摘要
关节软骨缺损是临床的难题之一,自体关节软骨移植存在来源有限、创伤大、费用高等缺点,异体关节软骨移植难以排除免疫源性的问题。随着组织工程的发展,利用人工再生组织来修复关节软骨缺损,为临床解决这一难题提供了新的思路和方法。虽然关节软骨组织工程的研究已经取得了很大进展,但仍有大量的问题等待解决。尤其是软骨和软骨下骨的分层、断裂问题,和宿主的整合问题,关节软骨修复的长期效果等。
研究目的
将胶原、壳聚糖与β-磷酸三钙(Tricalcium Phosphate,TCP)有机复合,模仿体内关节软骨的分层结构,增加支架材料的力学强度,构建胶原/壳聚糖/β-TCP层状梯度支架材料。分别将扩增的经成骨诱导的兔骨髓间充质干细胞(BoneMesenchymal Stem Cells,BMSCs)和软骨细胞植入其中体外微重力下培养,对照组采用普通培养板培养,观察成骨细胞和软骨细胞在胶原/壳聚糖/β-TCP层状梯度修复体中的黏附、增殖及生物学性状变化,以评价该复合材料作为关节软骨组织工程支架的可行性,并观察模拟微重力对细胞及构建组织的效用。
研究方法
以细胞和支架材料为实验对象,以普通培养和微重力培养分两组对照。MTT实验分组:5个时间组:1天、3天、5天、7天、9天。5个平行孔/组,两培养组共50孔。
1.成骨细胞和材料复合:用全骨髓贴壁筛选法培养新西兰兔原代BMSCs,体外扩增后进行成骨诱导,3周后检测细胞碱性磷酸酶活性和钙结节生成情况,诱导成功后和修复体复合体外普通培养和旋转培养仪模拟微重力培养,进行MTT检测比较两组细胞增殖情况并观察模拟微重力对细胞的影响;培养1周后行扫描电镜检查观察成骨细胞在修复体材料内黏附生长情况;4周后,细胞和材料复合物脱钙、石蜡包埋、切片,行苏木精一伊红染色及Ⅰ型胶原免疫组织化学染色,观察成骨细胞的生物学性状变化及模拟微重力对复合物的力学影响。
2.软骨细胞和材料复合:取新西兰幼兔的关节软骨消化培养软骨细胞体外培养扩增,取2、3代细胞行s—100细胞免疫化学染色,收集前4代软骨细胞作为种子细胞和修复体复合体外普通培养和旋转培养仪模拟微重力培养,进行MTT检测比较两组细胞增殖情况并观察模拟微重力对软骨细胞的影响;培养7天后行扫描电镜检查观察软骨细胞在修复体材料内黏附生长情况;4周后,细胞和材料复合物进行脱钙、石蜡包埋、切片,行苏木精—伊红染色及Ⅱ型胶原免疫组织化学染色,观察软骨细胞的生物学性状变化及模拟微重力对复合物的力学影响。
统计学分析:MTT实验中,相同时间点两组之间的比较用两独立样本t检验;同组不同时间点比较采用完全随机设计单因素方差分析(One-way ANOVA);两组整体之间比较采用重复测量数据的方差分析。α=0.05为统计学检验水准。
研究结果
1.细胞形态:初接种时BMSCs呈球形,悬浮在培养液中,24h后开始贴壁,细胞呈长梭形,少量多角形,约1周左右细胞铺满培养瓶。加入成骨诱导培养液后细胞生长相对缓慢,其形态发生了改变,细胞逐渐增大,进而出现重叠生长。2周后细胞间可见圆形或卵圆形的钙化结节,结节周围细胞呈放射状分布。碱性磷酸酶染色阳性,Von Kossa法检测出钙化结节。体外单层培养的软骨细胞呈球形,悬浮在培养液中,5h后开始贴壁,细胞呈扁平状,大多数三角形,少量多角形,约1周细胞铺满培养瓶,呈“铺路石”样。第2、3代细胞S—100免疫细胞化学染色胞浆内呈阳性反应。
2.细胞/支架光镜下观察:细胞悬液滴加于经预湿处理的复合支架材料上,支架材料迅速膨胀,证明细胞扩散到支架内。接种24h后,倒置显微镜下见材料不透明,无法观察其内细胞生长情况,但可见大量成骨细胞或软骨细胞贴附在材料旁培养板内。
3.MTT检测:成骨细胞和材料复合后,除第1天两组之间无显著性差异外(P=0.706),其余时间点两组之间有显著性差异(P<0.05),模拟微重力组明显比培养板组细胞增殖力高。两组之间整体比较也有统计学意义(P=0.000),证明模拟微重力对成骨细胞在修复体内的增殖比普通培养起到了更好的促进作用。软骨细胞和材料复合后,不同时间点两组之间有显著性差异(P<0.05),模拟微重力组明显比培养板组细胞增殖力高。两组之间整体比较也有统计学意义(P=0.000),证明模拟微重力对软骨细胞在修复体内增殖比普通培养起到了更好的促进作用。
4.扫描电镜:培养7d后,细胞成团地吸附于修复体表面及孔隙侧壁,并可见细胞间通过突起相互连接,或伸出伪足贴附于支架孔隙壁上;从复合支架材料中间切开,普通培养组见其内部有少量细胞贴附于孔隙侧壁,模拟微重力组则可见到大量细胞,细胞数量比普通培养组明显增多。
5.HE染色和免疫组化结果:成骨细胞和材料:4周后,材料表面可见大量细胞,内部可见部分细胞聚集成团;细胞呈长梭形,细胞团中有基质分泌,沉积于细胞周围;Ⅰ型胶原免疫组织化学染色示胞浆内呈阳性反应。软骨细胞和材料:4周后,材料表面可见大量细胞黏附成层状,内部亦可见细胞聚集成团;细胞呈圆形或椭圆形,细胞团中有基质分泌,沉积于细胞周围,Ⅱ型胶原免疫组织化学染色示胞浆内呈阳性反应。普通培养组的细胞多聚集在修复体表面,细胞数量明显多于修复体内部,而模拟微重力组材料表面和材料内部细胞数量相差不大,内部细胞数量明显比普通培养组多。
结论
1.成功培养出兔原代软骨细胞和BMSCs,并成功诱导BMSCs为成骨细胞,为进一步的细胞和材料的接种提供了数量充足和生物学性能良好的种子细胞。
2.胶原/壳聚糖/β-TCP层状梯度复合体模拟了正常关节软骨分层结构,细胞相容性好,能提供细胞生长的三维环境。
3.转壁式旋转培养仪产生的模拟微重力可以使软骨细胞和成骨细胞在修复体内高密度生长,能更好的促进细胞增殖和细胞外基质分泌,提高了组织工程化关节软骨体外培养的质量,为关节软骨组织工程的研究开辟了新的道路。
Articular cartilage defects is one of the orthopedics clinical problems, articular cartilage autotransplantation limited by its shortcomings such as sources, trauma, the high cost. Articular cartilage allograft transplantation difficult to rule out the possibility of endogenous immune problems. Along with the development of tissue engineering, using artificial regeneration organizations to repair articular cartilage defects, providing new ideas and methods to solve the problem . Although the articular cartilage tissue engineering research has made great progress, there is still a large number of issues to be resolved. Particularly cartilage and subchondral bone tiered, fracture, the host integration, and the long-term effects of articular cartilage repairing.
Objective: Collagen, chitosan andβ-tricalcium phosphate are compounded organically to building layered gradient composite, in order to imitate the hierarchical structure of articular cartilage and increase the mechanical strength of scaffold. The osteoblasts were induced from rabbit bone marrow-derived mesenchymal stem cells and chondrocytes were seeded onto the porous collagen-chitosan-β-tricalcium phosphate composite scaffold and were cultured in a stimulated microgravity environment respectly, The effects of the composite scaffold on the cell adhesivity, proliferation and morphological changes were observed, to evaluate the feasibility of composite material as articular cartilage tissue engineering scaffolds and observe the effectiveness of simulated microgravity on cells and constructs.
Methods: Put the cells and scaffold as experimental object, The experiment were given to two groups: the general culture and microgravity culture, the general culture as a control. MTT assay subgroups: five time groups: first day, third day, fifth day, seventh days,ninth day. Five parallel hole / group, 50 holes totally.
1.BMSCs composite materials: the rabbit primary BMSCs were cultured by whole bone marrow adherent screening method and induced to osteoblasts,the result improved by detecting the activity of alkaline phosphatase and formation of calcium nodule after three weeks culture,then the BMSCs were seeded onto the scaffolds and cultured in vitro, The effects of the composite scaffold between the two groups on the cell adhesivity, proliferation, morphological changes, and synthesis of the extracellular matrix were observed by MTT assay, scanning electron microscopy one week later, hematoxylin- eosin stain and collagen type I immunohistochemical staining after four weeks.2. Chondrocytes composite materials: the New Zealand immature rabbit primary chondrocytes were cultured in vitro amplification, the second and third generation cells were improved by S-100 immunocytochemistry staining ,then the first four generation chondrocytes were seeded onto the scaffolds and cultured in vitro, The effects of the composite scaffold between the two groups on the cell adhesivity, proliferation, morphological changes, and synthesis of the extracellular matrix were observed by MTT assay, scanning electron microscopy one week later, hematoxylin- eosin stain and collagen type II immunohistochemical staining after four weeks.
Statistical analysis: MTT assay, the same time point of comparison between the two groups using two independent samples t-test .the same group at different time points compared using the completely randomized design of single-factor analysis of variance (One-way ANOVA); two overall comparison between the two groups using analysis of variance of repeated measures data. a= 0.05 is the level for the statistical test.
Results: 1. Cell morphology: BMSCs were suspended in culture medium with spherical shape at the beginning stage, then adhered culture flask and elongated after 24 h with its fusiform shape in majority and polygonal in minority. The cells covered culture flask about one week . After be induced, the cells grew slowly and changed its form into more large shape ,then overlapped one another. There were a mount of round or oval calcified nodules among cells two weeks later and distributed in radiat, the cells' Alkaline Phosphatase staining was positive and Von Kossa method staining of calcified nodules was positive. The chondrocytes were suspended in culture medium with spherical shape at the beginning stage, then adhered culture flask and elongated after 5h with its triangle shape in majority and polygonal in minority. The cells covered culture flask about one week with "paving stones" appearance. The S-100 immunocytochemical staining of chondrocytes ware positive.2. Cell / scaffold microscopy: After dropping cells suspension in the wet pretreatment scaffold, the scaffold expansion rapidly revealed that the cells spread in it. The result of inverted microscope revealed that the scaffolds were opaque and a large number of osteoblast and chondrocytes adhered the adjacent material. 3. MTT assay: After osteoblasts were seeded onto the scaffolds , There was a significant difference between two groups at different time points (P <0.05) except the first day (P = 0.706), and group-factors was also statistically significant (P = 0.000).
After chondrocytes were seeded onto the scaffolds , There was a significant difference between the two groups at different time points (P <0.05), and group-factors was also statistically significant (P = 0.000).4. Scaning Electron Microscope: After cultured 7 days, the cells agglomerate adsorbed on the surface and pore walls of scaffolds, linked with each other by cell processes or stretched out pseudo-foot attached to the pore wall. Discussion from the middle of scaffold, there were a small number of cells attached to pore wall.5. HE staining and immunohistochemical staining: Osteoblasts and scaffolds: After 4 weeks, there were a large number of cells with spherical shape at the surface and a small amount of cells overlapped in it , around which deposited much extra cell matrix .The typeⅠcollagen immunohistochemistry show positive response within the cytoplasm. Chondrocytes and scaffolds: After 4 weeks,there were a large number of cells with round shape at the surface and a small amount of cells overlapped in it, around which deposited much extra cell matrix . The typeⅡcollagen immunohistochemistry show positive response within the cytoplasm.
Conclusion: 1. We develope successfully New Zealand rabbit primary chondrocytes and BMSCs, and successfully induced BMSCs to osteoblasts, which provided sufficient quantity and better biological properties cells resource for further experiment. 2. Collagen / chitosan /β-TCP layered gradient composites simulate the hierarchical structure of normal articular cartilage, which can provide three-dimensional growth environment for cells with its better compatibility. 3. Simulated microgravity from rotating wall vessel bioreactor can promote osteoblasts and chondrocytes grow in high density in scaffold, and promote the secretion of extracellular matrix, which improve the quality of tissue-engineered articular cartilage in vitro and open a new way for the articular cartilage tissue engineering research.
引文
[1]SR Frenkel,PE Cesare.scaffolds for articular cartilage repair[J].Annals of Biomedical Engineering,2004;32,(1):26-34.
[2]Blanka Sharma,Jemmofer H.E.Engineering Structurally Organized Cartilage and Bone Tissues[J].Annals of Biomedical Engineering,2004;32(1):148-159.
[3]Hoemann,J.Sun,McKee,et al.Tissue engineering of cartilage using an injectable and adhesive chitosan-based cell-delivery vehicle[J].Osteoarthritis and Cartilage.2005;13(4):318-329.
[4]Frank A.M(u|¨)ller,Lenka M(u|¨)ller,Ingo Hofmann,et al.Cellulose-based scaffold materials for cartilage tissue engineering[J].Biomaterials.2006;27(21):3955-3963.
[5]Song HX,Li FB,Shen HL,et al.Repairing articular cartilage defects with tissue-engineering cartilage in rabbits[J].Chin J Traumatol.2006;9(5):266-271.
[6]tanworth SJ,Newland AC.Stem cells:progress in research and edging towards the clinical setting[J].Clin Med 2001;1(5):378-382.
[7]崔玉明,胡蕴玉,吕昌伟,等.诱导骨髓基质细胞向软骨细胞分化的实验研究[J].中国临床康复,2003,7(17):2397-2399.
[8]Fleming JE Jr,Comell CN,Muschler GF.Bone cells and matrices in orthopedic tissue engineering[J].Orthop Clin North Am 2000;31(3):357-374.
[9]许卫兵,吕刚,黄涛等.体外培养兔骨髓基质干细胞复合β-磷酸三钙的肌内成骨能力观察[J].中国临床康复,2005;9(22):43-45.
[10]Baddoo M,Hill K,Wilkinson R,et al.Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection[J].Journal of Cellular Biochemistry,2003,89:1235-1249.
[11]Deschaseaux F,Gindraux F,Saadi R,et al.Direct selection of human bone marrow mesenchymal stem cells using an anti-CD49a antibody reveals their CD45med,low phenotype[J].British Journal of Haematology,2003,122, 506-517.
[12]Majumdar MK,Bank SV,Peluso DP,et al.Isolation,characterization,and chondrogenic potential of human bone marrow-derived multipotential stromal cells[J].Journal of Cell(u|¨)lar Physiology,2000,185(1):98-106.
[13]Quirici N,Soligo D,Bossolasco P,et al.Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies[J].Experimential Hematology,2002,30:783-791.
[14]Hung SC,Nien-Jung C,Shie-Liang H et al.Isolation and characterization of size-sieved stem cells from human bone marrow[J].Stem Cells,2002,20:249-258.
[15]王玉学,毕郑钢,杨成林.兔骨髓基质干细胞诱导为骨髓源成骨细胞的时间及功能特征[J].中国临床康复,2005,9(46):24-26.
[16]余方圆.几种构建组织工程关节软骨方法的比较研究[D].中国人民解放军军医进修学院,2004:46-49.
[17]Brittberg M,Peterson L,Sjogren-Jansson E,et al.Articular cartilage engineering with autologous chondrocyte transplantation.A review of recent developments.J Bone Joint Surg(Am),2003,85(suppl 3):109-115.
[18]夏万尧,曹谊林,商庆新,等.组织工程化软骨组织形成的最佳细胞浓度和最佳形成时间的实验研究.中国修复重建杂志,1999,13(4):244-248.
[19]鄂征,刘流.医学组织工种技术与临床应用[M].北京:北京出版社,2003.
[20]李文辉,侯筱魁,汤亭亭,等.藻酸钙复合材料构建组织工程化骨及软骨的实验[J].中国临床康复,2005,9(26):237-239.
[21]王立春,张旗涛,张宏颖.纤维蛋白凝胶基质材料用于组织工程修复兔关节软骨缺损与Ⅰ型胶原凝胶和混合凝胶性能特征的比较[J].中国临床康复,2005,9(26):116-118.
[22]范宏斌,胡蕴玉,吴红,等.转化生长因子明胶微球制备及体外缓释研究[J].中国矫形外科杂志,2005,13(3):208-211.
[23]卢华定,蔡道章,黄冬梅,等.Ⅰ型胶原三维立体支架高密度培养软骨细胞的增 殖及其分泌功能[J].中国临床康复,2005,9(18):62-65.
[24]王身国,杨健,蔡晴,等.组织工程用生物材料及细胞支架研究进展[J].中华整形外科杂志,2000,16(6):328-330.
[25]Baek CH,Ko YJ.Characteris tics of tis sue-engineered cartilage on macroporous biodegradable PLGA scaffold.Laryngoscope 2006;116(10):1829-1834.
[26]田晋洪,艾玉峰,潘宝华,等.以聚羟基丁酸已酯为支架材料的组织工程隆鼻法的实验研究[J].中国美容医学,2006,15(4):376-379.
[27]van Susante,Buma P,van Beuningen HM,et al.Respons ivenes s of bovine chondrocytes to growth factors in medium with different serum concentrations.J Orthop Res 2000;18(1):68-77.
[28]王跃,杨志明,解慧琪,等.三种支架材料与软骨细胞复合构建组织工程化软骨的研究[J].中华实验外科杂志,2002,19(2):125-127.
[29]郑磊,王前.骨组织工程中细胞外基质材料的选择[J].中华外科杂志,2000;38(10):745-748.
[30]Jarman- Smith ML,Bodamyali T,Stevens C,et al.Porcine collagen Crosslinking degradation and its capability for fibroblast adhesion and proliferation[J].J Mater Sci Mater Med,2004,15(8):925-932.
[31]Madihally S V,Matthew H W T.Porous chitosan scaffolds for tissue engineering[J].Biomaterials,1999,20(12):1133-1142.
[32]Majeti N.V.Ravi Kumar.A review of chitin and chitosan applications[J].Reactive and Functional Polymers,2000,46(1):1-27.
[33]Gao Changyou,Wang Dengyong,Shen Jiacong.Fabrication of porous collagen/chitosan scaffolds with controlling microstructure for dermal equivalent.Polymers for Advanced Technologies[J],2003,14(6):373-379.
[34]Ma Jianbiao,Wang Hongjun,Binglin He,et al.A preliminary in vitro study on the fabrication and tissue engineering applications of a novel chitosan bilayer material as a scaffold of human neofetal dermal fibroblasts[J].Biomaterials, 2001,22(4):331-336.
[35]SHI De-hai,CAI Dao-zhang,Zhou Chang-ren,et al.Development and potential of a biomimetic chitosan /type Ⅱ collagen scaffold for cartilage tissue engineering[J].Chin Med J.2005;118(17):1436-1443.
[36]吴俊,孙俊英.软骨组织工程支架材料研究进展[J].中国矫形外科杂志.2004;12(3-4):282-284.
[37]韩祥永,傅远飞,张富强.β-磷酸三钙、胶原及其复合物在骨组织工程中的应用研究进展[J].上海口腔医学,2007;16(1):93-96.
[38]Vicente V,Meseguer L,Martinez F,et al.Ultrastructural study of the osteointegration of bioceramics(whitlockite and composite beta- TCP +collagen) in rabbit bone[J].Ultrastruct Pathol,1996,20(2):179-188.
[39]Vunjak-Novakovic G,Martin.I,Obradovic B,et al.Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineering cartilage[J].J Orthop Res,1999,17:130-138.
[40]蔡哲,张岚,舒峻等.空间生物技术在干细胞和组织工程学研究中的应用[J].中国康复理论与实践,2004,10(1):9-10.
[41]Licato LL,Grimm EA.Multiple intefleukin-2 signaling pathways differentially regulated by microgravity[J].Immunopharmacology,1999,44(3):273-279.
[42]Freed L E,Pellis N,Searby N,et al.Microgravity cultivation of cells and tissues[J].Gravit Space Biol Bull,1999,12(2):57-66.
[43]Valerie E Meyers,Majd Zayzafoon,Joanne T Douglas,et al.RhoA and Cytoskeletal Disruption Mediate Reduced Osteoblastogenesis and Enhanced Adipogenesis of Human Mesenchymal Stem Cells in Modeled Microgravity[J].J Bone Miner Res,2005,20(10):1858-1866.
[44]江青艳,张守全,傅伟龙.微重力组织工程的产生与发展[J].中国生物工程杂志,2002,22(3):37-9
[45]Eyrich D,Goepferich A,Blunk T.Fibrin in tissue engineering.Adv Exp Med Biol.2006;585:379-92.
[46]Demain A L,Fanga.Secondary metabolism in simulated microgravity[J]. Chem Rec,2001,1(4):333-346.
[47]Clejan S,O'Connor K,Rosensweign.Tri - dimensional prostate cell culture in simulated microgravity and induced changes in lipid second messengers and signal transduction[J].J Cell Mol Med,2001,5(1):60-73.
[48]Ohyabu Y,Kida N,Kojima H,et al.Cartilaginous tissue formation from bone marrow cells using rotating wall vessel(RWV) bioreactor.Biotechnol Bioeng.2006;95(5):1003-8.
[49]吴华,吴纪饶,朱立新,等.模拟微重力对改良纤维蛋白胶软骨细胞复合物培养的影响.中国运动医学杂志2007;26(5):547-551.
[50]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(14):2609-12.
[51]罗飞,许建中,王序全,等.模拟微重力环境促进人间充质干细胞体外高效增殖.第三军医大学学报.2005,27(16):1640-1643.
[52]Zhang Chunqiu,Zhang Xizheng,Wu Han.Direct compression as an appropriately mechanical environment in bone tissue reconstruction in vitro.Medical Hypotheses.2006:67:1414-1418.
[53]Freed LE,Vunjak-Novakovic G.Microgravity tissue engineering[J].In Vitro Cell Dev Biol Anim,1997,33(5):381-5.
[54]Zhang X,Li XB,Yang SZ,et al.The effects of simulated microgravity on cultured chicken embryonic chondrocytes.Adv Space Res.2003;32(8):1577-83.
[55]何清义,李起鸿.间歇性液态压力和连续灌注系统下的软骨细胞培养.中华骨科杂志,2000,20(9):545-547.
[56]Huang S,Ingber DE.The structural and mechanical complexity of cell-growth control.Nat Cell Biol,1999,1:E131-E138.
[57]Chen CS,Ingber DE.Tensegrity and mechanoregulation:from skeleton to cytoskeleton.Osteoarthritis Cartilage,1999,7:81-94.
[58]Huang S,Chen CS,Ingber DE.Control of cyclin D1,P27(Kipl)and cell cycle progression in human capillary endothelial cells by cell shape and cytoskeletal tension.Mol Biol Cell,1998,9:3179-3193.
[59]Ingber DE.Tensegrity:The architectural basis of celluar mechanoransduction[J].Ann Rev Physiol,1997,59:575-599.
[60]毛艳,张西正,郭勇,等.软骨组织工程中力学因素的影响及应用.国外医学生物医学工程分册,2005,28(6):336-339.
[1]Wang,X,Grogan,S.P,Rieser,F,et al.Tissue engineering of biphasic cartilage constructs using various biodegradable scaffolds:an invitro study[J].Biomaterials.2004;25(17),3681-3688.
[2]Tuli,R.,Nandi,S.,Li,W.J.,et al.Human mesenchymal progenitor cell-based tissue engineering of a singleunit osteochondral construct[J].Tissue Engineering;2004;10(7-8),1169-1179.
[3]Kandel,R.A.,Grynpas,M.,Pilliar,R.,et al.Repair of osteochondral defectswith biphasic cartilage-calcium polyphosphate constructs in a sheep model[J].Biomaterials,2006;27(22):4120-31.
[4]Schaefer,D.,Martin,I.,Shastri,P.,et al.In vitro generation of osteochondral composites[J].Biomaterials.2000;21(24):2599-2606.
[5]Gao,J.,Dennis,J.E.,Solchaga,L.A.,et al.Tissue-engineered fabrication of an osteochondral composite graft using rat bone marrow-derived mesenchymal stem cells[J].Tissue Engineering.2001;7(4):363-371.
[6]Schaefer,D.,Martin,I.,Jundt,G.,et al.Tissue-engineered composites for the repair of large osteochondral defects[J].Arthritis & Rheumatism.2002;46(9):2524-2534.
[7]Schek,R.M.,Taboas,J.M.,Segvich,S.J.,et al.Engineered osteochondral grafts using biphasic composite solid free-form fabricated scaffolds[J]. Tissue Engineering .2004;10 (9-10):1376-1385.
[8] Sherwood, J.K., Riley, S.L., Palazzolo, R, et al. A three-dimensional osteochondral composite scaffold for articular cartilage re'pair[J]. Biomaterials 2002.23 (24), 4739-4751.
[9] Cao, T., Ho, K.H., Teoh, S.H. Scaffold design and in vitro study of osteochondral coculture in a three-dimensional porous polycaprolactone scaffold fabricated by fused deposition modeling[J]. Tissue Engineering,2003;9 (Suppl. 1), S103-S112.
[10] Alhadlaq, A., Elisseeff, J.H., Hong, L. et al. Adult stem cell driven genesis of human-shaped articular condyle[J]. Annals of Biomedical Engineering 2004;32 (7):911-923.
[11] Fukuda, A., Kato, K., Hasegawa, M., et al. Enhanced repair of large osteochondral defects using a combination of artificial cartilage and basic fibroblast growth factor[J]. Biomaterials.2005;26 (20):4301-4308.
[12] Benya, P.D., Shaffer, J.D., Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels[J]. Cell. 1982;30(1):215-224.
[13] Binette, F, McQuaid, D.P, Haudenschild, D.R, et al. Expression of a stable articular cartilage phenotype without evidence of hypertrophy by adult human articular chondrocytes in vitro[J]. Journal of Orthopaedic Research. 1998; 16 (2):207-216.
[14] Barbero, A., Ploegert, S., Heberer, M., et al. Plasticity of clonal populations of dedifferentiated adult human articular chondrocytes[J]. Arthritis & Rheumatism.2003;48(5):1315-1325.
[15] Jakob, M., Demarteau, O., Schafer, D., et al. Specific growth factors during the expansion and redifferentiation of adult human articular chondrocytes enhance chondrogenesis and cartilaginous tissue formation in vitro[J]. Journal of Cellular Biochemistry. 2001;81 (2) :368-377.
[16] Barbero, A., Grogan, S., Schafer, D., et al. Age related changes in human articular chondrocyte yield, proliferation and post-expansion chondrogenic capacity[J]. Osteoarthritis Cartilage. 2004;12 (6) :476-484.
[17] Jakob, M., Demarteau, O, Suetterlin, R. et al. Chondrogenesis of expanded adult human articular chondrocytes is enhanced by specific prostaglandins[J].Rheumatology .2004;43 (7) :852-857.
[18] Lee, C.R., Grodzinsky, A.J., Hsu, H.P. et al. Effects of harvest and selected cartilage repair procedures on the physical and biochemical properties of articular cartilage in the canine knee[J]. Journal of Orthopaedic Research.2000; 18 (5) :790-799.
[19] Kafienah, W., Jakob, M., Demarteau, O. et al. Three-dimensional tissue engineering of hyaline cartilage: comparison of adult nasal and articular chondrocytes[J]. Tissue Engineering. 2002;8 (5) :817-826.
[20] Tay, A.G., Farhadi, J., Suetterlin, R. et al. Cell yield, proliferation, and postexpansion differentiation capacity of human ear, nasal, and rib chondrocytes[J]. Tissue Engineering. 2004; 10 (5-6) :762-770.
[21] Van Osch, G.J., Mandl, E.W., Jahr, H. et al. Considerations on the use of ear chondrocytes as donor chondrocytes for cartilage tissue engineering[J].Biorheology.2004;41 (3-4) :411-421.
[22] Tuan, R.S., Boland, G., Tuli, R. Adult mesenchymal stem cells and cell-based tissue engineering[J]. Arthritis Research & Therapy. 2003;5(1) :32-45.
[23] Winter, A., Breit, S., Parsch, D. et al. Cartilage-like gene expression in differentiated human stem cell spheroids: a comparison of bone marrow-derived and adipose tissue-derived stromal cells[J]. Arthritis & Rheumatism .2003 ;48(2) :418-429.
[24] Oshima, Y., Watanabe, N., Matsuda, K. et al. Fate of transplanted bone-marrow-derived mesenchymal cells during osteochondral repair using transgenic rats to simulate autologous transplantation[J]. Osteoarthritis Cartilage. 2004;12 (10) :811-817.
[25] Uematsu, K., Hattori, K., Ishimoto, Y. et al. Cartilage regeneration using mesenchymal stem cells and a three-dimensional poly lactic-glycolic acid (PLGA) scaffold[J]. Biomaterials.2005;26 (20) :4273-4279.
[26] Wakitani, S., Mitsuoka, T., Nakamura, N. et al. Autologous bone marrow stromal cell transplantation for repair of full-thickness articular cartilage defects in human patellae: two case reports[J]. Cell Transplantation. 2004;13 (5) :595-600.
[27] Hendriks, J., De Bruijn, E., Riesle, J. et al. A powerful tool in cartilage tissue engineering: coculturing primary chondrocytes with expanded chondrocytes enhances chondrogenesis. Transactions of the 51st Annual Meeting of the Orthopaedic Research Society[C]. 2005. 30:1792.
[28] Frisbie, D., Lu, Y., Colhoun, H. et al. In vivo evaluation of a one step autologous cartilage resurfacing technique in a long term equine model.Transactions of the 51st Annual Meeting of the Orthopaedic Research Society[C], 2005. 30:1355.
[29] Lu, Y., Dhanaraj, S., Wang. Z. et al. A novel intra-operative approach to treat full thickness articular cartilage defects with chondrocyte-loaded implants.Transactions of the 51st Annual Meeting of the Orthopaedic Research Society[C], 2005. 30:1363.
[30] Davisson, T., Kunig, S., Chen, A., et al., Static and dynamic compression modulate matrix metabolism in tissue engineered cartilage[J]. Journal of Orthopaedic Research. 2002a;20 (4):842-848
[31] Lee, C.R., Grodzinsky, A.J., Spector, M. Biosynthetic response of passaged chondrocytes in a type II collagen scaffold to mechanical compression[J].Journal of Biomedical Materials Research A. 2003;64 (3) :560-569.
[32] Kisiday, J.D., Jin, M., DiMicco, M.A. et al. Effects of dynamic compressive loading on chondrocyte biosynthesis in self-assembling peptide scaffolds[J].Journal of Biomechanics 2004;37 (5) :595-604.
[33] Mauney, J.R., Sjostorm, S., Blumberg, J. et al. Mechanical stimulation promotes osteogenic differentiation of human bone marrow stromal cells on 3-D partially demineralized bone scaffolds in vitro[J]. Calcified Tissue International.2004;74 (5) :458-468.
[34] Waldman, S.D., Spited, C.G., Grynpas, M.D. et al. Effect of biomechanical conditioning on cartilaginous tissue formation in vitro[J]. The Journal of Bone & Joint Surgery. American 2003;85 (Suppl. 2) :101-105.
[35] Lima, E.G., Mauck, R.L., Han, S.H. et al. Functional tissue engineering of chondral and osteochondral constructs[J]. Biorheology. 2004;41 (3-4) :577-590.
[36] Hung, C.T., Mauck, R.L., Wang, C.C. et al. A paradigm for functional tissue engineering of articular cartilage via applied physiologic deformational loading[J]. Annals of Biomedical Engineering. 2004;32 (1) :35-49.
[37] Malda, J., Rouwkema, J., Martens, D.E. et al., Oxygen gradients in tissue-engineered PEGT/PBT cartilaginous constructs: measurement and modeling[J]. Biotechnology and Bioengineering.2004;86 (1) :9—18.
[38] Lewis, M.C., Macarthur, B.D., Malda, J. et al. Heterogeneous proliferation within engineered cartilaginous tissue: the role of oxygen tension[J].Biotechnology and Bioengineering. 2005.91 (5), 607-615.
[39] Martin, I., Obradovic, B., Freed, L.E. et al.Method for quantitative analysis of glycosaminoglycan distribution in cultured natural and engineered cartilage[J].Annals of Biomedical Engineering. 1999;27 (5) :656-662.
[40] Vunjak-Novakovic, G., Martin, I., Obradovic, B. et al. Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage[J]. Journal of Orthopaedic Research. 1999; 17 (1):130-138.
[41] Chang, C.H., Lin, F.H., Lin, C.C. et al. Cartilage tissue engineering on the surface of a novel gelatincalcium-phosphate biphasic scaffold in a double-chamber bioreactor[J].Journal of Biomedical Materials Research. 2004; 71B (2) :313-321.
[42] Davisson, T., Sah, R.L., Ratcliffe, A. Perfusion increases cell content and matrix synthesis in chondrocyte three-dimensional cultures [J]. Tissue Engineering. 2002b;8 (5) :807-816.
[43] Pazzano, D., Mercier, K.A., Moran, J.M. et al. Comparison of chondrogenesis in static and perfused bioreactor culture. Biotechnology Progress. 2000; 16(5) :893-896.
[44] Wendt, D., Stroebel, S., Jakob, M. et al. Uniform tissues engineered by seeding and culturing cells in 3D scaffolds under perfusion at defined oxygen tensions[J].Biorheology.2006;43(3-4):481-8
[45] Bancroft, G.N., Sikavitsas, V.I., van den, D.J. et al. Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner. Proceedings of the National Academy of Sciences of the United States of America[C]. 2002;99 (20) :12600-12605.
[46] Goldstein, A.S., Juarez, T.M., Helmke, C.D. et al. Effect of convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds[J]. Biomaterials. 2001;22(11) :1279-1288.
[47] Sikavitsas, V.I., Bancroft, G.N., Lemoine, J.J. et al. Flow perfusion enhances the calcified matrix deposition of marrow stromal cells in biodegradable nonwoven fiber mesh scaffolds[J]. Annals of Biomedical Engineering. 2005;33 (1):63-70.
[48] Braccini, A., Wendt, D., Jaquiery, C. et al. Threedimensional perfusion culture of human bone marrow cells and generation of osteoinductive grafts[J]. Stem Cells. 2005;23 (8): 1066-1072.
[49] Kreklau, B., Sittinger, M., Mensing, M.B. et al. Tissue engineering of biphasic joint cartilage transplants[J]. Biomaterials. 1999;20:1743-1749.
[50] Cioffi, M., Boschetti, F., Raimondi, M.T. et al. Modeling evaluation of the fluid-dynamic microenvironment in tissueengineered constructs: A micro-CT based model[J]. Biotechnology & Bioengineering.2006;93 (3) :500-510.
[51] Porter, B., Zauel, R., Stockman, H. et al.3-D computational modeling of media flow through scaffolds in a perfusion bioreactor[J]. Journal of Biomechanics .2005;38 (3) :543-549.
[52] Raimondi, M.T., Boschetti, F., Falcone, L. et al. Mechanobiology of engineered cartilage cultured under a quantified fluid-dynamic environment [J]. Biomechanics and Modeling in Mechanobiology. 2002;1(1) :69-82.
[53] Raimondi,M.T, Boschetti,F, Falcone,L. et al. The effect of media perfusion on three dimensional cultures of human chondrocytes : integration of experimental and computational approaches[J].. Biorheology.2004;41(3-4):401-10.
[54] Sucosky.P, Osorio,D.F, Brown, J.B. et al. Fluid mechanics of a spinner-flask bioreactor[J]. Biotechnology and Bioengineering.2004;85(1):, 34-46.
[55] Williams, K.A., Saini, S., Wick, T.M., Computational fluid dynamics modeling of steady-state momentum and mass transport in a bioreactor for cartilage tissue engineering[J]. Biotechnology Progress. 2002; 18 (5) :951-963.