Thickness-wise growth technique for human articular chondrocytes to fabricate three-dimensional cartilage grafts.

Abstract

IntroductionCutting 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.MethodsNormal 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.ResultsNHACs 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.ConclusionsSpontaneous thickness-wise growth might provide a simple tissue-engineering method for manufacturing cartilaginous 3D tissues.