Cultivation of human tenocytes in high-density culture

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.

Synergistic effects of dexamethasone and quinolones on human-derived tendon cells

“…Embedded in this network of fibrils are tenocytes. These cells produce all components of the extracellular matrix and consequently play an important role in the structure and function of the tendon [16,17]. Cell-cell and cell-matrix interactions are mediated via transmembrane signal transduction receptors such as 1 -integrins.…”

In vitro study comparing two collageneous membranes in view of their clinical application for rotator cuff tendon regeneration

“…9,20 When the tendon fibers have been altered by severe chronic degeneration, primary repair is not a sure method of treatment and re-tear rates are discouraging. In fact, the poor tissue quality creates difficulties in achieving secure fixation with primary repair.…”

“…9 In the present study, cells derived from tendons were used and were characterized by means of cell morphology and ECM protein production as performed in many other studies. 12,13,21,23,26,29,30,33,35 Moreover, in our study in order to maintain the phenotype we used the explant technique, in which cells are permitted to grow from small fragments of tissue instead of enzymatic digestion that affects cell membrane and destroys the cell-cell and cell-matrix relationship.…”

“…8 Tendons are composed of highly specialized mesenchyme-derived cells (tenocytes) embedded in a three-dimensional network of extracellular matrix (ECM) consisting predominantly of collagen type I (CICP; >95%), other types of collagens (type III and V), proteoglycans (PG), elastin, and fibronectin (FBN). 9 Tendons have very poor spontaneous regenerative capabilities, 8 and despite intensive remodeling, complete regeneration is never achieved. 10 Finally, it is also known that tendon ruptures are closely associated with other concomitant diseases or related therapies that cause tendon degeneration.…”

Regenerative Medicine for Tendon Regeneration and Repair: The Role of Bioscaffolds and Mechanical Loading

“…Moreover, experimental studies have demonstrated that cultured cells can be switched between different fates including growth, differentiation, apoptosis, directional motility or different stem cell lineages, by modulating cell shape. (Barkhausen T et al 2003) (Brown RA et al 1998) (Wang JH et al 2004) (Schulze-Tanzil G et al 2004) Externally applied cyclic strain under in vitro conditions has enormous effects on various functions of tenocytes, such as their metabolism, proliferation, orientation and matrix deposition (Screen HRC et al 2005) (Yamamoto E et al 2005) Kessler et al (Kessler et al 2001) demonstrated that collagen fibres and tendon cells can be oriented along the direction of the stress and can upregulate synthesis of tissue inhibitor matrix metalloproteinases-1 and -3 as well as of collagen type I, the main component of tendinous extracellular matrix. It is known that cyclic strain can affect cell morphology and induce uniaxial cellular alignment.…”