Abstract
Tendons are dense connective tissues, which are critical for the integrity and function of our musculoskeletal system. During tendon aging and degeneration, tendon stem/progenitor cells (TSPCs) experience profound phenotypic changes with declined cellular functions that can be linked to the known increase in complications during tendon healing process in elderly patients. Tissue engineering is a promising approach for achieving a complete recovery of injured tendons. However, use of autologous cells from aged individuals would require restoring the cellular fitness prior to implantation. In this study, we applied an established cell sheet model for in vitro tenogenesis and compared the sheet formation of TSPC derived from young/healthy (Y-TSPCs) versus aged/degenerative (A-TSPCs) human Achilles tendon biopsies with the purpose to unravel differences in their potential to form self-assembled three-dimensional (3D) tendon organoids. Using our three-step protocol, 4 donors of Y-TSPCs and 9 donors of A-TSPCs were subjected to cell sheet formation and maturation in a period of 5 weeks. The sheets were then cross evaluated by weight and diameter measurements; quantification of cell density, proliferation, senescence and apoptosis; histomorphometry; gene expression of 48 target genes; and collagen type I protein production. The results revealed very obvious and significant phenotype in A-TSPC sheets characterized by being fragile and thin with poor tissue morphology, and significantly lower cell density and proliferation, but significantly higher levels of the senescence-related gene markers and apoptotic cells. Quantitative gene expression analyses at the mRNA and protein levels, also demonstrated abnormal molecular circuits in the A-TSPC sheets. Taken together, we report for the first time that A-TSPCs exhibit profound deficits in forming 3D tendon tissue organoids, thus making the cell sheet model suitable to investigate the molecular mechanisms involved in tendon aging and degeneration, as well as examining novel pharmacologic strategies for rejuvenation of aged cells.
Highlights
Tendons are dense connective tissues, which are critical for the integrity and function of our musculoskeletal system (Schiele et al, 2013; Docheva et al, 2015; Costa-Almeida et al, 2019)
In order to investigate how aging affects tendon stem/progenitor cells (TSPCs), we compared the phenotypic difference between Achilles tendon-derived young tendon stem/progenitor cell (Y-TSPC) from young/healthy and aged tendon stem/progenitor cell (A-TSPC) from aged/degenerative human donors
As well as to minimize costs, we designed our study with administration of only one growth factor from the TGFβ family. It will be of great importance in follow up research to investigate the synergistic action of Connective Tissue Growth Factor (CTGF) and TGFβ in the 3D tendon cell sheet model, which may result in quicker maturation of the cell sheets
Summary
Tendons are dense connective tissues, which are critical for the integrity and function of our musculoskeletal system (Schiele et al, 2013; Docheva et al, 2015; Costa-Almeida et al, 2019). Tendons need a long period for rehabilitation, especially in aged patients, accompanied with higher failure risk and unsatisfactory repair outcomes (Hirzinger et al, 2014). Tissue aging involves many intrinsic and extrinsic processes and it is frequently linked with tissue degeneration, tendon rupture incidence as well as reduced healing capacity (Gumucio et al, 2014; Schneider et al, 2018; Steinmann et al, 2020). Aging is reflected by a decline in organ and tissue homeostasis (de Lucas et al, 2018). Many studies on age-related diseases have revealed that a diminished stem cell pool is responsible for adult tissue degeneration (Zhang et al, 2018). The exact molecular mechanism behind tendon aging and degeneration remain by large unclear
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