关节软骨细胞和骨髓间充质干细胞不同共培养方式对细胞增殖与分化的影响
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摘要
背景:在关节软骨细胞和骨髓间充质干细胞共培养环境中,软骨细胞功能活跃,增殖速度加快,骨髓间充质干细胞促进了软骨细胞的表型维持,而且少量软骨细胞提供的微环境能够诱导骨髓间充质干细胞成软骨分化,降低了软骨分化过程中出现的肥大表型和软骨内骨化,但是影响共培养系统中细胞增殖分化的因素和细胞间相互作用的机制尚不明确。目的:观察软骨细胞和骨髓间充质干细胞接触共培养对细胞增殖分化的影响,进一步优化共培养方式,为探讨共培养系统中二者的相互作用机制提供依据。方法:分离和培养新西兰兔(南京医科大学实验动物中心提供)软骨细胞和骨髓间充质干细胞,取第2代软骨细胞和骨髓间充质干细胞,以1∶3的比例直接混合在24孔板进行接触共培养为接触共培养组,以1∶3比例分别在Transwell的上室和下室进行非接触共培养为非接触共培养组,共培养21d。细胞爬片进行甲苯胺蓝染色、COL2a1及Cx43细胞免疫化学染色。应用CCK8检测共培养系统中细胞的增殖情况,ELISA检测共培养细胞上清液中糖胺聚糖和转化生长因子β3水平,Western blot检测共培养细胞COL2a1和Cx43的表达。然后添加转化生长因子β3进行软骨细胞和骨髓间充质干细胞接触共培养和非接触共培养,比较细胞增殖、糖胺聚糖水平、COL2a1及Cx43蛋白表达的变化。结果与结论:(1)随着共培养时间的延长,细胞增殖能力逐渐增高,接触共培养组细胞吸光度值明显高于非接触共培养组(P <0.05),糖胺聚糖和转化生长因子β3水平以及COL2a1和Cx43的蛋白表达明显高于非接触共培养组;(2)添加10μg/L转化生长因子β3共培养21 d,接触共培养组细胞增殖、糖胺聚糖水平、COL2a1及Cx43的表达与未添加转化生长因子β3比较无明显变化(P>0.05),而非接触共培养组的细胞增殖、糖胺聚糖水平、COL2a1及Cx43蛋白表达明显提高(P<0.05),但仍明显低于未添加转化生长因子β3的接触共培养组(P <0.05);(3)结果表明,接触共培养比非接触共培养明显促进细胞的增殖和分化,Cx43在其中起重要作用。
BACKGROUND: Coculture of articular chondrocytes with bone marrow mesenchymal stem cells(BMSCs) can contribute to proliferation and phenotype maintenance of articular chondrocytes and chondrogenic differentiation of BMSCs as a result of the microenvironment provided by the small number of articular chondrocytes. Furthermore, the coculture system decreases hypertrophic phenotype and enchondral ossification during BMSCs chondrogenic differentiation. However, the mechanisms underlying the cell-cell interactions in the coculture system of articular chondrocytes with BMSCs have not been fully clarified and necessitate further studies. OBJECTIVE: To investigate the effect of the contact coculture of articular chondrocytes with BMSCs on the cell proliferation and differentiation in vitro, and to further optimize the coculture pattern providing a basis for further exploring the interaction mechanism of articular chondrocytes and BMSCs in the coculture system. METHODS: Articular chondrocytes and BMSCs were isolated from New Zealand white rabbits(provided by the Animal Core Facility of Nanjing Medical University, Nanjing, China) and expanded in vitro. The articular chondrocytes and BMSCs both at passage 2 were harvested and cocultured at the ratio of 1:3 for 21 days. The coculture patterns included the cell-cell contact coculture in the 24-well plates as experimental group and cell-cell noncontact coculture through a Transwell chamber as control group. Cytochemistry staining and immunocytochemistry staining were performed to observe cell morphology and to evaluate distribution of related proteins. Cell proliferation was determined using Cell Counting Kit-8. Levels of glycosaminoglycans and transforming growth factor-β3(TGF-β3) in the medium supernatants of the experimental and control groups were detected by ELISA. Expression of type II collagen alpha 1(COL2 a1) and connexin 43(Cx43) in the coculture system of articular chondrocytes with BMSCs were analyzed using western blot. Cell proliferation, glycosaminoglycan level and expression levels of COL2 a1 and Cx43 in the two groups were evaluated following TGF-β3 supplementation in each group and articular chondrocytes were subsequently cocultured with BMSCs in the contact or noncontact manner. RESULTS AND CONCLUSION:(1) The ability of cell proliferation gradually increased with prolonged coculture time, the mean absorbance value of the experimental group was significantly higher that that of the control group(P < 0.05). The mean levels of glycosaminoglycan and TGF-β3 and expression levels of COL2 a1 and Cx43 in the experimental group were significantly higher than those in the control group respectively.(2) Following addition of 10 μg/L TGF-β3, the aforementioned indicators showed no significant changes in the experimental group(P > 0.05), whereas increased significantly in the control group, but still lower than those in the experimental group with no TGF-β3 supplementation(P < 0.05). Our results indicate that the contact coculture of articular chondrocytes with BMSCs significantly promotes cell proliferation and differentiation as compared with the noncontact coculture, and Cx43 plays an important role in cell proliferation and differentiation.
BACKGROUND: Coculture of articular chondrocytes with bone marrow mesenchymal stem cells(BMSCs) can contribute to proliferation and phenotype maintenance of articular chondrocytes and chondrogenic differentiation of BMSCs as a result of the microenvironment provided by the small number of articular chondrocytes. Furthermore, the coculture system decreases hypertrophic phenotype and enchondral ossification during BMSCs chondrogenic differentiation. However, the mechanisms underlying the cell-cell interactions in the coculture system of articular chondrocytes with BMSCs have not been fully clarified and necessitate further studies. OBJECTIVE: To investigate the effect of the contact coculture of articular chondrocytes with BMSCs on the cell proliferation and differentiation in vitro, and to further optimize the coculture pattern providing a basis for further exploring the interaction mechanism of articular chondrocytes and BMSCs in the coculture system. METHODS: Articular chondrocytes and BMSCs were isolated from New Zealand white rabbits(provided by the Animal Core Facility of Nanjing Medical University, Nanjing, China) and expanded in vitro. The articular chondrocytes and BMSCs both at passage 2 were harvested and cocultured at the ratio of 1:3 for 21 days. The coculture patterns included the cell-cell contact coculture in the 24-well plates as experimental group and cell-cell noncontact coculture through a Transwell chamber as control group. Cytochemistry staining and immunocytochemistry staining were performed to observe cell morphology and to evaluate distribution of related proteins. Cell proliferation was determined using Cell Counting Kit-8. Levels of glycosaminoglycans and transforming growth factor-β3(TGF-β3) in the medium supernatants of the experimental and control groups were detected by ELISA. Expression of type II collagen alpha 1(COL2 a1) and connexin 43(Cx43) in the coculture system of articular chondrocytes with BMSCs were analyzed using western blot. Cell proliferation, glycosaminoglycan level and expression levels of COL2 a1 and Cx43 in the two groups were evaluated following TGF-β3 supplementation in each group and articular chondrocytes were subsequently cocultured with BMSCs in the contact or noncontact manner. RESULTS AND CONCLUSION:(1) The ability of cell proliferation gradually increased with prolonged coculture time, the mean absorbance value of the experimental group was significantly higher that that of the control group(P < 0.05). The mean levels of glycosaminoglycan and TGF-β3 and expression levels of COL2 a1 and Cx43 in the experimental group were significantly higher than those in the control group respectively.(2) Following addition of 10 μg/L TGF-β3, the aforementioned indicators showed no significant changes in the experimental group(P > 0.05), whereas increased significantly in the control group, but still lower than those in the experimental group with no TGF-β3 supplementation(P < 0.05). Our results indicate that the contact coculture of articular chondrocytes with BMSCs significantly promotes cell proliferation and differentiation as compared with the noncontact coculture, and Cx43 plays an important role in cell proliferation and differentiation.
引文
[1]Ko CY, Ku KL, Yang SR, et al. In vitro and in vivo co-culture of chondrocytes and bone marrow stem cells in photocrosslinked PCL-PEG-PCL hydrogels enhances cartilage formation. J Tissue Eng Regen Med. 2016;10(10):E485-E496.
[2]Acharya C, Adesida A, Zajac P, et al. Enhanced chondrocyte proliferation and mesenchymal stro-mal cells chondrogenesis in coculture pellets mediate improved cartilage formation. J Cell Physiol.2012;227(1):88-97.
[3]冯万文,莱浙军,李小民,等.骨髓间充质干细胞和软骨细胞共培养种子细胞特征及体内成软骨活性[J].中国组织工程研究与临床康复, 2008,12(3):442-446.
[4]Bian L, Zhai DY, Mauck RL, et al. Coculture of human mesenchymal stem cells and articular chondrocytes reduces hypertrophy and enhances functional properties of engineered cartilage. Tissue Eng Part A. 2011;17(7-8):1137-1145.
[5]Steck E, Fischer J, Lorenz H, et al. Mesenchymal stem cell differentiation in an experimental cartilage defect:restriction of hypertrophy to bone-close neocartilage. Stem Cells Dev. 2009;18(7):969-978.
[6]冯万文,夏亚一,孙正义,等.骨髓间充质干细胞和软骨细胞共培养复合同种异体脱钙骨基质修复关节软骨全层缺损[J].第四军医大学学报, 2006,27(18):1683-1686.
[7]Zuo Q, Cui W, Liu F, et al. Co-cultivated mesenchymal stem cells support chondrocytic differentiation of articular chondrocytes. Int Orthop. 2013;37(4):747-752.
[8]Kang N, Liu X, Yan L, et al. Different ratios of bone marrow mesenchymal stem cells and chon-drocytes used in tissue-engineered cartilage and its application for human ear-shaped substitutes in vitro.Cells Tissues Organs. 2013;198(5):357-366.
[9]陈宗雄,刘晓强,王万宗.骨髓间充质干细胞和关节软骨细胞共培养与转化生长因子β1诱导的比较[J].中国组织工程研究与临床康复, 2010,14(27):5037-5040.
[10]Wu L, Leijten JC, Georgi N, et al. Trophic effects of mesenchymal stem cells increase chondro-cyte proliferation and matrix formation. Tissue Eng Part A. 2011;17(9-10):1425-1436.
[11]Meretoja VV, Dahlin RL, Kasper FK, et al. Enhanced chondrogenesis in co-cultures with articu-lar chondrocytes and mesenchymal stem cells. Biomaterials. 2012;33(27):6362-6369.
[12]Yao Y, Huang Y, Qian D, et al. Effect of Various Ratios of Co-Cultured ATDC5 Cells and Chondrocytes on the Expression of Cartilaginous Phenotype in Microcavitary Alginate Hydrogel. J Cell Biochem.2017;118(11):3607-3615.
[13]Knight MM, McGlashan SR, Garcia M, et al. Articular chondrocytes express connexin 43 hemi-channels and P2 receptors-a putative mechanoreceptor complex involving the primary cilium. J Anat. 2009;214(2):275-283.
[14]Mayan MD, Carpintero-Fernandez P, Gago-Fuentes R, et al. Human articular chondrocytes ex-press multiple gap junction proteins:differential expression of connexins in normal and osteoarthritic cartilage. Am J Pathol. 2013;182(4):1337-1346.
[15]Zhang X, Sun Y, Wang Z, et al. Up-regulation of connexin-43expression in bone marrow mes-enchymal stem cells plays a crucial role in adhesion and migration of multiple myeloma cells. Leuk Lymphoma. 2015;56(1):211-218.
[16]De Windt TS, Saris DB, Slaper-Cortenbach IC, et al. Direct Cell-Cell Contact with Chondrocytes Is a Key Mechanism in Multipotent Mesenchymal Stromal Cell-Mediated Chondrogenesis. Tissue Eng Part A. 2015;21(19-20):2536-2547.
[17]Yang YH, Lee AJ, Barabino GA. Coculture-driven mesenchymal stem cell-differentiated articu-lar chondrocyte-like cells support neocartilage development. Stem Cells Transl Med. 2012;1(11):843-854.
[18]Dahlin RL, Ni M, Meretoja VV, et al. TGF-β3-induced chondrogenesis in co-cultures of chon-drocytes and mesenchymal stem cells on biodegradable scaffolds. Biomaterials. 2014;35(1):123-132.
[19]Chen FM, Zhang M, Wu ZF. Toward delivery of multiple growth factors in tissue engineering. Biomaterials. 2010;31(24):6279-6308.
[20]Dahlin RL, Meretoja VV, Ni M, et al. Chondrogenic phenotype of articular chondrocytes in monoculture and co-culture with mesenchymal stem cells in flow perfusion. Tissue Eng Part A.2014;20(21-22):2883-2891.
[21]Schrobback K, Klein TJ, Woodfield TB. The importance of connexin hemichannels during chondroprogenitor cell differentiation in hydrogel versus microtissue culture models. Tissue Eng Part A. 2015;21(11-12):1785-1794.
[22]Willebrords J, Maes M, Crespo Yanguas S, et al. Inhibitors of connexin and pannexin channels as potential therapeutics. Pharmacol Ther.2017;180:144-160.
[23]Zhang YD, Zhao SC, Zhu ZS, et al. Cx43-and Smad-Mediated TGF-β/BMP Signaling Pathway Promotes Cartilage Differentiation of Bone Marrow Mesenchymal Stem Cells and Inhibits Osteoblast Differentiation. Cell Physiol Biochem. 2017;42(4):1277-1293.
[2]Acharya C, Adesida A, Zajac P, et al. Enhanced chondrocyte proliferation and mesenchymal stro-mal cells chondrogenesis in coculture pellets mediate improved cartilage formation. J Cell Physiol.2012;227(1):88-97.
[3]冯万文,莱浙军,李小民,等.骨髓间充质干细胞和软骨细胞共培养种子细胞特征及体内成软骨活性[J].中国组织工程研究与临床康复, 2008,12(3):442-446.
[4]Bian L, Zhai DY, Mauck RL, et al. Coculture of human mesenchymal stem cells and articular chondrocytes reduces hypertrophy and enhances functional properties of engineered cartilage. Tissue Eng Part A. 2011;17(7-8):1137-1145.
[5]Steck E, Fischer J, Lorenz H, et al. Mesenchymal stem cell differentiation in an experimental cartilage defect:restriction of hypertrophy to bone-close neocartilage. Stem Cells Dev. 2009;18(7):969-978.
[6]冯万文,夏亚一,孙正义,等.骨髓间充质干细胞和软骨细胞共培养复合同种异体脱钙骨基质修复关节软骨全层缺损[J].第四军医大学学报, 2006,27(18):1683-1686.
[7]Zuo Q, Cui W, Liu F, et al. Co-cultivated mesenchymal stem cells support chondrocytic differentiation of articular chondrocytes. Int Orthop. 2013;37(4):747-752.
[8]Kang N, Liu X, Yan L, et al. Different ratios of bone marrow mesenchymal stem cells and chon-drocytes used in tissue-engineered cartilage and its application for human ear-shaped substitutes in vitro.Cells Tissues Organs. 2013;198(5):357-366.
[9]陈宗雄,刘晓强,王万宗.骨髓间充质干细胞和关节软骨细胞共培养与转化生长因子β1诱导的比较[J].中国组织工程研究与临床康复, 2010,14(27):5037-5040.
[10]Wu L, Leijten JC, Georgi N, et al. Trophic effects of mesenchymal stem cells increase chondro-cyte proliferation and matrix formation. Tissue Eng Part A. 2011;17(9-10):1425-1436.
[11]Meretoja VV, Dahlin RL, Kasper FK, et al. Enhanced chondrogenesis in co-cultures with articu-lar chondrocytes and mesenchymal stem cells. Biomaterials. 2012;33(27):6362-6369.
[12]Yao Y, Huang Y, Qian D, et al. Effect of Various Ratios of Co-Cultured ATDC5 Cells and Chondrocytes on the Expression of Cartilaginous Phenotype in Microcavitary Alginate Hydrogel. J Cell Biochem.2017;118(11):3607-3615.
[13]Knight MM, McGlashan SR, Garcia M, et al. Articular chondrocytes express connexin 43 hemi-channels and P2 receptors-a putative mechanoreceptor complex involving the primary cilium. J Anat. 2009;214(2):275-283.
[14]Mayan MD, Carpintero-Fernandez P, Gago-Fuentes R, et al. Human articular chondrocytes ex-press multiple gap junction proteins:differential expression of connexins in normal and osteoarthritic cartilage. Am J Pathol. 2013;182(4):1337-1346.
[15]Zhang X, Sun Y, Wang Z, et al. Up-regulation of connexin-43expression in bone marrow mes-enchymal stem cells plays a crucial role in adhesion and migration of multiple myeloma cells. Leuk Lymphoma. 2015;56(1):211-218.
[16]De Windt TS, Saris DB, Slaper-Cortenbach IC, et al. Direct Cell-Cell Contact with Chondrocytes Is a Key Mechanism in Multipotent Mesenchymal Stromal Cell-Mediated Chondrogenesis. Tissue Eng Part A. 2015;21(19-20):2536-2547.
[17]Yang YH, Lee AJ, Barabino GA. Coculture-driven mesenchymal stem cell-differentiated articu-lar chondrocyte-like cells support neocartilage development. Stem Cells Transl Med. 2012;1(11):843-854.
[18]Dahlin RL, Ni M, Meretoja VV, et al. TGF-β3-induced chondrogenesis in co-cultures of chon-drocytes and mesenchymal stem cells on biodegradable scaffolds. Biomaterials. 2014;35(1):123-132.
[19]Chen FM, Zhang M, Wu ZF. Toward delivery of multiple growth factors in tissue engineering. Biomaterials. 2010;31(24):6279-6308.
[20]Dahlin RL, Meretoja VV, Ni M, et al. Chondrogenic phenotype of articular chondrocytes in monoculture and co-culture with mesenchymal stem cells in flow perfusion. Tissue Eng Part A.2014;20(21-22):2883-2891.
[21]Schrobback K, Klein TJ, Woodfield TB. The importance of connexin hemichannels during chondroprogenitor cell differentiation in hydrogel versus microtissue culture models. Tissue Eng Part A. 2015;21(11-12):1785-1794.
[22]Willebrords J, Maes M, Crespo Yanguas S, et al. Inhibitors of connexin and pannexin channels as potential therapeutics. Pharmacol Ther.2017;180:144-160.
[23]Zhang YD, Zhao SC, Zhu ZS, et al. Cx43-and Smad-Mediated TGF-β/BMP Signaling Pathway Promotes Cartilage Differentiation of Bone Marrow Mesenchymal Stem Cells and Inhibits Osteoblast Differentiation. Cell Physiol Biochem. 2017;42(4):1277-1293.