The Effects of Hypoxia-Reoxygenation in Mouse Digital Flexor Tendon-Derived Cells

Ischemia-reperfusion injury refers to the exacerbated and irreversible tissue damage caused by blood flow restoration after a period of ischemia. The hypoxia-reoxygenation (H/R) model in vitro is ideal for studying ischemia-reperfusion injury at the cellular level. We employed this model and investigated the effects of cobalt chloride- (CoCl₂-) induced H/R in cells derived from mouse digital flexor tendons.

Various H/R conditions were simulated via treatment of tendon-derived cells with different concentrations of CoCl₂ for 24 h, followed by removal of CoCl₂ to restore a normal oxygen state for up to 96 h. Cell viability was measured using the Cell Counting Kit-8 (CCK-8) assay. Cell growth was determined via observation of cell morphology and proliferation. Oxidative stress markers and mitochondrial activity were detected. The expression levels of hypoxia-inducible factor- (HIF-) 1α, vascular endothelial growth factor-A (VEGF-A), collagen I, and collagen III were determined using Western blot (WB), real-time PCR, and immunofluorescence staining. Cellular apoptosis was analyzed via flow cytometry, and the expression of apoptosis-related proteins Bax and bcl-2 was examined using WB.

The cells treated with low concentrations of CoCl₂ showed significantly increased cell viability after reoxygenation. The increase in cell viability was even more pronounced in cells that had been treated with high concentrations of CoCl₂. Under H/R conditions, cell morphology and growth were unchanged, while oxidative stress reaction was induced and mitochondrial activity was increased. H/R exerted opposite effects on the expression of HIF-1α mRNA and protein. Meanwhile, the expression of VEGF-A was upregulated, whereas collagen type I and type III were significantly downregulated. The level of cellular apoptosis did not show significant changes during H/R, despite the significantly increased Bax protein and reduced bcl-2 protein levels that led to an increase in the Bax/bcl-2 ratio during reoxygenation.

Tendon-derived cells were highly tolerant to the hypoxic environments induced by CoCl₂. Reoxygenation after hypoxia preconditioning promoted cell viability, especially in cells treated with high concentrations of CoCl₂. H/R conditions caused oxidative stress responses but did not affect cell growth. The H/R process had a notable impact on collagen production and expression of apoptosis-related proteins by tendon-derived cells, while the level of cellular apoptosis remained unchanged.