Macromolecular crowding alone or in combination with other in vitro microenvironment modulators to maintain tenogenic phenotype in vitro

Tendon injuries are very common among the different musculoskeletal disorders affecting millions of people annually. Given that current treatments have failed to restore injured tendons to the prior to injury state, cell-based therapies hold a great promise to engineer functional tissue surrogates in vitro in order to restore the damaged tissues. However, the development of a rich in extracellular matrix tenocyte-assembled tendon equivalent requires prolonged in vitro culture, which is associated with phenotypic drift. Recapitulation of tendon tissue microenvironment in vitro with cues that enhance and accelerate extracellular matrix synthesis and deposition, whilst maintaining tenocyte phenotype, may lead to functional tissue engineering cell-based therapies. Herein, we assessed the synergistic effect of macromolecular crowding with low oxygen tension or growth factor supplementation or surface topography as in vitro microenvironmental modulators on the behaviour of human tenocytes. Firstly, it was demonstrated that human tenocytes cultured at 2 % oxygen tension and with 50 μg/ml carrageenan (macromolecular crowder used) significantly increased synthesis and deposition of collagen types I, III, V and VI. Gene analysis at day 7 illustrated that human tenocytes at 2 % oxygen tension and with 50 μg/ml carrageenan significantly increased expression of the extracellular matrix-related genes prolyl 4-hydroxylase subunit alpha 1, procollagen-lysine 2-oxoglutarate 5-dioxygenase 2, the tendon-related markers scleraxis, tenomodulin and elastin, whilst chondrogenic (e.g. runt-related transcription factor 2, cartilage oligomeric matrix protein, aggrecan) and osteogenic (e.g. secreted phosphoprotein 1, bone gamma-carboxyglutamate protein) trans-differentiation markers were significantly down-regulated or remained unchanged. Then, we assessed the synergistic effect of simultaneous and serial growth factor (insulin growth factor-1, platelet-derived growth factor ββ, growth differentiation factor 5 and transforming growth factor β3) supplementation to carrageenan in human tenocyte function, collagen synthesis and deposition and gene expression. Transforming growth factor β3 supplementation (without / with carrageenan) induced the highest (among all groups) DNA content. In all cases, tenocyte proliferation was significantly increased as a function of time in culture, whilst metabolic activity was not affected. Carrageenan supplementation induced significantly higher collagen deposition than groups without carrageenan (without / with any growth factor). Of all the growth factors used, transforming growth factor β3 induced the highest collagen XXVI deposition when used together with carrageenan in both simultaneous and serial fashion. At day 13, gene expression analysis revealed that transforming growth factor β3 in serial supplementation to carrageenan upregulated the most and downregulated the least collagen-and tendon-related genes and upregulated the least and downregulated the most osteo-, chondro-, fibrosis-and adipose-related trans-differentiation genes. Finally, the combining effect of surface topography and macromolecular crowding on human tenocyte culture was analysed. Our data demonstrated that bidirectionally aligned electrospun fibres induced physiological tenocyte growth and alignment (without / with carrageenan), whilst macromolecular crowding enhanced and accelerated tendon-specific extracellular matrix deposition, which was further oriented to the direction of the electrospun fibres, recapitulating extracellular matrix orientation in native tendons. Collectively, these data advocate the use of multifactorial approaches for the accelerated development of functional tissue-like surrogates in vitro