Kikuchi Tetsutaro, Shimizu Tatsuya
Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University (TWIns), 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.
Regen Ther. 2020 Jan 17;14:119-127. doi: 10.1016/j.reth.2019.12.001. eCollection 2020 Jun.
Cutting the cost of manufacturing is important for extending the use of tissue-engineered therapeutic products. The present study aimed to develop a simple method for fabrication of cartilaginous tissues for regenerative therapy, utilizing the phenomenon where human articular chondrocytes grow thickness-wise and spontaneously form three-dimensionally thick tissues.
Normal human articular chondrocytes (NHACs) were cultured with varying concentrations of transforming growth factor beta 1 (TGF-β1) and/or fibroblast growth factor-2 (FGF-2) to optimize the culture condition for thickness-wise growth of chondrocytes. Next, the tissues grown in the optimal condition were subjected to re-differentiation culture in attached and detached states to assess differentiation capacity by evaluating secreted factors, histological analysis, and a gene expression assay.
NHACs grew thickness-wise efficiently in the presence of 1 ng/mL TGF-β1 and 10 ng/mL FGF-2. After two weeks of culture, NHACs grew with 11-fold higher thickness and 16-fold higher cell number compared to cells which were neither treated with TGF-β1 nor with FGF-2. These thickness-wise-grown chondrocytes could be re-differentiated by a differentiation medium according to the increase in melanoma inhibitory activity (MIA) and positive safranin-O staining. Interestingly, the cartilaginous gene expression was considerably different between the attached and detached conditions even in the same culture medium, indicating the necessity of detachment and shrinkage to achieve further differentiation.
Spontaneous thickness-wise growth might provide a simple tissue-engineering method for manufacturing cartilaginous 3D tissues.
降低制造成本对于扩大组织工程治疗产品的应用至关重要。本研究旨在开发一种简单的方法来制造用于再生治疗的软骨组织，利用人类关节软骨细胞沿厚度方向生长并自发形成三维厚组织的现象。
将正常人关节软骨细胞（NHACs）与不同浓度的转化生长因子β1（TGF-β1）和/或成纤维细胞生长因子-2（FGF-2）一起培养，以优化软骨细胞沿厚度方向生长的培养条件。接下来，将在最佳条件下生长的组织在附着和分离状态下进行再分化培养，通过评估分泌因子、组织学分析和基因表达测定来评估分化能力。
在1 ng/mL TGF-β1和10 ng/mL FGF-2存在下，NHACs能有效地沿厚度方向生长。培养两周后，与未用TGF-β1和FGF-2处理的细胞相比，NHACs的生长厚度增加了11倍，细胞数量增加了16倍。这些沿厚度方向生长的软骨细胞可通过分化培养基根据黑色素瘤抑制活性（MIA）的增加和番红O染色阳性进行再分化。有趣的是，即使在相同的培养基中，附着和分离条件下的软骨基因表达也有很大差异，这表明需要分离和收缩以实现进一步分化。
自发的沿厚度方向生长可能为制造软骨三维组织提供一种简单的组织工程方法。
