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Introduction The role of hypoxia in connective tissue repair and regeneration is unclear. Intact tendon, ligament and cartilage are relatively avascular tissues compared to bone, and demonstrate oxygen tension of 1-7%. Healing tendon, ligament and cartilage tissues are accompanied by increased vascularity, as are certain disease states including tendinosis. Given this stark contrast between the native connective tissue state and the increased vascularity observed in healing and diseased tissues, we hypothesized that the increased oxygen tension present in hypervascular tissues will affect the connective tissue cell phenotype. As our prior studies of the effects of hypoxia (1%0₂) on human tenocyte differentiation have revealed a chondrocyte phenotype in hypoxia, we subsequently examined the role of hypoxia on human chondrocyte differentiation.

Methods Human chondrocytes were isolated from patients (36-49y) undergoing total hip replacement for avascular necrosis. Human chondrocytes were isolated by collagenase digestion, and were passaged at low density in hypoxic (1%0₂) and normoxic (21%0₂) culture conditions. Low passage (3-5) chondrocytes were used. Human chondrocytes were cultured at low, medium, and high densities in hypoxic or normoxic conditions, and harvested at week 1, 2, 4, 6, 8, 10, and 12. Cell lysates were analyzed for collagen 1, 2, 3 and 10 expression. qRTPCR of chondrocyte markers sox9 and aggrecan was also performed. Cells were subjected to light microscopy assays of alcian blue staining of proteoglycans, as well as immunofluorescence of collagen 1 and 2. Pharmacologic manipulation of human chondrocytes was performed using Rac1 inhibitor NSC23766 as well as the ROCK inhibitor Y27632.

Results

1. Human chondrocytes dedifferentiate in normoxic culture, as demonstrated by increased collagen 1 and decreased collagen 2 expression.

2. Human chondrocytes retain the chondrocyte phenotype in hypoxic culture, as observed by decreased collagen 1 and increased collagen 2 expression.

3. A hypertrophic chondrocyte phenotype was observed in normoxic culture at low density over time, as observed by increased collagen 10 expression.

4. Reversion of the chondrocyte phenotype in normoxia was observed with decreased Rac1 and increased RhoA activity, suggesting a role of decreased tension coupled with increased compressive forces in maintaining the human chondrocyte phenotype.

The significance of these findings is as follows: human chondrocytes dedifferentiate in normoxic environments, a process which is reversed by altering cellular forces. These results suggest that a key role of tissue vascularity - via increased oxygen tension - is to control human connective tissue cell differentiation, a process key to regeneration and repair.