Cultivation of human tenocytes in high-density culture

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Abstract
Limited supplies of tendon tissue for use in reconstructive surgery require development of phenotypically stable tenocytes cultivated in vitro. Tenocytes in monolayer culture display an unstable phenotype and tend to dedifferentiate, but those in three-dimensional culture may remain phenotypically and functionally differentiated. In this study we established a three-dimensional high-density culture system for cultivation of human tenocytes for tissue engineering. Human tenocytes were expanded in monolayer culture before transfer to high-density culture. The synthesis of major extracellular matrix proteins and the ultrastructural morphology of the three-dimensional cultures were investigated for up to 2 weeks by electron microscopy, immunohistochemistry, immunoblotting and quantitative, real-time PCR. Differentiated tenocytes were able to survive over a period of 14 days in high-density culture. During the culture period tenocytes exhibited a typical tenocyte morphology embedded in an extensive extracellular matrix containing cross-striated collagen type I fibrils and proteoglycans. Moreover, expression of the tendon-specific marker scleraxis underlined the tenocytic identity of these cells. Taken together, we conclude that the three-dimensional high-density cultures may be useful as a new approach for obtaining differentiated tenocytes for autologous tenocyte transplantation to support tendon and ligament healing and to investigate the effect of tendon-affecting agents on tendon in vitro.