Aged Tendon Stem/Progenitor Cells Are Less Competent to Form 3D Tendon Organoids Due to Cell Autonomous and Matrix Production Deficits

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.

Tendon Stem/Progenitor Cell Subpopulations and Their Implications in Tendon Biology

“…Additionally, downregulation of nuclear regulator CITED2 and Aquaporin 1(AQP1) are also reported to be correlated with TSPCs aging ( Hu et al, 2017 ; Chen et al, 2020 ). Functionally incompetent aged TSPCs will assemble into a less cell-populated, poorly organized and biomechanically inferior three-dimensional tendon organoids ( Yan et al, 2020 ). With advancing age, osteogenic differentiation potential and BMP expression are enhanced in TSPCs, which consequently contribute to increased heterotopic ossification in tendons ( Dai et al, 2020 ).…”

Dental follicle cell differentiation towards periodontal ligament-like tissue in a self-assembly three-dimensional organoid model

“…Alternatively, selfassembly models such as pellet and cell sheet cultures have gained a lot of interest, because they parallel the natural tissue formation process and secure native cell-to-cell and cell-to-matrix interactions, which initiate the appropriate and inherent cell signalling cascades of the used cell types (Yan et al, 2018). Previous studies have shown that mesenchymederived stem/progenitor cells, subjected to pellet and cell sheet culture protocols, were able to produce tenogenic (Hsieh et al, 2018;Yan et al, 2020) and chondrogenic scaffold-free tissues (Pattappa et al, 2019a;Pattappa et al, 2019b).…”

“…In this study, the PDL tissue-forming ability of DFCs was investigated, in comparison to the previously established and characterised PDL-hTERT cell line (Docheva et al, 2010), by using a novel self-assembly 3D organoid model for tenogensis/ ligamentogensis (Hsieh et al, 2018;Yan et al, 2020) and by carrying out detailed histomorphometric, quantitative PCR and protein analyses.…”

“…This three-step model is based on a self-assembly of a cell sheet, which is rolled into a 3D rod-like organoid that is subjected to static axial stretch of 10 % and maturation of 2 weeks (Hsieh et al, 2018;Yan et al, 2020). In the first, 2D expansion, step both cell types were plated into cell culture Petri dish (100 mm diameter, Falcon, USA) with a density of 8 × 10 4 cells/cm 2 until reaching full confluence by day 5 in low-glucose DMEM medium (Gibco) supplemented with 10 % FBS, glutamine (365.3 mg/L) and 1× MEM amino acids (all from Sigma-Aldrich), In the second, 2D stimulation, step both cell types were cultured in high-glucose DMEM (Gibco) 10 % FBS, 1× MEM amino acids and 50 µg/mL ascorbic acid (Sigma-Aldrich) for 14 d. Afterwards, monolayered cell sheets were detached from the dish using a cell scraper and manually rolled to form approx.…”

Rebuilding Tendons: A Concise Review on the Potential of Dermal Fibroblasts

“…Our previous study proved that human TSPCs have great potential to form well-aligned and collagen-rich cell sheets [ 95 ]. Moreover, our further research revealed that aged/degenerative TSPCs exhibit defective cell sheets compared to young/healthy TSPCs [ 96 ], demonstrating that the cell sheet model is suitable to mimic in vitro tenogenic differentiation process as well as to investigate pathomechanisms involved in tendon aging. Hence, it will be of great interest to perform a follow up study with the aim to subject DFs to the tendon cell sheet protocol.…”