6. HYPOXIC REGULATION OF CHONDROCYTE DIFFERENTIATION AND ITS APPLICATION TO CARTILAGE REPAIR

Abstract

Purpose: Chondral injuries of the knee are commonly seen at arthroscopy, yet there is no consensus on the most appropriate treatment method. However, untreated cartilage injury predisposes to osteoarthritis contributing to pain and disability. For cell-based cartilage repair strategies, an ex-vivo expansion phase is required to obtain sufficient numbers of cells needed for therapy. Although recent reports demonstrated the central role of oxygen for the function and differentiation of chondrocytes, little is known of the effect of physiological low oxygen concentrations during the expansion of the cells and whether this alters their chondrogenic capacity.

Method: Initial studies of chondrocyte expansion were performed in mature mice, with cells expanded at either atmospheric oxygen tension (21%) or 5% 02 in monolayer cultures. Chondrogenic differentiation was subsequently assessed via micromass culture. Having determined that oxygen tension influences murine chondrocyte expansion and differentiation, similar studies were conducted using adult human chondrocytes taken from knee arthroplasty off-cuts, with mRNA expression of select genes involved in the chondrogenic program analyzed by q-PCR.

Results: Cellular morphology was improved in hypoxic culture, with a markedly more fibroblastic appearance seen after greater than 2 passages in 21% O2. Micromass cultures maintained in hypoxic conditions demonstrated stronger staining with Alcian blue, indicating stronger expression of cartilaginous glycosaminoglycans. Collagen type II mRNA expression was two-fold higher in cells expanded at 5% as compared to expansion at 21% O2. Micromass cultures grown at 21% O2 showed up to a twofold increase in the tissue content of glycosaminoglycans when formed with cells expanded at 5% instead of 21% O2. However, no differences in the mRNA expression or staining for collagen type II protein were observed in these micromass cultures. Hypoxia (5% O2) applied during micromass cultures gave rise to tissues with low contents of glycosaminoglycans.

Conclusion: In-vivo, chondrocytes are adapted to a hypoxic environment. Taking this into account, applying 5% O2 in the expansion phase in the course of cell-based cartilage repair strategies, may result in a repair tissue with higher quality by increasing the content of glycosaminoglycans.