Restoring the normal structure and function of injured tendons is a major challenge in orthopaedics. The discovery of tendon-derived stem cells (TDSCs) provides a novel perspective to treat tendon injuries, which is expected to be an efficient approach to promote tendon repair and regeneration (Li et al., 2021).
Isolation of TDSCs
The isolation of tendon derived stem cells (TDSC), both from the sheath and from fibres, was performed following a published protocol (Yang et al., 2018). In brief, tendon tissues were dissected from the Achilles tendon of a pig and washed with antibiotic. After washing, the tissue was cut into small pieces and digested with type II collagenase overnight at 37˚C. The enzymatic activity was neutralized with fetal bovine serum and tissue pieces passed through a 70 µm cell strainer to yield single-cell suspension and then centrifuged. The released cells were resuspended in L‑DMEM; Lonza, 15% FBS, 1% Penicillin-Streptomycin-Amphotericin B solution, 1% L-glutamine, 0.4 ng/ml epidermal growth factor, 2.5 ng/ml basic fibroblast growth factor and 2.5 ng/ml stem cell factor. Cells were seeded at 50,000 cells/well seeding density and cultured at 37˚C in two different oxygen conditions (2% and 21%). After 48 h of initial plating, the cells were washed twice to remove remaining non-adherent bodies. After one week, when cells reached confluence, they were split at 5000 cells/cm2. Starting from passage P1, conditioned media (serum-free) was collected in order to isolate and characterize Extracellular Vesicles (EVs).
Isolation of Tenocytes
The isolation of tenocytes was performed using the same pig used for TDSC isolation, only by plating directly into 6-well plates small pieces of pig tendon fibres covered with DMEM, 10% FBS, 1% PSA, 1% L-glutamine and 1% Nonessential amino acids.
TDSCs from tendon fibres displayed significantly enhanced rates of proliferation when compared to both TDSC from the tendon sheath and tenocytes. This was further influenced by culture in a physiological 2% O2 condition where all cells proliferated more rapidly than their air oxygen cultured counterparts.
Flow cytometry analysis was performed to confirm the expression levels of surface markers: CD14, CD19, CD34, CD44, CD45, HLA-DR, CD73, CD90, CD105. Subsequent molecular analysis confirmed the expression of tenocyte-specific markers: Scleraxis, Tenomodulin, and Thrombospondin 4, and of stem cells markers: Nanog, Oct-4 and Nestin. The multi-lineage differentiation potential of porcine TDSCs was then performed for osteogenic, chondrogenic and adipogenic induction media to confirm stem cell properties. The extraction and characterization of EVs from TDSCs and tenocytes was performed to confirm their cell-instructive-effect with protein quantification by Bradford assay, size distribution determination via Zetasizer, and a THP-1 based immunological model.
TDSCs from tendon fibres display an enhanced proliferative potential than those from sheath or tenocytes. Immunophenotype, molecular analysis, and differentiation potential are consistent across all cell types while EV functionality differences remain to be determined."