Contact or noncontact cocultures of articular chondrocytes with bone marrow mesenchymal stem cells: cell proliferation and differentiation

doi:10.3969/j.issn.2095-4344.1521

Abstract

Abstract:

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 (COL2a1) 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 COL2a1 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 COL2a1 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.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Chondrocytes, Bone Marrow, Mesenchymal Stem Cells, Coculture Techniques, Transforming Growth Factor beta3, Connexin 43, Tissue Engineering

CLC Number:

Zhao Wenhui, Pi Hongtao, Feng Wanwen, Li Xiangdong, Wang Jianwei, Liu Yuepeng, Jiang Yang, Ma Jianxin, Xia Yayi, Wang Cuifang, Shao Linlin, Li Chunhui, Yu Hongyang, Liu Shanglin, Dong Yanbin, Ma Yahui. Contact or noncontact cocultures of articular chondrocytes with bone marrow mesenchymal stem cells: cell proliferation and differentiation[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(1): 24-29.

Figures/Tables 2

2.1 共培养细胞的形态变化分离培养的第2代BMSCs呈纺锤形或长梭形，细胞折光性强，与成纤维细胞形态相似，排列具有方向性，呈漩涡样或辐射状，见图1A。第2代ACs呈多角形、三角形或不规则的卵圆形，核为圆形或椭圆形，可见成簇生长现象，细胞核居细胞中心，胞浆丰富，细胞增殖融合为单层，呈多边形或星形，排列紧密为“铺路石”样，见图1B。接触共培养组共培养4 h细胞开始贴壁，随着ACs和BMSCs共培养时间的延长，长梭形细胞逐渐变为多角形、类圆形，呈聚集生长，形态接近软骨细胞。接触共培养21 d，甲苯胺蓝染色显示共培养细胞及其周围基质浓染，细胞核质分界清晰，细胞核被染成深蓝色，胞质染成蓝紫色，见图2A。细胞免疫化学COL2a1染色后胞浆染成棕黄色，胞核不着色，为强阳性，见图2B。非接触共培养组共培养21 d，观察Transwell下室的BMSCs，发现仅有部分细胞变为多角形或类圆形，大部分细胞仍呈长梭形或短梭形，甲苯胺蓝染色显示共培养细胞及其周围基质染色较浅，COL2a1染色为弱阳性，而ACs体积和核浆比例变化不大，见图3。 2.2 ACs和BMSCs接触共培养对细胞增殖分化的影响随着ACs和BMSCs共培养时间的延长，共培养细胞平均吸光度值逐渐增高，共培养21 d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)，见图4。培养上清液中糖胺多糖和转化生长因子β3水平，见表1。共培养细胞COL2a1和Cx43的表达明显高于非接触共培养组(P < 0.05)，见图5。 2.3 外源性转化生长因子β3对共培养细胞增殖分化的影响接触共培养组和非接触共培养组添加10 µg/L转化生长因子β3进行共培养21 d，接触共培养组添加转化生长因子β3前后细胞增殖无明显变化(P > 0.05)，见图4，培养上清液中糖胺多糖水平和共培养细胞COL2a1及Cx43的表达增高也无显著性意义(P > 0.05)；而非接触共培养组添加转化生长因子β3后细胞的增殖、培养上清液糖胺多糖水平和共培养细胞COL2a1及Cx43的表达则明显提高(P < 0.05)，但仍明显低于接触共培养组(P < 0.05)，见表1，图5和图6。"

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