosphate porous ceramic materials.This material not onlyhas good biocompatibility and high mechanical strength,but aslo can caterto the cartilage tissue engineering reconstruction needs. With thecollaboration of Shanghai Bio-lu biomaterialcompany,by the means of theobservation of the results of chondrocytes cultured in differentmicrostructure bioceramic scaffolds in vitro,A suitable micro-structureof bioceramic scaffolds can be found. Through the measurement ofanatomical structure of rabbits,the rabbit model of articular cartilagedefects can be built up. Via animal experiments, the cartilagereconstruction effect ofβ-TCP material was observated and assessed,sothat suitable microstructure bioceramic scaffold can be found in vivo.The aim of this study is to provide an experimental basis for furtherclinical application ofβ-TCP bioceramic scaffold in articular cartilagedefect reconstruction.
【Methods】:
(1) Select the healthy New Zealand white rabbits age 2 months,ears fateof intravenous air embolism death,take bilateral femur,humerus and tibia,remove soft tissue respectively,and use of vernier caliper and 64-rowCT to to obtain the anatomical data.(2) With the patented technology andon the basis of preliminary studies, the sintering of suitabletissue-engineered cartilage constructed with appropriate micro-structure ofβ-TCP bioceramic scaffolds.(3) Isolated adult rabbitcostal cartilage and articular cartilage cells and cultured in vitro toobserve the two types of cartilage cells to obtain the rate,survival rate,the rate of cell adhesion,morphological changes,MTT determined cellproliferation rate,and to carry out a toluidine blue staining,saffron"0" stain, methylene blue dimethyl sulfate glycosaminoglycandetermination.Optimize the cartilage suitable algebra cartilage cellsand their mass in order to fit the study of tissue-engineering. (4)Composite of costal cartilage cell biology of different micro-structureof ceramic materials in vitro, the calculation of cartilage cellsproliferation of different growth trends within stent,optimize thesuitable microstructure ofβ-TCP bioceramic scaffold for the bettergrowth of cartilage cells.(5) Put composite of the scaffolds with optimalmicrostructure for growth of cartilage cells and costal cartilage cellsinto rabbit articular cartilage defect model in vitro,to verify thefeasibility of the micro-structure of the animals to repair articularcartilage defect.
【Results】:
(1) Anatomic measurement results obtained in rabbit femur,humerus andtibia of the data and conducted a three-dimensional reconstruction of CT,the measurement results show that humeral head cartilage from theepiphyseal line to 4.5±0.2mm.Medial tibial platform width of 6.1±0.4mm,width of lateral platform 6.4±0.3m m,but the surface coverageof the tibial plateau have meniscus tissue,surgery can not be revealed,the two parts of the body can not meet the required tests.Medial femoralcondyle width of 5.0±0.2mm,lateral condylar width of 4.0±0.1mm,the width of both sides of the condyle is smaller and less structuredsurface,the same test does not meet the needs of the body.Patellarsurface width of 6.5±0.5m m,the surface of condylar cartilage to marrowdistance of 9.1±0.6 mm,and more regular surface morphology,theseresults can fit the needs of the body.
(2) The use of patented technology is suitable to the study oftissue-engineered cartilage.
(3) The results of two types of chondrocytes cultured in vitro showed thatthe articular cartilage:digest time 6.49±1.87h,cells were 5.79±1.7×105/100 mg.Costal cartilage:digest time 4.14±1.45h,cells were4.76±1.2×105/100 mg,costal cartilage cells and articular cartilagecells in the cell to obtain the rate of survival rate on the wall did nothave statistical significance,and digestion time of costal cartilagecell was less than that of articular cartilage cells,P value of 0.0032.Dimethyl methylene blue determination of cell culture medium in passageof the GAG content showed that first generation of cartilage cells andsecond generation cartilage cells in thea mount of GAG was similar withoutsignificant difference,but there is statistical difference from the 3rdgeneration of cartilage cells.
(4) The cartilage cells were cultured in the scaffolds with differentmicro-structure,and the results showed that ,in the first week of culture,rib chondrocytes cultured in different pore and interconnection had nosignificant difference in growth rate,but from the beginning of thesecond week,,the growth rate of the scaffold with the 120μm diameterinterconnection was quicker than that of other interconnections withinthe scaffold material,and this situation lasted until the fourth week.
(5) The results of animal experiment verification in vivo show that costalChondrocytes compounded withβ-TCP ceramic scaffold can repair rabbitarticular cartilage defects.4 months later,the histology score of 20.76±2.13,toluidine blue staining,saffron "0" stain,and staining of typeⅡcollagen stain all showed a good complete repair of cartilage tissue.
【Conclusion】:Cartilage defect in rabbit femoral trocheae groove is a suitable animalmodel for cartilage tissue engineering research.
Resultes of optimization accounted that costal chondrocytes culturedin vitro had more rapid growth rate, secreted more amount ofglycosaminoglycans. Second generation costal chondrocytes are moresuitable for cartilage tissue engineering.
The costal chondrocytes cultured in bioceramic scaffolds with different microstructure had different results.The bioceramic scaffolds withmicrostructures of pore 500-630μm and interconnection 120μm were moresuitable for the growth of chondrocytes in vitro.
The results of animal experiments in vivo accounted that composite indifferent ways produced different results of the cartilage reconstruction.Second generation rib chondrocytes cultured in scaffolds were better thanthe first generation without culture.
Hard materials of bioceramics can be used as the scaffold for cartilageengineering.
The chondrocytes of the upper level of bioceramic scaffolddifferentiated to cartilage tissue.The chondrocytes of the lower levelof bioceramic scaffold did not differentiate to cartilage tissue and hadthe possibility to differentiate to bone tissue.
引文
1. Dong J, Uemura T, Shirasaki Y, Tateishi T. Promotion of bone formation using highly pure porous beta-TCP combined with bone marrow-derived osteoprogenitor cells. Biomaterials 2002;23:4493-502.
2. Sachlos E, Reis N, Ainsley C, Derby B, Czemuszka JT. Novel collagen scaffolds with predefined internal morphology made by solid freeform fabrication. Biomaterials 2003;24:1487-97.
3. Holmes TC. Novel peptide-based biomaterial scaffolds for tissue engineering. Trends Biotechnol 2002;20:16-21.
4. Schaefer D, Martin I, Jundt G, et al. Tissue-engineered composites for the repair of large osteochondral defects. Arthritis Rheum 2002;46:2524-34.
5. Peterson L, Minas T, Brittberg M, Nilsson A, Sjogren-Jansson E, Lindahl A. Two- to 9-year outcome after autologous chondrocyte transplantation of the knee. Clin Orthop Relat Res 2000:212-34.
6. Saadeh PB, Brent B, Mehrara BJ, et al. Human cartilage engineering: chondrocyte extraction, proliferation, and characterization for construct development. Ann Plast Surg 1999;42:509-13.
7. Falsafi S, Koch RJ. Growth of tissue-engineered human nasoseptal cartilage in simulated microgravity. Arch Otolaryngol Head Neck Surg 2000;126:759-65.
8. Watt FM. Effect of seeding density on stability of the differentiated phenotype of pig articular chondrocytes in culture. J Cell Sci 1988;89 ( Pt 3):373-8.
9. Isogai N, Kusuhara H, Ikada Y, et al. Comparison of different chondrocytes for use in tissue engineering of cartilage model structures. Tissue Eng 2006;12:691-703.
10. O'Driscoll SW, Fitzsimmons JS. The role of periosteum in cartilage repair. Clin Orthop Relat Res 2001:S190-207.
11. Homminga GN, Bulstra SK, Bouwmeester PS, van der Linden AJ. Perichondral grafting for cartilage lesions of the knee. J Bone Joint Surg Br 1990;72:1003-7.
12. Steimberg N, Viengchareun S, Biehlmann F, et al. SV40 large T antigen expression driven by col2al regulatory sequences immortalizes articular chondrocytes but does not allow stabilization of type Ⅱ collagen expression. Exp Cell Res 1999;249:248-59.
13. Verbruggen G, Malfait AM, Almqvist KF, et al. Development of immortalized human articular cartilage cell lines. Agents Actions SuppI 1993;39:267-72.
14. Bentzon JF, Sondergaard CS, Kassem M, Falk E. Smooth muscle cells healing atherosclerotic plaque disruptions are of local, not blood, origin in apolipoprotein E knockout mice. Circulation 2007;116:2053-61.
15. Wakitani S, Imoto K, Yamamoto T, Saito M, Murata N, Yoneda M. Human autologous culture expanded bone marrow mesenchymal cell transplantation for repair of cartilage defects in osteoarthritic knees. Osteoarthritis Cartilage 2002;10:199-206.
16. Kramer J, Bohrnsen F, Schlenke P, Rohwedel J. Stem cell-derived chondrocytes for regenerative medicine. Transplant Proc 2006;38:762-5.
17. Worster AA, Nixon AJ, Brower-Toland BD, Williams J. Effect of transforming growth factor betal on chondrogenic differentiation of cultured equine mesenchymal stem cells. Am J Vet Res 2000;61:1003-10.
18. Rubio D, Garcia-Castro J, Martin MC, et al. Spontaneous human adult stem cell transformation. Cancer Res 2005;65:3035-9.
19. Adachi N, Sato K, Usas A, et al. Muscle derived, cell based ex vivo gene therapy for treatment of full thickness articular cartilage defects. J Rheumatol 2002;29:1920-30.
20. Kramer J, Hegert C, Guan K, Wobus AM, Muller PK, Rohwedel J. Embryonic stem cell-derived chondrogenic differentiation in vitro: activation by BMP-2 and BMP-4. Mech Dev 2000;92:193-205.
21. Hong Z, Zhang P, He C, et al. Nano-composite of poly(L-lactide) and surface grafted hydroxyapatite: mechanical properties and biocompatibility. Biomaterials 2005;26:6296-304.
22. Svensson A, Nicklasson E, Harrah T, et al. Bacterial cellulose as a potential scaffold for tissue engineering of cartilage. Biomaterials 2005;26:419-31.
23. Lee CR, Grad S, Gorna K, Gogolewski S, Goessl A, Alini M. Fibrin-polyurethane composites for articular cartilage tissue engineering: a preliminary analysis. Tissue Eng 2005;11:1562-73.
24.Subramanian A,Vu D,Larsen GF,Lin HY.Preparation and evaluation of the electrospun chitosan/PEO fibers for potential applications in cartilage tissue engineering.J Biomater Sci Polym Ed 2005;16:861-73.
25.Engin,C T.Manufacture of macroporous calcium hydroxyapatite bioceramics [J].Journal of the European.Ceramic Society 1999;19:2569-72.
26．吴建锋，徐晓虹．多孔磷酸三钙生物陶瓷的研制．陶瓷学报1999；20：104-7．
27．周大利，郑昌琼，尹光福．高性能多孔β-磷酸三钙生物陶瓷制备新工艺研究．生物医学工程学杂志1999；16：52-5．
28.Moran JM,Pazzano D,Bonassar U.Characterization of polylactic acid-polyglycolic acid composites for cartilage tissue engineering.Tissue Eng 2003;9:63-70.
29.Guo X,Wang C,Zhang Y,et al.Repair of large articular cartilage defects with implants of autologous mesenchymal stem cells seeded into beta-tricalcium phosphate in a sheep model.Tissue Eng 2004;10:1818-29.
30.Vacanti CA.The history of tissue engineering.J Cell Mol Med 2006;10:569-76.
1.Dong J,Uemura T,Shirasaki Y,Tateishi T.Promotion of bone formation using highly pure porous beta-TCP combined with bone marrow-derived osteoprogenitor cells.Biomaterials 2002;23:4493-502.
2.Sachlos E,Reis N,Ainsley C,Derby B,Czernuszka JT.Novel collagen scaffolds with predefined internal morphology made by solid freeform fabrication.Biomaterials 2003;24:1487-97.
3.Holmes TC.Novel peptide-based biomaterial scaffolds for tissue engineering.Trends Biotechnol 2002;20:16-21.
4．丛锐，王全平，吴道澄．骨缺损修复材料可降解速固化骨水泥结构特征及力学性能．中国临床康复2002；6：2065．
5．丛锐，王全平，冯立宁．骨缺损修复材料可降解速固化骨水泥的生物相容性研究．中国临床康复2002；6：502-3．
6.Moran JM,Pazzano D,Bonassar LJ.Characterization of polylactic acid-polyglycolic acid composites for cartilage tissue engineering.Tissue Eng 2003;9:63-70.
7.Guo X,Wang C,Zhang Y,et al.Repair of large articular cartilage defects with implants of autologous mesenchymal stem cells seeded into beta-tricalcium phosphate in a sheep model.Tissue Eng 2004;10:1818-29.
8.Lu JX,Flautre B,Anselme K,et al.Role of interconnections in porous bioceramics on bone recolonization in vitro and in vivo.J Mater Sci Mater Med 1999;10:111-20.
9.Lu JX,Gallur A,Flautre B,et al.Comparative study of tissue reactions to calcium phosphate ceramics among cancellous,cortical,and medullar bone sites in rabbits.J Biomed Mater Res 1998;42:357-67.
10.Lu JX,Huang ZW,Tropiano P,et al.Human biological reactions at the interface between bone tissue and polymethylmethacrylate cement.J Mater Sci Mater Med 2002:13:803-9.
11.Guo X,Wang C,Duan C,et al.Repair of osteochondral defects with autologous chondrocytes seeded onto bioceramic scaffold in sheep.Tissue Eng 2004：10：1830-40．
12．谢峰，张文杰，陈凡凡，et al．成熟软骨细胞促进小鼠胚胎干细胞向软骨分化．中国科学C辑：生命科学2008；38：783-6．
13.PedrozoHA,Schwartz Z,Gomez R,et al.Growth plate chondrocytes store latent transforming growth factor (TGF)-beta 1 in their matrix through latent TGF-beta 1 binding protein-1.J Cell Physiol 1998;177:343-54.
14.Kramer J,Hegert C,Guan K,Wobus AM,Muller PK,Rohwedel J.Embryonic stem cell-derived chondrogenic differentiation in vitro:activation by BMP-2 and BMP-4.Mech Dev 2000;92:193-205.
15.Kawaguchi J,Mee PJ,Smith AG.Osteogenic and chondrogenic differentiation of embryonic stem cells in response to specific growth factors.Bone 2005;36:758-69.
16.Chen CW,Tsai YH,Deng WP,et al.Type Ⅰ and Ⅱ collagen regulation of chondrogenic differentiation by mesenchymal progenitor cells.J Orthop Res 2005;23:446-53.
1.Dong J,Uemura T,Shirasaki Y,Tateishi T.Promotion of bone formation using highly pure porous beta-TCP combined with bone marrow-derived osteoprogenitor cells.Biomaterials 2002;23:4493-502.
2．鄂征，刘流．医学组织工种技术与临床应用【M】．北京出版社2003．
3.Engin,C T.Manufacture of macroporous calcium hydroxyapatite bioceramics [J].Journal of the European.Ceramic Society 1999;19:2569-72.
4.Moran JM,Pazzano D,Bonassar LJ.Characterization of polylactic acid-polyglycolic acid composites for cartilage tissue engineering.Tissue Eng 2003;9:63-70.
5．吴建锋，徐晓虹．多孔磷酸三钙生物陶瓷的研制．陶瓷学报1999；20：104-7．
6．周大利，郑昌琼，尹光福．高性能多孔β-磷酸三钙生物陶瓷制备新工艺研究．生物医学工程学杂志1999；16：52-5．
7.Shapiro F,Koide S,Glimcher MJ.Cell origin and differentiation in the repair of full-thickness defects of articular cartilage.J Bone Joint Surg Am 1993;75:532-53.
8.Puelacher WC,Wisser J,Vacanti CA,Ferraro NF,Jaramillo D,Vacanti JP.Temporomandibular joint disc replacement made by tissue-engineered growth of cartilage.J Oral Maxillofac Surg 1994;52:1172-7;discussion 7-8.
9.Hong Z,Zhang P,He C,et al.Nano-composite of poly(L-lactide) and surface grafted hydroxyapatite:mechanical properties and biocompatibility.Biomaterials 2005;26:6296-304.
10.Svensson A,Nicklasson E,Harrah T,et al.Bacterial cellulose as a potential scaffold for tissue engineering of cartilage.Biomaterials 2005;26:419-31.
11.Lee CR,Grad S,Gorna K,Gogolewski S,Goessl A,Alini M.Fibrin-polyurethane composites for articular cartilage tissue engineering:a preliminary analysis.Tissue Eng 2005;11:1562-73.
12.Subramanian A,Vu D,Larsen GF,Lin HY.Preparation and evaluation of the electrospun chitosan/PEO fibers for potential applications in cartilage tissue engineering.J Biomater Sci Polym Ed 2005;16:861-73.
13. Lu JX, Flautre B, Anselme K, et al. Role of interconnections in porous bioceramics on bone recolonization in vitro and in vivo. J Mater Sci Mater Med 1999;10:111-20.
1. Bentzon JF, Sondergaard CS, Kassem M, Falk E. Smooth muscle cells healing atherosclerotic plaque disruptions are of local, not blood, origin in apolipoprotein E knockout mice. Circulation 2007; 116:2053-61.
2. Murphy JM, Dixon K, Beck S, Fabian D, Feldman A, Barry F. Reduced chondrogenic and adipogenic activity of mesenchymal stem cells from patients with advanced osteoarthritis. Arthritis Rheum 2002;46:704-13.
3. Rubio D, Garcia-Castro J, Martin MC, et al. Spontaneous human adult stem cell transformation. Cancer Res 2005;65:3035-9.
4. Adachi N, Sato K, Usas A, et al. Muscle derived, cell based ex vivo gene therapy for treatment of full thickness articular cartilage defects. J Rheumatol 2002;29:1920-30.
5. Huang JI, Beanes SR, Zhu M, Lorenz HP, Hedrick MH, Benhaim P. Rat extramedullary adipose tissue as a source of osteochondrogenic progenitor cells. Plast Reconstr Surg 2002;109:1033-41; discussion 42-3.
6. Kramer J, Hegert C, Guan K, ffobus AM, Muller PK, Rohwedel J. Embryonic stem cell-derived chondrogenic differentiation in vitro: activation by BMP-2 and BMP-4. Mech Dev 2000;92:193-205.
7. Schaefer D, Martin I, Jundt G, et al. Tissue-engineered composites for the repair of large osteochondral defects. Arthritis Rheum 2002:46:2524-34.
8. Isogai N, Kusuhara H, Ikada Y, et al. Comparison of different chondrocytes for use in tissue engineering of cartilage model structures. Tissue Eng 2006;12:691-703.
9. Benya PD, Shaffer JD. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 1982;30:215-24.
10. Peterson L, Minas T, Brittberg M, Nilsson A, Sjogren-Jansson E, Lindahl A. Two- to 9-year outcome after autologous chondrocyte transplantation of the knee. Clin Orthop Relat Res 2000:212-34.
11.Anderson DE,AthanasiouKA.Passaged goat costal chondrocytes provide a feasible cell source for temporomandibular joint tissue engineering.Ann Biomed Eng 2008;36:1992-2001.
12.Passaretti D,Silverman RP,Huang W,et al.Cultured chondrocytes produce injectable tissue-engineered cartilage in hydrogel polymer.Tissue Eng 2001;7:805-15.
13.Lee JW,Qi WN,Scully SP.The involvement of betal integrin in the modulation by collagen of chondrocyte-response to transforming growth factor-betal.J Orthop Res 2002;20:66-75.
14.Chesterman PJ,Smith AU.Homotransplantation of articular cartilage and isolated chondrocytes.An experimental study in rabbits.J Bone Joint Surg Br 1968;50:184-97.
15.Gooch KJ,Blunk T,Courter DL,et al.IGF-Ⅰ and mechanical environment interact to modulate engineered cartilage development.Biochem Biophys Res Commun 2001;286:909-15.
16.Hu DN,Yang PY,Ku MC,Chu CH,Lim AY,Hwang MH.Isolation and cultivation of human articular chondrocytes.Kaohsiung J Med Sci 2002;18:113-20.
17.Kawasaki K,Ochi M,Uchio Y,Adachi N,Matsusaki M.Hyaluronic acid enhances proliferation and chondroitin sulfate synthesis in cultured chondrocytes embedded in collagen gels.J Cell Physiol 1999;179:142-8.
18．陈富林，毛天球．兔关节软骨细胞得体外分离、纯化及鉴定．现代口腔医学杂志1998；12：263-5．
19.Koh KB,George J.Radiological parameters of bleeding renal angiomyolipoma.Scand J Urol Nephrol 1996;30:265-8.
20.Shapiro F,Koide S,Glimcher MJ.Cell origin and differentiation in the repair of full-thickness defects of articular cartilage.J Bone Joint Surg Am 1993;75:532-53.
21.Bruns J,Kersten P,Silbermann M,Lierse W.Cartilage-flow phenomenon and evidence for it in perichondrial grafting.Arch Orthop Trauma Surg 1997;116:66-73.
22. Hunziker EB, Rosenberg LC. Repair of partial-thickness defects in articular cartilage: cell recruitment from the synovial membrane. J Bone Joint Surg Am 1996:78:721-33.
23. Nevo Z, Robinson D, Horowitz S, Hasharoni A, Yayon A. The manipulated mesenchymal stem cells in regenerated skeletal tissues. Cell Transplant 1998:7:63-70.
24. Quarto R, Campanile G, Cancedda R, Dozin B. Modulation of commitment, proliferation, and differentiation of chondrogenic cells in defined culture medium. Endocrinology 1997;138:4966-76.
25. Adkisson HD, Gillis MP, Davis EC, Maloney W, Hruska KA. In vitro generation of scaffold independent neocartilage. Clin Orthop Relat Res 2001:S280-94.
26. Chu CR, Dounchis JS, Yoshioka M, Sah RL, Coutts RD, Amiel D. Osteochondral repair using perichondrial cells. A 1-year study in rabbits. Clin Orthop Relat Res 1997:220-9.
27. Brittberg M. Autologous chondrocyte transplantation. Clin Orthop Relat Res 1999:S147-55.
28. Minas T, Peterson L. Advanced techniques in autologous chondrocyte transplantation. Clin Sports Med 1999;18:13-44, v-vi.
29. Gillogly SD, Voight M, Blackburn T. Treatment of articular cartilage defects of the knee with autologous chondrocyte implantation. J Orthop Sports Phys Ther 1998:28:241-51.
30. Breinan HA, Minas T, Hsu HP, Nehrer S, Sledge CB, Spector M. Effect of cultured autologous chondrocytes on repair of chondral defects in a canine model. J Bone Joint Surg Am 1997;79:1439-51.
31. Nehrer S, Breinan HA, Ramappa A, et al. Chondrocyte-seeded collagen matrices implanted in a chondral defect in a canine model. Biomaterials 1998;19:2313-28.
32. van Susante JL, Buma P, Homminga GN, van den Berg WB, Veth RP. Chondrocyte-seeded hydroxyapatite for repair of large articular cartilage defects. A pilot study in the goat. Biomaterials 1998:19:2367-74.
33. Wakitani S, Goto T, Young RG, Mansour JM, Goldberg VM, Caplan AI. Repair of large full-thickness articular cartilage defects with allograft articular chondrocytes embedded in a collagen gel. Tissue Eng 1998:4:429-44.
34. Rahfoth B, Weisser J, Sternkopf F, Aigner T, von der Mark K, Brauer R. Transplantation of allograft chondrocytes embedded in agarose gel into cartilage defects of rabbits. Osteoarthritis Cartilage 1998:6:50-65.
35. Kawamura S, Wakitani S, Kimura T, et al. Articular cartilage repair. Rabbit experiments with a collagen gel-biomatrix and chondrocytes cultured in it. Acta Orthop Scand 1998:69:56-62.
36. Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 1998:238:265-72.
37. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997:276:71-4.
38. Kadiyala S, Young RG, Thiede MA, Bruder SP. Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro. Cell Transplant 1997:6:125-34.
39. Fortier LA, Nixon AJ, Williams J, Cable CS. Isolation and chondrocytic differentiation of equine bone marrow-derived mesenchymal stem cells. Am J Vet Res 1998:59:1182-7.
40. Butnariu-Ephrat M, Robinson D, Mendes DG, Halperin N, Nevo Z. Resurfacing of goat articular cartilage by chondrocytes derived from bone marrow. Clin Orthop Relat Res 1996:234-43.
41. Hendrickson DA, Nixon AJ, Grande DA, et al. Chondrocyte-fibrin matrix transplants for resurfacing extensive articular cartilage defects. J Orthop Res 1994:12:485-97.
42.van Susante JL,Buma P,Schuman L,Homminga GN,van den Berg WB,Veth RP.Resurfacing potential of heterologous chondrocytes suspended in fibrin glue in large full-thickness defects of femoral articular cartilage:an experimental study in the goat.Biomaterials 1999;20:1167-75.
43.Grande DA,Breitbart AS,Mason J,Paulino C,Laser J,Schwartz RE.Cartilage tissue engineering:current limitations and solutions.Clin Orthop Relat Res 1999:S176-85.
44．谢峰，张文杰，陈凡凡，et al．成熟软骨细胞促进小鼠胚胎干细胞向软骨分化．中国科学C辑：生命科学2008；38：783-6．
45.PedrozoHA,Schwartz Z,Gomez R,et al.Growth plate chondrocytes store latent transforming growth factor (TGF)-beta 1 in their matrix through latent TGF-beta 1 binding protein-1.J Cell Physiol 1998;177:343-54.
46.Kawaguchi J,Mee PJ,Smith AG.Osteogenic and chondrogenic differentiation of embryonic stem cells in response to specific growth factors.Bone 2005;36:758-69.
47.Chen CW,Tsai YH,Deng WP,et al.Type Ⅰ and Ⅱ collagen regulation of chondrogenic differentiation by mesenchymal progenitor cells.J Orthop Res 2005;23:446-53.
48.Gilbert JE.Current treatment options for the restoration of articular cartilage.Am J Knee Surg 1998;11:42-6.
49.Schreiber RE,Ilten-Kirby BM,Dunkelman NS,et al.Repair of osteochondral defects with allogeneic tissue engineered cartilage implants.Clin Orthop Relat Res 1999:S382-95.
1.Lu JX,Flautre B,Anselme K,et al.Role of interconnections in porous bioceramics on bone recolonization in vitro and in vivo.J Mater Sci Mater Med 1999;10:111-20.
2.Guo X,Wang C,Duan C,et al.Repair of osteochondral defects with autologous chondrocytes seeded onto bioceramic scaffold in sheep.Tissue Eng 2004;10:1830-40.
3．马超，王臻，卢建熙，et al．球形多孔支架材料体内血管化的量化分析．
4．谢峰，张文杰，陈凡凡，et al．成熟软骨细胞促进小鼠胚胎干细胞向软骨分化．中国科学C辑：生命科学2008；38：783-6．
5.PedrozoHA,Schwartz Z,Gomez R,et al.Growth plate chondrocytes store latent transforming growth factor (TGF)-beta 1 in their matrix through latent TGF-beta 1 binding protein-1.J Cell Physiol 1998;177:343-54.
6.Kramer J,Hegert C,Guan K,WobusAM,Muller PK,Rohwedel J.Embryonic stem cell-derived chondrogenic differentiation in vitro:activation by BMP-2 and BMP-4.Mech Dev 2000;92:193-205.
7.Kawaguchi J,Mee PJ,Smith AG.Osteogenic and chondrogenic differentiation of embryonic stem cells in response to specific growth factors.Bone 2005;36:758-69.
8.ChenCW,Tsai YH,Deng WP,et al.Type Ⅰ and Ⅱ collagen regulation of chondrogenic differentiation by mesenchymal progenitor cells.J Orthop Res 2005;23:446-53.
9.Zaucke F,Dinser R,Maurer P,Paulsson M.Cartilage oligomeric matrix protein (COMP) and collagen Ⅸ are sensitive markers for the differentiation state of articular primary chondrocytes.Biochem J 2001;358:17-24.
10.Vandenberg GW,De La Noue J.Evaluation of protein release from chitosan-alginate microcapsules produced using external or internal gelation.J Microencapsul 2001;18:433-41.
11.Hench LL,Wilson J.Biocompatibility of silicates for medical use.Ciba Found Symp 1986;121:231-46.
1.Jones AC,Arns CH,Hutmacher DW,Milthorpe BK,Sheppard AP,Knackstedt MA.The correlation of pore morphology,interconnectivity and physical properties of 3D ceramic scaffolds with bone ingrowth.Biomaterials 2009:30:1440-51.
2．谢峰，张文杰，陈凡凡，et al．成熟软骨细胞促进小鼠胚胎干细胞向软骨分化．中国科学C辑：生命科学2008；38：783-6．
3.PedrozoHA,Schwartz Z,Gomez R,et al.Growth plate chondrocytes store latent transforming growth factor (TGF)-beta 1 in their matrix through latent TGF-beta 1 binding protein-1.J Cell Physiol 1998;177:343-54.
4.Kramer J,Hegert C,Guan K,Wobu sAM,Muller PK,Rohwedel J.Embryonic stem cell-derived chondrogenic differentiation in vitro:activation by BMP-2 and BMP-4.Mech Dev 2000;92:193-205.
5.Hoshikawa A,Nakayama Y,Matsuda T,Oda H,Nakamura K,Mabuchi K.Encapsulation of chondrocytes in photopolymerizable styrenated gelatin for cartilage tissue engineering.Tissue Eng 2006;12:2333-41.
6.Kawaguchi J,Mee PJ,Smith AG.Osteogenic and chondrogenic differentiation of embryonic stem cells in response to specific growth factors.Bone 2005;36:758-69.
7.Chen CW,Tsai YH,Deng WP,et al.Type Ⅰ and Ⅱ collagen regulation of chondrogenic differentiation by mesenchymal progenitor cells.J Orthop Res 2005;23:446-53.
8.Homminga GN,Bulstra SK,Bouwmeester PS,van der Linden AJ.Perichondral grafting for cartilage lesions of the knee.J Bone Joint Surg Br 1990;72:1003-7.
9.Wernike E,Li Z,Alini M,Grad S.Effect of reduced oxygen tension and long-term mechanical stimulation on chondrocyte-polymer constructs.Cell Tissue Res 2008;331:473-83.
10．陈金武，秦瑞峰，李建虎，刘洪臣．关节腔环境诱导骨髓基质干细胞-磷酸三钙复合体中干细胞向软骨细胞表型的分化．
11. Peterson L, Brittberg M, Kiviranta I, Akerlund EL, Lindahl A. Autologous chondrocyte transplantation. Biomechanics and long-term durability. Am J Sports Med 2002;30:2-12.
12. Kessler MW, Ackerman G, Dines JS, Grande D. Emerging technologies and fourth generation issues in cartilage repair. Sports Med Arthrosc 2008;16:246-54.
13. Hettrich CM, Crawford D, Rodeo SA. Cartilage repair: third-generation cell-based technologies-basic science, surgical techniques, clinical outcomes. Sports Med Arthrosc 2008:16:230-5.
14. Isogai N, Kusuhara H, Ikada Y, et al. Comparison of different chondrocytes for use in tissue engineering of cartilage model structures. Tissue Eng 2006:12:691-703.
15. O'Driscoll SW. Preclinical cartilage repair: current status and future perspectives. Clin Orthop Relat Res 2001:S397-401.
16. Endres M, Neumann K, Schroder SE, et al. Human polymer-based cartilage grafts for the regeneration of articular cartilage defects. Tissue Cell 2007:39:293-301.
17. Gabler F, Frauenschuh S, Ringe J, et al. Emulsion-based synthesis of PLGA-microspheres for the in vitro expansion of porcine chondrocytes. Biomol Eng 2007:24:515-20.
18. Moran JM, Pazzano D, Bonassar LJ. Characterization of polylactic acid-polyglycolic acid composites for cartilage tissue engineering. Tissue Eng 2003:9:63-70.
19. Guo X, Wang C, Zhang Y, et al. Repair of large articular cartilage defects with implants of autologous mesenchymal stem cells seeded into beta-tricalcium phosphate in a sheep model. Tissue Eng 2004:10:1818-29.
1.Dong J,Uemura T,Shirasaki Y,Tateishi T.Promotion of bone formation using highly pure porous beta-TCP combined with bone marrow-derived osteoprogenitor cells.Biomaterials 2002;23:4493-502.
2.Sachlos E,Reis N,Ainsley C,Derby B,Czernuszka JT.Novel collagen scaffolds with predefined internal morphology made by solid freeform fabrication.Biomaterials 2003;24:1487-97.
3.Holmes TC.Novel peptide-based biomaterial scaffolds for tissue engineering.Trends Biotechnol 2002;20:16-21.
4． 鄂征，刘流．医学组织工种技术与临床应用【M】．北京出版社2003．
5.Kim BS,Mooney DJ.Development of biocompatible synthetic extracellular matrices for tissue engineering.Trends Biotechnol 1998;16:224-30.
6.Bryant S J,Anseth KS.The effects of scaffold thickness on tissue engineered cartilage in photocrosslinked poly(ethylene oxide) hydrogels.Biomaterials 2001;22:619-26.
7.Marijnissen WJ,van Osch G J,Aigner J,et al.Alginate as a chondrocyte-delivery substance in combination with a non-woven scaffold for cartilage tissue engineering.Biomaterials 2002;23:1511-7.
8.Wakitani S,Kimura T,Hirooka A,et al.Repair of rabbit articular surfaces with allograft chondrocytes embedded in collagen gel.J Bone Joint Surg Br 1989;71:74-80.
9.Stone KR,Steadman JR,Rodkey WG,Li ST.Regeneration of meniscal cartilage with use of a collagen scaffold.Analysis of preliminary data.J Bone Joint Surg Am 1997;79:1770-7.
10.Homminga G,Buma P,Koot H,van der Kraan P,van den Berg W.Chondrocyte behavior in fibrin glue in vitro.Acta Orthop Scand 1993 Aug;64:441-5.
11.Hendrickson DA,Nixon AJ,Erb HN,Lust G.Phenotype and biological activity of neonatal equine chondrocytes cultured in a three-dimensional fibrin matrix.Am J Vet Res 1994;55:410-4.
12.Hendrickson DA,Nixon A J,Grande DA,et al.Chondrocyte-fibrin matrix transplants for resurfacing extensive articular cartilage defects.J Orthop Res 1994;12:485-97.
13.Brolin K,Halldin P.Development of a finite element model of the upper cervical spine and a parameter study of ligament characteristics.Spine 2004;29:376-85.
14.Xia W,Liu W,Cui L,et al.Tissue engineering of cartilage with the use of chitosan-gelatin complex scaffolds.J Biomed Mater Res B Appl Biomater 2004;71:373-80.
15.Nettles DL,Elder SH,Gilbert JA.Potential use of chitosan as a cell scaffold material for cartilage tissue engineering．Tissue Eng 2002；8：1009-16．
16．Hoemann CO,Sun J，Legare A，McKee M D,Buschmann MD．Tissue engineering of cartilage using an injectable and adhesive chitosan-based cell-delivery vehicle．Osteoarthritis Cartilage 2005；13：318-29．
17．Zhao X，Yu SB，Wu EL,Mao ZB,Yu CL．Transfection of primary chondrocytes using chitosan-pEGFP nanoparticles．J Control Release 2006；112：223-8．
18．王彦君，孔维佳．软骨组织工程天然支架材料的研究进展．国外医学：耳鼻喉科学分册2004；28：179．
19．陈书军，雷德林，杨维军．以藻酸钙为载体的可注射软骨．解放军医学杂志2001；26：241-4．
20.Li Z,Ramay HR,Hauch KD,Xiao D,Zhang M.Chitosan-alginate hybrid scaffolds for bone tissue engineering.Biomaterials 2005;26:3919-28.
21.Guo JF,Jourdian GW,MacCallum DK.Culture and growth characteristics of chondrocytes encapsulated in alginate beads.Connect Tissue Res 1989;19:277-97.
22.Paige KT,Cima LG,Yaremchuk M J,Vacanti JP,Vacanti CA.Injectable cartilage.Plast Reconstr Surg 1995;96:1390-8;discussion 9-400.
23．杨维东，李石保，陈富林．可注射性组织工程骨研究．中华口腔医学杂志2001；36：102．
24.Paige KT,Cima LG,Yaremchuk M J,Schloo BL,Vacanti JP,Vacanti CA.De novo cartilage generation using calcium alginate-chondrocyte constructs.Plast Reconstr Surg 1996;97:168-78;discussion 79-80.
25.Lee JY,Hall R,Pelinkovic D,et al.New use of a three-dimensional pellet culture system for human intervertebral disc cells:initial characterization and potential use for tissue engineering.Spine 2001;26:2316-22.
26.Lindenhayn K,Perka C,Spitzer R,et al.Retention of hyaluronic acid in alginate beads:aspects for in vitro cartilage engineering.J Biomed Mater Res 1999;44:149-55.
27.Sittinger M,Perka C,Schultz O,Haupl T,Burmester GR.Joint cartilage regeneration by tissue engineering.Z Rheumatol 1999;58:130-5.
28.Kelly TA,Ng KW,Wang CC,Ateshian GA,Hung CT.Spatial and temporal development of chondrocyte-seeded agarose constructs in free-swelling and dynamically loaded cultures.J Biomech 2006;39:1489-97.
29．王跃，杨志明，解慧琪．组织工程化软骨的应力应变研究．生物医学工程学杂志 2001;18:181-184
30.Zhang Q,Yao K,Liu L,et al.Evaluation of porous collagen membrane in guided tissue regeneration.Artif Cells Blood Substit Immobil Biotechnol 1999;27:245-53.
31.Kawasaki K,Ochi M,Uchio Y,Adachi N,Matsusaki M.Hyaluronic acid enhances proliferation and chondroitin sulfate synthesis in cultured chondrocytes embedded in collagen gels.J Cell Physiol 1999;179:142-8.
32.Yoo HS,Lee EA,Yoon J J,Park TG.Hyaluronic acid modified biodegradable scaffolds for cartilage tissue engineering.Biomaterials 2005;26:1925-33.
33.Yamane S,Iwasaki N,Majima T,et al.Feasibility of chitosan-based hyaluronic acid hybrid biomaterial for a novel scaffold in cartilage tissue engineering.Biomaterials 2005;26:611-9.
34.Puelacher WC,Wisser J,Vacanti CA,Ferraro NF,Jaramillo D,Vacanti JP.Temporomandibular joint disc replacement made by tissue-engineered growth of cartilage.J Oral Maxillofac Surg 1994;52:1172-7;discussion 7-8.
35．王得春，陈峥嵘，宋后燕．聚乙醇酸负载同种异体软骨细胞移植修复兔关节软骨缺损．中华创伤杂志1997；13：228-31．
36.Sherwood JK,Riley SL,Palazzolo R,et al.A three-dimensional osteochondral composite scaffold for articular cartilage repair.Biomaterials 2002;23:4739-51.
37.Cao Y,Rodriguez A,Vacanti M,Ibarra C,Arevalo C,Vacanti CA.Comparative study of the use of poly(glycolic acid),calcium alginate and pluronics in the engineering of autologous porcine cartilage.J Biomater Sci Polym Ed 1998;9:475-87.
38.Boyan BD,Hummert TW,Dean DD,Schwartz Z.Role of material surfaces in regulating bone and cartilage cell response.Biomaterials 1996;17:137-46.
39.Malicev E,Radosavljevic D,Velikonja NK.Fibrin gel improved the spatial uniformity and phenotype of human chondrocytes seeded on collagen scaffolds.Biotechnol Bioeng 2007;96:364-70.
40.Lu H,Cai D,Fen Z,et al.[Culture of chondrocytes using collagen-hydroxyapatite composite scaffolds in vitro].Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 2006;20:144-7.
41．李忠，杨柳，陈光必．胶原凝胶复合cPPf／PLLA支架与软骨细胞相容性研究．四川医学2005；26：597-3．
42.Mohan N,Nair PD,Tabata Y.A 3D biodegradable protein based matrix for cartilage tissue engineering and stem cell differentiation to cartilage.J Mater Sci Mater Med 2008.
43.Bhattarai SR,Bhattarai N,Yi HK,Hwang PH,Cha DI,Kim HY.Novel biodegradable electrospun membrane:scaffold for tissue engineering.Biomaterials 2004;25:2595-602.
44.Park GE,Pattison MA,Park K,Webster TJ.Accelerated chondrocyte functions on NaOH-treated PLGA scaffolds.Biomaterials 2005;26:3075-82.
45.Muller FA,Muller L,Hofmann I,Greil P,Wenzel MM,Staudenmaier R.Cellulose-based scaffold materials for cartilage tissue engineering.Biomaterials 2006;27:3955-63.
46.Ma Z,Gao C,Gong Y,Shen J.Cartilage tissue engineering PLLA scaffold with surface immobilized collagen and basic fibroblast growth factor.Biomaterials 2005;26:1253-9.
47.Engin,C T.Manufacture of macroporous calcium hydroxyapatite bioceramics [J].Journal of the European.Ceramic Society 1999;19:2569-72.
48．吴建锋，徐晓虹．多孔磷酸三钙生物陶瓷的研制．陶瓷学报1999；20：104-7．
49．周大利，郑昌琼，尹光福．高性能多孔β-磷酸三钙生物陶瓷制备新工艺研究．生物医学工程学杂志1999；16：52-5．
50.Moran JM,Pazzano D,Bonassar LJ.Characterization of polylactic acid-polyglycolic acid composites for cartilage tissue engineering.Tissue Eng 2003;9:63-70.
51.Guo X,Wang C,Zhang Y,et al.Repair of large articular cartilage defects with implants of autologous mesenchymal stem cells seeded into beta-tricalcium phosphate in a sheep model.Tissue Eng 2004;10:1818-29.
52.Hong Z,Zhang P,He C,et al.Nano-composite of poly(L-lactide) and surface grafted hydroxyapatite:mechanical properties and biocompatibility.Biomaterials 2005;26:6296-304.
53.Svensson A,Nicklasson E,Harrah T,et al.Bacterial cellulose as a potential scaffold for tissue engineering of cartilage.Biomaterials 2005;26:419-31.
54.Lee CR,Grad S,Gorna K,Gogolewski S,Goessl A,Alini M.Fibrin-polyurethane composites for articular cartilage tissue engineering:a preliminary analysis.Tissue Eng 2005;11:1562-73.
55.Subramanian A,Vu D,Larsen GF,Lin HY.Preparation and evaluation of the electrospun chitosan/PEO fibers for potential applications in cartilage tissue engineering.J Biomater Sci Polym Ed 2005;16:861-73.
1.Schaefer D,Martin I,Jundt G,et al.Tissue-engineered composites for the repair of large osteochondral defects.Arthritis Rheum 2002;46:2524-34.
2.Peterson L,Minas T,Brittberg M,Nilsson A,Sjogren-Jansson E,Lindahl A.Twoto 9-year outcome after autologous chondrocyte transplantation of the knee.Clin Orthop Relat Res 2000:212-34.
3.Saadeh PB,Brent B,Mehrara BJ,et al.Human cartilage engineering:chondrocyte extraction,proliferation,and characterization for construct development.Ann Plast Surg 1999;42:509-13.
4.van Osch GJ,van der Veen SW,Verwoerd-Verhoef HL.In vitro redifferentiation of culture-expanded rabbitand human auricularchondrocytes for cartilage reconstruction.P1ast Reconstr Surg 2001;107:433-40.
5.Smith RL,Lin J,Trindade MC,et al.Time-dependent effects of intermittent hydrostatic pressure on articular chondrocyte type Ⅱ collagen and aggrecan mRNA expression.J Rehabil Res Dev 2000;37:153-61.
6.Falsafi S,Koch RJ.Growth of tissue-engineered human nasoseptal cartilage in simulated microgravity.Arch Otolaryngol Head Neck Surg 2000;126:759-65.
7.Watt FM.Effect of seeding density on stability of the differentiated phenotype of pig articular chondrocytes in cuiture.J Cell Sci 1988;89 ( Pt 3):373-8.
8．孙天威，杨志明．天然生物支架材料在软骨修复中的研究进展．中国矫形外科杂志2006；14：461-5．
9.Isogai N,Kusuhara H,Ikada Y,et al.Comparison of different chondrocytes for use in tissue engineering of cartilage model structures.Tissue Eng 2006;12:691-703.10.Anderson DE,AthanasiouKA.Passaged goat costal chondrocytes provide a feasible cell source for temporomandibular joint tissue engineering.Ann Biomed Eng 2008;36:1992-2001.
11．夏万尧，曹谊林，商庆林．同种异体组织工程化软骨组织形成的初步实验研究．中华整形外科杂志2000；16：264．
12.Wakitani S,Goto T,Young RG,Mansour JM,Goldberg VM,Caplan AI.Repair of large full-thickness articular cartilage defects with allograft articular chondrocytes embedded in a collagen gel. Tissue Eng 1998;4:429-44.
13. Wong MW, Qin L, Tai JK, Lee SK, Leung KS, Chan KM. Engineered allogeneic chondrocyte pellet for reconstruction of fibrocartilage zone at bone-tendon junction-a preliminary histological observation. J Biomed Mater Res B Appl Biomater 2004;70:362-7.
14. O'Driscoll SW, Fitzsimmons JS. The role of periosteum in cartilage repair. Clin Orthop Relat Res 2001:S190-207.
15. Homminga GN, Bulstra SK, Bouwmeester PS, van der Linden AJ. Perichondral grafting for cartilage lesions of the knee. J Bone Joint Surg Br 1990;72:1003-7.
16. Wakitani S, Goto T, Pineda SJ, et al. Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. J Bone Joint Surg Am 1994;76:579-92.
17. Steimberg N, Viengchareun S, Biehlmann F, et al. SV40 large T antigen expression driven by col2al regulatory sequences immortalizes articular chondrocytes but does not allow stabilization of type Ⅱ collagen expression. Exp Cell Res 1999;249:248-59.
18. Oyajobi BO, Frazer A, Hollander AP, et al. Expression of type Ⅹ collagen and matrix calcification in three-dimensional cultures of immortalized temperature-sensitive chondrocytes derived from adult human articular cartilage. J Bone Miner Res 1998:13:432-42.
19. Brower-Toland BD, Saxer RA, Goodrich LR, et al. Direct adenovirus-mediated insulin-like growth factor Ⅰ gene transfer enhances transplant chondrocyte function. Hum Gene Ther 2001;12:117-29.
20. Verbruggen G, Malfait AM, Almqvist KF, et al. Development of immortalized human articular cartilage cell lines. Agents Actions Suppl 1993:39:267-72.
21. Bentzon JF, Sondergaard CS, Kassem M, Falk E. Smooth muscle cells healing atherosclerotic plaque disruptions are of local, not blood, origin in apolipoprotein E knockout mice. Circulation 2007;116:2053-61.
22. Wakitani S, Imoto K, Yamamoto T, Saito M, Murata N, Yoneda M. Human autologous culture expanded bone marrow mesenchymal cell transplantation for repair of cartilage defects in osteoarthritic knees. Osteoarthritis Cartilage 2002;10:199-206.
23. Kramer J, Bohrnsen F, Schlenke P, Rohwedel J. Stem cell-derived chondrocytes for regenerative medicine. Transplant Proc 2006:38:762-5.
24. Worster AA, Nixon AJ, Brower-Toland BD, Williams J. Effect of transforming growth factor betal on chondrogenic differentiation of cultured equine mesenchymal stem cells. Am J Vet Res 2000:61:1003-10.
25. Darling EM, Athanasiou KA. Growth factor impact on articular cartilage subpopulations. Cell Tissue Res 2005:322:463-73.
26. Kuroda R, Usas A, Kubo S, et al. Cartilage repair using bone morphogenetic protein 4 and muscle-derived stem cells. Arthritis Rheum 2006:54:433-42.
27. Griffith LG, Naughton G. Tissue engineering-current challenges and expanding opportunities. Science 2002:295:1009-14.
28. Gao JJ, Min BS, Ahn EM, Nakamura N, Lee HK, Hattori M. New triterpene aldehydes, lucialdehydes A~C, from Ganoderma lucidum and their cytotoxicity against murine and human tumor cells. Chem Pharm Bull (Tokyo) 2002:50:837-40.
29. Murphy JM, Dixon K, Beck S, Fabian D, Feldman A, Barry F. Reduced chondrogenic and adipogenic activity of mesenchymal stem cells from patients with advanced osteoarthritis. Arthritis Rheum 2002:46:704-13.
30. Rubio D, Garcia-Castro J, Martin MC, et al. Spontaneous human adult stem cell transformation. Cancer Res 2005:65:3035-9.
31. Nevo Z, Robinson D, Horowitz S, Hasharoni A, Yayon A. The manipulated mesenchymal stem cells in regenerated skeletal tissues. Cell Transplant 1998:7:63-70.
32. Urist MR, Terashima Y, Nakagawa M, Stamos C. Cartilage tissue differentiation from mesenchymal cells derived from mature muscle in tissue culture. In Vitro 1978:14:697-706.
33. Adachi N, Sato K, Usas A, et al. Muscle derived, cell based ex vivo gene therapy for treatment of full thickness articular cartilage defects. J Rheumatol 2002:29:1920-30.
34. Erickson GR, Gimble JM, Franklin DM, Rice HE, Awad H, Guilak F. Chondrogenic potential of adipose tissue-derived stromal cells in vitro and in vivo. Biochem Biophys Res Commun 2002:290:763-9.
35. Huang JI, Beanes SR, Zhu M, Lorenz HP, Hedrick MH, Benhaim P. Rat extramedullary adipose tissue as a source of osteochondrogenic progenitor cells. Plast Reconstr Surg 2002;109:1033-41;discussion 42-3.
36.Kramer J,Hegert C,Guan K,Wobus AM,Muller PK,Rohwedel J.Embryonic stem ce11-derived chondrogenic differentiation in vitro:activation by BMP-2and BMP-4.Mech Dev 2000;92:193-205.
37．晏杰，刘玲蓉，张其清．诱导骨髓问充质干细胞向软骨细胞分化的体外研究中国修复重建外科杂志2006；20：1114-8．
38.Kim IY,Seo SJ,Moon HS,et al.Chitosanand its derivatives for tissue engineering applications.Biotechnol Adv 2008;26:1-21.
39.Gerard C,Catuogno C,Amargier-Huin C,et al.The effect of alginate,hyaluronate and hyaluronate derivatives biomaterials on synthesis of non-articular chondrocyte extracellular matrix.J Mater Sci Mater Med 2005;16:541-51.
40.Endres M,Neumann K,Schroder SE,et al.Human polymer-based cartilage grafts for the regeneration of articular cartilage defects.Tissue Cell 2007;39:293-301.
41.Gabler F,Frauenschuh S,Ringe J,et al.Emulsion-based synthesis of PLGA-microspheres for the in vitro expansion of porcine chondrocytes.Biomol Eng 2007;24:515-20.
42.Ringe J,Kaps C,Burmester GR,Sittinger M.Stem cells for regenerative medicine:advances in the engineering of tissues and organs.Naturwissenschaften 2002;89:338-51.
43．戴刚，李起鸿，周强．关节软骨组织工程种子细胞的优化获取．第三军医大学学报 2002：24：129-31．
44.Iwasa J,Ochi M,Uchio Y,Katsube K,Adachi N,Kawasaki K.Effects of cell density on proliferation and matrix synthesis of chondrocytes embedded in atelocollagen gel.Artif Organs 2003;27:249-55.
45.Saini S,Wick TM.Concentric cylinder bioreactor for production of tissue engineered cartilage:effect of seeding densityand hydrodynamic loading on construct development.Biotechnol Prog 2003;19:510-21.
46.Mauck RL,Wang CC,Oswald ES,Ateshian GA,Hung CT.The role of cell seeding density and nutrient supply for articular cartilage tissue engineering with deformational loading.Osteoarthritis Cartilage 2003;11:879-90.
47.Millward-Sadler SJ,Wright MO,Lee H,et al.Integrin-regulated secretion of interleukin 4:A novel pathway of mechanotransduction in human articularchondrocytes.J Cell Biol 1999;145:183-9.
48.Salter DM,Robb JE,Wright MO.Electrophysiological responses of human bone cells to mechanical stimulation:evidence for specific integrin function in mechanotransduction.J Bone Miner Res 1997;12:1133-41.
49.Durrant LA,Archer CW,Benjamin M,Ralphs JR.Organisation of the chondrocyte cytoskeletonand its response to changing mechanical conditions in organ culture.J Anat 1999;194 ( Pt 3):343-53.
50.Vunjak-Novakovic G,Martin I,Obradovic B,et al.Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage.J Orthop Res 1999;17:130-8.
51.O'Driscoll SW,Fitzsimmons JS,CommissoCN.Role of oxygen tension during cartilage formation by periosteum.J Orthop Res 1997;15:682-7.
52.Wernike E,Li Z,Alini M,Grad S.Effect of reduced oxygen tension and long-term mechanical stimulation on chondrocyte-polymer constructs.Cell Tissue Res 2008;331:473-83.
53.Freed LE,Vunjak-Novakovic G.Microgravity tissue engineering.In Vitro Cell Dev Biol Anim 1997;33:381-5.
54.Schulz RM,Bader A.Cartilage tissue engineering and bioreactor systems for the cultivation and stimulation of chondrocytes.Eur Biophys J 2007;36:539-68.
55.Chen HC,Hu YC.Bioreactors for tissue engineering.Biotechnol Lett 2006;28:1415-23.
56．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．中国组织工程研究与临床康复2007；11：2609-12．