关节软骨细胞诱导骨髓间充质干细胞向软骨样细胞分化的研究
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摘要
目的:(1)分离、培养及鉴定骨髓源性间充质干细胞( Bone Marrow Mesenchymal Stem Cells,BMSCs),并获取高纯度的BMSCs;(2)分离、培养及鉴定关节软骨细胞(Articular Cartilage Cells,ACs),并获取高纯度的ACs;(3)BMSCs与ACs共同培养诱导,并检测其诱导结果;(4)对共培养生物诱导与TGF-β1化学诱导的结果进行比较。
方法:(1)获取并培养、增殖高纯度骨髓间充质干细胞:从SD大鼠(4周龄)的股骨及胫骨干中取得骨髓,联合利用密度梯度离心法及差异贴壁法分离、纯化BMSCs,体外扩增并传代后,相差显微镜下观察细胞的形态及流式细胞仪检测细胞表面标记:CD29、CD90、CD34及CD45,再通过成脂肪样、成骨样及成神经样细胞多向分化诱导,检测其诱导结果,确定所得细胞为BMSCs,为下一步实验打下基础。(2)获取并培养、增殖高纯度的关节软骨细胞:从SD大鼠(4周龄)的正常股骨头表面取得关节软骨片,先后运用0.25%的胰蛋白酶和0.2%Ⅱ型胶原酶消化软骨片,当相差显微镜下见有大量软骨细胞游离后,离心、弃上清液,加入含15%FBS的软骨细胞培养液进行培养、扩增传代,并取样行台盼蓝染色计数。当细胞传至第三代时,运用甲苯胺蓝染色及HE染色检查、鉴别该细胞,确定所得细胞为关节软骨细胞,为下一步实验打下基础。(3)BMSCs与ACs在Transwell共培养:分别吸取第三代BMSCs和ACs,按1:1的细胞比例分别植于Transwell共培养系统中,下室植入BMSCs,上室植入ACs,同时设置相同浓度单独BMSCs空白对照组,分别在用含10%FBS的高糖DMEM培养基培养,相差显微镜下观察细胞的增殖和基质合成情况,并对各组细胞爬片进行Ⅱ型胶原免疫组化染色检查。(4)共培养生物诱导与TGF-β1化学诱导的比较:将BMSCs用于TGF-β1(10 ng/m1)诱导,同时与1:2,1:1,2:1(关节软骨细胞:BMSCs)浓度的细胞共培养诱导组比较对照。并运用MTT比色法检查、氨基聚糖(GAG)含量检测及WESTERN BLOT检测各组细胞Ⅱ型胶原基因表达量,确定其诱导情况。
结果:(1)获取并培养、增殖高纯度BMSCs:相差显微镜下观察结果:原代细胞接种12h后可见部分细胞贴壁,24h后细胞大量贴壁,外观呈现纺锤形或多角形。72h后细胞数量开始增多,大部分细胞形态呈长梭形、似成纤维细胞。培养10d左右细胞铺满培养瓶底达80%左右,细胞呈极性排列,部分集落可呈漩涡状。细胞表面标记物经流式细胞仪检测结果:BMSCs均一地表达CD29及CD90,阳性率分别为92.5%及94.4%;而CD34和CD45为阴性,阴性率分别为99.3%和99.7%。成脂肪样、成骨样及成神经样细胞诱导均获得成功。(2)获取并培养高纯度的ACs:运用上述方法可获得一定纯度的ACs。在倒置相差显微镜下观察发现:软骨细胞接种12h后开始贴壁,48h完成贴壁。原代培养的ACs呈圆形及多角形,胞浆丰富,胞核圆形,居中,细胞长成一片可呈明显的铺路石样外观,与长梭形的成纤维细胞较易区分。试验证明:经本方法消化培养后仅见单一的软骨细胞生长。(3)BMSCs与ACs共培养:共培养组BMSCs数量增多,细胞外基质合成丰富,其基质能被Ⅱ型胶原免疫组化染色,细胞染色呈现黄色。(4)共培养生物诱导与TGF-β1化学诱导的比较:共培养细胞生物诱导的MTT比色法检查、氨基聚糖(GAG)含量检测及Ⅱ型胶原WESTERN BLOT检测结果显示:各组细胞共培养诱导结果均不差于TGF-β1 10ng/m的l化学诱导结果,但1:1、2:1共培养组诱导统计结果差异不明显。
结论:(1)密度梯度离心和差异贴壁法可以获取纯度较高的BMSCs;(2)高纯度的ACs可以经胰蛋白酶、Ⅱ型胶原酶联合消化后,过滤、离心、培养获得;(3)BMSCs与ACs共培养可作为修复关节软骨的种子细胞。
Objectives: (1) To isolate, culture and identificate Bone marrow Mesenchymal stem cells, (BMSCs),obtaining high- concentration and high -purity BMSCs;(2)To isolate, culture and identificate articular cartilage cells (ACs),obtaining high- concentration and high -purity ACs;(3)To Induce BMSCs co-cultured with the ACs and detect the induction ;(4)To compare co-cultured biological induction and chemical induction。
Methods:(1)Obtain, culture and proliferate high-purity BMSCs: Bone marrow were obtained from femoral shaft of SD rats (4 weeks) and BMSCs were isolated by gradient centrifugation and adherent separation and passaged after Amplification in vitro. The cell shape was observed under phase contrast microscope and the expression of CD 29、CD90、CD34 and CD 45 of the cells were analyzed by flow cytometry. Make sure the cells obtained were BMSCs by inducing of fat-like, osteoblast-like and adult neural-like cellstolay the foundation for the next step experiment. (2)Obtain, culture and passage high- concentration and high–purity ACs:Articular cartilage were obtained from the surface of normal femoral head of SD rats(4-week),digested by trypsin(0.25%)and CollageaseⅡ(0.2%).After observing numerous ACs free under phase contrast microscope,centrifuge, deposit Supernatant and place the cells into cartilage cell culture medium to Cultivate, proliferate, and passage; sample, count with trypan blue staining and detect and identify ACs with Toluidine blue and HE staining methods to lay the foundation for the next step experiment.。(3)Co-culture BMSCs and ACs in Transwell:Aspire the third passage BMSCs and ACs and place them respectively into Transwell co-culture system by1:1,BMSCs were placed in the bottom and in the upper ACs。Meanwhile set the same concentration of BMSCs control group separately, cultured in culture medium of NMEM containing 10% FBS respectly. Then Cell proliferation and matrix synthesis would be observed under phase contrast microscopy and Envision immunohistochemical stain typeⅡcollagen was used to analyze each group of cell-seeded cover slips (.4)Comparison of Co-culture bio-induced and TGF-β1 Comparison of chemically induced:Use TGF-β1(10 ng/m1)induction and compare with co-culture induced group of BMSCs of 1:2,1:1,2:1 . MTT assay, glucose amino glycan (GAG) content detection and typeⅡcollagen WESTERN BLOT test were adopted to check the induction of each group cells.
Result:(1)Obtain, culture and proliferate high-purity BMSCs:Some cells anchored were observed under Inverted phase contrast , after 24 hours,most wall-adhering cells were seen , which appeared as a fusiform polygonal appearance ,and cell nucleus were observed too。72h later the number of cells increased and their shapes were fibroblast-like, as with fibroblastoid cells. Meanwhile pairs of nucleolar could be seen in most nucleus and Cells were colony-like growth. Till 10d the cells covered the bottom of culture bottle and up to 90%,Polar cells were arranged, and the colony was swirling. Cell surface markers were tested by flow cytometry: BMSCs uniformly express CD29 and CD90, the positive rates were 92.5%and 94.4%,; while CD34 and CD45 were negative, the negative rates were 99.3%and 99.7%。Fat-like, osteoblast-like and adult neural-like cells induction succeeded. (2) acquire and develop high-grade ACs: the use of the above method can get a certain purity of ACs. Observation under the inverted phase contrast microscope showed: ACs vaccinated after 12h began to adhere, 48h completed adherence. Primary cultured chondrocytes was round and polygonal with abundant cytoplasm, round nucleus which exists in the center; the cells grew into a clear cobblestone appearance, distinguished with long spindle-shaped fibroblasts easily.Only single cartilage cell could be seen growing after digestion of this method.(3)BMSCs were co-cultured with ACs:The number of co-cultured BMSCs increased ,synthesis of extracellular matrix was rich ,which can be stained by typeⅡcollagen immunohistochemical staining and cells staining took on yellow.(4)Comparison of Co-culture bio-induction and TGF-β1 chemical induction: MTT colorimetric examination of Co-cultured cell bio-induction, glucose amino glycan (GAG) content detection and typeⅡcollagen WESTERN BLOT test results showed that:The induction of cell co-culture results were better than the results of TGF-β1 chemical induction,but no significant differences appeared in statistical results of1:1,1:2 co-culture induction.
Conclusion:(1) High-purity BMSCs can be obtained with density gradient centrifugation and differential adhesion method ;(2)High-purity ACs can be obtained by filtrating, centrifugating and culturing after digestion in trypsin and CollagenⅡ. (3)BMSCs co-cultured with ACs can act as seed cells repairing cartilago articularis.
方法:(1)获取并培养、增殖高纯度骨髓间充质干细胞:从SD大鼠(4周龄)的股骨及胫骨干中取得骨髓,联合利用密度梯度离心法及差异贴壁法分离、纯化BMSCs,体外扩增并传代后,相差显微镜下观察细胞的形态及流式细胞仪检测细胞表面标记:CD29、CD90、CD34及CD45,再通过成脂肪样、成骨样及成神经样细胞多向分化诱导,检测其诱导结果,确定所得细胞为BMSCs,为下一步实验打下基础。(2)获取并培养、增殖高纯度的关节软骨细胞:从SD大鼠(4周龄)的正常股骨头表面取得关节软骨片,先后运用0.25%的胰蛋白酶和0.2%Ⅱ型胶原酶消化软骨片,当相差显微镜下见有大量软骨细胞游离后,离心、弃上清液,加入含15%FBS的软骨细胞培养液进行培养、扩增传代,并取样行台盼蓝染色计数。当细胞传至第三代时,运用甲苯胺蓝染色及HE染色检查、鉴别该细胞,确定所得细胞为关节软骨细胞,为下一步实验打下基础。(3)BMSCs与ACs在Transwell共培养:分别吸取第三代BMSCs和ACs,按1:1的细胞比例分别植于Transwell共培养系统中,下室植入BMSCs,上室植入ACs,同时设置相同浓度单独BMSCs空白对照组,分别在用含10%FBS的高糖DMEM培养基培养,相差显微镜下观察细胞的增殖和基质合成情况,并对各组细胞爬片进行Ⅱ型胶原免疫组化染色检查。(4)共培养生物诱导与TGF-β1化学诱导的比较:将BMSCs用于TGF-β1(10 ng/m1)诱导,同时与1:2,1:1,2:1(关节软骨细胞:BMSCs)浓度的细胞共培养诱导组比较对照。并运用MTT比色法检查、氨基聚糖(GAG)含量检测及WESTERN BLOT检测各组细胞Ⅱ型胶原基因表达量,确定其诱导情况。
结果:(1)获取并培养、增殖高纯度BMSCs:相差显微镜下观察结果:原代细胞接种12h后可见部分细胞贴壁,24h后细胞大量贴壁,外观呈现纺锤形或多角形。72h后细胞数量开始增多,大部分细胞形态呈长梭形、似成纤维细胞。培养10d左右细胞铺满培养瓶底达80%左右,细胞呈极性排列,部分集落可呈漩涡状。细胞表面标记物经流式细胞仪检测结果:BMSCs均一地表达CD29及CD90,阳性率分别为92.5%及94.4%;而CD34和CD45为阴性,阴性率分别为99.3%和99.7%。成脂肪样、成骨样及成神经样细胞诱导均获得成功。(2)获取并培养高纯度的ACs:运用上述方法可获得一定纯度的ACs。在倒置相差显微镜下观察发现:软骨细胞接种12h后开始贴壁,48h完成贴壁。原代培养的ACs呈圆形及多角形,胞浆丰富,胞核圆形,居中,细胞长成一片可呈明显的铺路石样外观,与长梭形的成纤维细胞较易区分。试验证明:经本方法消化培养后仅见单一的软骨细胞生长。(3)BMSCs与ACs共培养:共培养组BMSCs数量增多,细胞外基质合成丰富,其基质能被Ⅱ型胶原免疫组化染色,细胞染色呈现黄色。(4)共培养生物诱导与TGF-β1化学诱导的比较:共培养细胞生物诱导的MTT比色法检查、氨基聚糖(GAG)含量检测及Ⅱ型胶原WESTERN BLOT检测结果显示:各组细胞共培养诱导结果均不差于TGF-β1 10ng/m的l化学诱导结果,但1:1、2:1共培养组诱导统计结果差异不明显。
结论:(1)密度梯度离心和差异贴壁法可以获取纯度较高的BMSCs;(2)高纯度的ACs可以经胰蛋白酶、Ⅱ型胶原酶联合消化后,过滤、离心、培养获得;(3)BMSCs与ACs共培养可作为修复关节软骨的种子细胞。
Objectives: (1) To isolate, culture and identificate Bone marrow Mesenchymal stem cells, (BMSCs),obtaining high- concentration and high -purity BMSCs;(2)To isolate, culture and identificate articular cartilage cells (ACs),obtaining high- concentration and high -purity ACs;(3)To Induce BMSCs co-cultured with the ACs and detect the induction ;(4)To compare co-cultured biological induction and chemical induction。
Methods:(1)Obtain, culture and proliferate high-purity BMSCs: Bone marrow were obtained from femoral shaft of SD rats (4 weeks) and BMSCs were isolated by gradient centrifugation and adherent separation and passaged after Amplification in vitro. The cell shape was observed under phase contrast microscope and the expression of CD 29、CD90、CD34 and CD 45 of the cells were analyzed by flow cytometry. Make sure the cells obtained were BMSCs by inducing of fat-like, osteoblast-like and adult neural-like cellstolay the foundation for the next step experiment. (2)Obtain, culture and passage high- concentration and high–purity ACs:Articular cartilage were obtained from the surface of normal femoral head of SD rats(4-week),digested by trypsin(0.25%)and CollageaseⅡ(0.2%).After observing numerous ACs free under phase contrast microscope,centrifuge, deposit Supernatant and place the cells into cartilage cell culture medium to Cultivate, proliferate, and passage; sample, count with trypan blue staining and detect and identify ACs with Toluidine blue and HE staining methods to lay the foundation for the next step experiment.。(3)Co-culture BMSCs and ACs in Transwell:Aspire the third passage BMSCs and ACs and place them respectively into Transwell co-culture system by1:1,BMSCs were placed in the bottom and in the upper ACs。Meanwhile set the same concentration of BMSCs control group separately, cultured in culture medium of NMEM containing 10% FBS respectly. Then Cell proliferation and matrix synthesis would be observed under phase contrast microscopy and Envision immunohistochemical stain typeⅡcollagen was used to analyze each group of cell-seeded cover slips (.4)Comparison of Co-culture bio-induced and TGF-β1 Comparison of chemically induced:Use TGF-β1(10 ng/m1)induction and compare with co-culture induced group of BMSCs of 1:2,1:1,2:1 . MTT assay, glucose amino glycan (GAG) content detection and typeⅡcollagen WESTERN BLOT test were adopted to check the induction of each group cells.
Result:(1)Obtain, culture and proliferate high-purity BMSCs:Some cells anchored were observed under Inverted phase contrast , after 24 hours,most wall-adhering cells were seen , which appeared as a fusiform polygonal appearance ,and cell nucleus were observed too。72h later the number of cells increased and their shapes were fibroblast-like, as with fibroblastoid cells. Meanwhile pairs of nucleolar could be seen in most nucleus and Cells were colony-like growth. Till 10d the cells covered the bottom of culture bottle and up to 90%,Polar cells were arranged, and the colony was swirling. Cell surface markers were tested by flow cytometry: BMSCs uniformly express CD29 and CD90, the positive rates were 92.5%and 94.4%,; while CD34 and CD45 were negative, the negative rates were 99.3%and 99.7%。Fat-like, osteoblast-like and adult neural-like cells induction succeeded. (2) acquire and develop high-grade ACs: the use of the above method can get a certain purity of ACs. Observation under the inverted phase contrast microscope showed: ACs vaccinated after 12h began to adhere, 48h completed adherence. Primary cultured chondrocytes was round and polygonal with abundant cytoplasm, round nucleus which exists in the center; the cells grew into a clear cobblestone appearance, distinguished with long spindle-shaped fibroblasts easily.Only single cartilage cell could be seen growing after digestion of this method.(3)BMSCs were co-cultured with ACs:The number of co-cultured BMSCs increased ,synthesis of extracellular matrix was rich ,which can be stained by typeⅡcollagen immunohistochemical staining and cells staining took on yellow.(4)Comparison of Co-culture bio-induction and TGF-β1 chemical induction: MTT colorimetric examination of Co-cultured cell bio-induction, glucose amino glycan (GAG) content detection and typeⅡcollagen WESTERN BLOT test results showed that:The induction of cell co-culture results were better than the results of TGF-β1 chemical induction,but no significant differences appeared in statistical results of1:1,1:2 co-culture induction.
Conclusion:(1) High-purity BMSCs can be obtained with density gradient centrifugation and differential adhesion method ;(2)High-purity ACs can be obtained by filtrating, centrifugating and culturing after digestion in trypsin and CollagenⅡ. (3)BMSCs co-cultured with ACs can act as seed cells repairing cartilago articularis.
引文
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8. Takahashi,-I; Onodera,-K; Sasano,-Y; Effect of stretching on gene expression of beta1 integrin and focal adhesion kinase and on chondrogenesis through cell-extracellular matrix interactions[J]. Eur-J-Cell-Biol. 2003 Apr; 82(4): 182-92.
9. Wu,-Y-N; Yang,-Z; Hui,-J-H; Cartilaginous ECM componentmodification of the micro-bead culture system for chondrogenic differentiation of mesenchymal stem cells[J]. Biomaterials. 2007 Oct; 28(28): 4056-67.
10.刘媛媛,苗春雷,唐胜建.软骨细胞上清液诱导B MS C向软骨细胞转化的实验研究[ J ] .细胞与分子免疫学杂志, 2007 .2 3 ( 7 )603 -605 .
11. Ni Yu n -f e n g. L i Xi a o -f e i . I n vivo chondrogensis by co-culture of rabbit bonf marrow-derived mesenchymal stem cells and chondrocytes[J].Journal of Clinical Rehabilitative Tissue Engineering Research April 15,2008 Vol.12.No.16 3185-3188.
12. Liu,-X; Zhou,-G-D; Lu,-X-J; Potential of chondrogenesis of bone marrow stromal cells co-cultured with chondrocytes on biodegradable scaffold: in vivo experiment with pigs and mice[J]. Zhonghua-Yi-Xue-Za-Zhi. 2007 Jul 17; 87(27): 1929-33.
13.Dorotka,-R; Windberger,-U; Macfelda,-K; Repair of articular cartilage defects treated by microfracture and a three-dimensional collagen matrix[J]. Biomaterials. 2005 Jun; 26(17): 3617-29.
14. Khillan,-J-S. Generation of chondrocytes from embryonic stem cells[J]. Methods-Mol-Biol. 2006; 330: 161-70.
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34. Cui,-X; Chen,-J; Zacharek,-A; Li,-Y; Nitric oxide donor upregulation of stromal cell-derived factor-1/chemokine (CXC motif) receptor 4 enhances bone marrow stromal cell migration into ischemic brain after stroke[J]. Stem-Cells. 2007 Nov; 25(11): 2777-85 .
35. Mauney,-J-R; Jaquiery,-C; Volloch,-V; In vitro and in vivo evaluation of differentially demineralized cancellous bone scaffolds combined with human bone marrow stromal cells for tissue engineering[J]. Biomaterials. 2005 Jun; 26(16): 3173-85.
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39. Violetta Zujovic, Veronique Taupin. Use of cocultured cell systems to elucidate chemokine-dependent neuronal/microglial interactions: control of microglial activation[J]. Methods. 2003 Apr;29(4):345-50.
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41. Inga Sidén-Kiamos, Dina Vlachou, Gabrielle Margos. Distinct roles for Pbs21 and Pbs25 in the in vitro ookinete to oocyst transformation of Plasmodium berghei[J]. Journal of Cell Science. 2000 Jul 113;3419-3426 .
42. Takigawa,-M; Nakanishi,-T; Kubota,-S; Role of CTGF/HCS24/ecogenin in skeletal growth control[J]. J-Cell-Physiol. 2003 Mar; 194(3): 256-66.
43. Alfaqeh,-H; Chua,-K-H; Aminuddin,-B-S; Effects of different media on the in vivo chondrogenesis of sheep bone marrow stem cells: histological assessment[J]. Med-J-Malaysia. 2008 Jul; 63 Suppl A: 119-20
44. Ge,-W; Jiang,-W-X; Li,-C-H; Transforming growth factor-beta1-loaded fibrin sealant promote bone marrow Mesenchymal stem cells to contract injectable tissue engineering cartilage in vivo[J]. Zhongguo-Yi-Xue-Ke-Xue-Yuan-Xue-Bao. 2005 Dec; 27(6): 692-5.
45.De-Bari,-C; Dell'ACio,-F; Luyten,-F-P. Failure of in vitro-differentiated mesenchymal stem cells from the synovial membrane to form ectopic stable cartilage in vivo[J]. Arthritis-Rheum. 2004 Jan; 50(1): 142-50.
46. Verbruggen,-G; Wang,-J; Wang,-L; Analysis of chondrocyte functional markers and pericellular matrix components by flow cytometry[J]. Methods-Mol-Med. 2004; 100: 183-208.
47. Sui,-Y; Clarke,-T; Khillan,-J-S. Limb bud progenitor cells induce differentiation of pluripotent embryonic stem cells into chondrogenic li neage[J]. Differentiation. 2003 Dec; 71(9-10): 578-85.
2.Tu-Q, Valverde-P, Chen-J. Osterix enhances proliferation and osteogenic potential of bone marrow stromal cells[J]. Biochem Biophys Res Commun. 2006,341(4):1257-1265.
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9.Honczarenko-M, Le-Y, Swierkowski-M, et al. Human bone marrow stromal cells express a distinct set of biologically functional chemokine receptors[J]. Stem cell. 2005,10:1-30.
10. Eliopoulos-N, Stagg-J, Lejeune-L, et al. Allogeneic marrow stromal cells are immune rejected by MHC class I- and class II-mismatched recipient mice[J]. Blood. 2005 ,106(13):4057-4065.
11. Miura-M, Miura-Y, Padilla-Nash-HM, et al. Accumulated Chromosomal Instability in Murine Bone Marrow Mesenchymal Stem Cells Leads to Malignant Transformation[J]. Stem Cells. 2005 Nov 10; [Epub ahead of print].
12. Fuentes-Boquete,-I; Lopez-Armada,-M-J; Maneiro,-E; Pig chondrocyte xenoimplants for human chondral defect repair: an in vitro model[J]. Wound-Repair-Regen. 2004 Jul-Aug; 12(4): 444-52.
13. Temenoff-JS,Mikos-AG.Tissue engineering for regeneration of articular cartilage[J].Biomaterials,2000,21(3):431-440.
14. Nerem-RM,Sambanis-A.Tissue engineering:from biology to biological substitutes [J].Tissue Eng,1995,1(1):3-13.
15.Albisetti,-W; de-Girolamo,-L; De-Bartolomeo,-O; Brini,-A-T. Traditional techniques and experimental therapeutic approach for the restore of degenerate cartilages. Ann-Ital-Chir[J]. 2006 Sep-Oct; 77(5): 433-9.
16. Mankin,H.J.The response of articular cartilage to mechanical injury[J].Bone Joint Surg,1982,64,460.
17. Trzeciak,-T; Kruczynski,-J; Value of autologous chondrocyte transplantation in experimental cartilage defects reconstruction[J]. Chir-Narzadow-Ruchu-Ortop-Pol. 2004; 69(3): 153-7.
18. ChanCK,Anastassiades TP.Anionie glyeoeon jugates from differentiated and Dedifferentiated cultures of bovine articular chondroeytes : modulation by TGF-beta[J」.In Vitro Cell Dev Biol Anim,1998,34(6):492一498.
19. Jasionowski,-M; Krzyminski,-K; Thermally-reversible gel for 3-D cell culture of chondrocytes[J]. J-Mater-Sci-Mater-Med. 2004 May; 15(5): 575-82.
20. Kajikawa,-A; Hirabayashi,-S; Harii,-K:An experimental study on the growth of condylar cartilage, using a new vascularized mandible heterotopic transplant model[J]. J-Oral-Maxillofac-Surg. 2003 Feb; 61(2): 239-45.
21. Gooch KJ,BlunkT,Courter DL,et al. Mechanical response of bovine articular cartilage under dynamic unconfined compression loading at physiological stress levels[J].Osteoarthritis and Cartilage.2004:12:909一915.
22. Passaretti D,Silverman RP,Huang W,et al. Cultured chondrocytes produce injectable tissue-engineered cartilage in hydrogel polymer[J]. Tissue Eng,2001:7(6):805一815.
23. Zhu,-L; Jiang,-H; Zhou,-G-D; Prelimilary study of constructing tissue-engineered cartilage with the endoskeletal scaffold of HDPE by bone marrow stromal cells[J]. Zhonghua-Zheng-Xing-Wai-Ke-Za-Zhi. 2008 Sep; 24(5): 377-81.
24. Kawaguchi,-J; Mee,-P-J; Smith,-A-G . Osteogenic and chondrogenic differentiation of embryonic stem cells in response to specific growth factors . Bone[J]. 2005 May; 36(5): 758-69.
25. Khillan,-J-S. Generation of chondrocytes from embryonic stem cells. Methods-Mol-Biol[J]. 2006; 330: 161-70.
26. Lee,-R-H; Kim,-B; Choi,-I; Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue[J]. Cell-Physiol-Biochem. 2004; 14(4-6): 311-24.
27. Winter,-A; Breit,-S; Parsch,-D; Cartilage-like gene expression in differentiated human stem cell spheroids: a comparison of bone marrow-derived and adipose tissue-derived stromal cells[J]. Arthritis-Rheum. 2003 Feb; 48(2): 418-29.
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