Cellular therapies play an important role in tendon tissue engineering with tenocytes being described as the most prominent cell population if available in large numbers. In vitro expansion of tenocytes in standard culture leads to phenotypic drift and cellular senescence. Maintenance of tenogenic phenotype in vitro can be achieved by recapitulating different aspects of the tendon microenvironment. One approach used to modulate in vitro microenvironment and enhance extracellular matrix (ECM) deposition is macromolecular crowding (MMC). In addition, as tendon has been described to be a relatively avascular and hypoxic tissue and low oxygen tension can stimulate collagen synthesis and cross-linking through the activation of hypoxia-inducible factor 1-alpha (HIF1-α), we venture to assess the synergistic effect of MMC and low oxygen tension on human tenocyte phenotype maintenance. SDS-PAGE and immunocytochemistry analysis demonstrated that human tenocytes treated with MMC at 2 % oxygen tension showed increased synthesis and deposition of collagen type I. Moreover, immunocytochemistry for the tendon-specific ECM proteins collagen type III, V, VI and fibronectin illustrated enhanced deposition when cells were treated with MMC at 2 % oxygen tension. In addition, western blot analysis revealed increased expression of tendon-specific protein Scleraxis, while a detailed gene analysis illustrated upregulation of tendon-specific genes and downregulation of trans-differentiation genes again when cells cultured with MMC under hypoxic conditions. Collectively, results suggest that the synergistic effect of MMC and low oxygen tension can accelerate the formation of ECM-rich substitutes, which stimulates tenogenic phenotype maintenance.

Cellular therapies play an important role in tendon tissue engineering and regenerative medicine with tenocytes being described as the most prominent cell population for these applications if available in large numbers. However, this is difficult to achieve, because in vitro expansion of tenocytes leads to phenotypic drift and loss of function. Recent work suggests that maintenance of tenogenic phenotype in vitro can be achieved by recapitulating different aspects of the native tendon microenvironment. One approach used to modulate in vitro microenvironment and enhance extracellular matrix (ECM) deposition is macromolecular crowding (MMC). MMC is based on the addition of inert macromolecules to the culture media to mimic the dense extracellular matrix and accelerate the production of ECM-rich substitutes. In addition, as tendon has been described to be a relatively avascular and hypoxic tissue and low oxygen tension can stimulate collagen synthesis and cross-linking through the activation of hypoxia-inducible factor 1-alpha (HIF1-α), we venture to assess the synergistic effect of MMC and low oxygen tension on human tenocyte phenotype maintenance by enhancing deposition of tissue-specific extracellular matrix.

SDS-PAGE and immunocytochemistry analysis, demonstrated that human tenocytes treated with the optimal MMC concentration at 2% oxygen tension showed increased collagen type I synthesis and deposition after 7 days. Moreover, immunocytochemistry for collagen type III, type V, VI, elastin and fibronectin illustrated enhanced deposition when cells were treated with MMC at 2% oxygen tension. In addition, it was shown that low oxygen tension and MMC did not affect the spindle-shape morphology, metabolic activity, proliferation and viability of human tenocytes Collectively, these results suggest that the synergistic effect of optimal macromolecular crowding concentration and low oxygen tension (2%) can accelerate the formation of ECM-rich substitutes, which may stimulate tenogenic phenotype maintenance. Further gene and protein analysis for tendon specific markers should be performed to validate our promising results.