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.

Introduction: 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 cells for therapeutic intervention. Although recent reports demonstrated the central role of oxygen in 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.

Methods: Articular mouse chondrocytes were prepared from the distal femoral condyles of adult mice and chondrocytes were liberated by collagenase type II treatment. Cells were cultured in RPMI 1640 media in monolayer under normoxic or hypoxic conditions (5% O2). Chondrogenic potential was subsequently assessed by plating the cells under micromass conditions and glycosaminoglycan deposition was determined by alcian blue staining. Having determined that oxygen tension infiuences murine chondrocyte expansion and differentiation, similar studies were conducted using adult human chondrocytes taken from knee arthroplasty off-cuts, and Aggrecan (ACAN) gene expression was analyzed using real-time quantitative PCR.

Results: Cellular morphology of cells from mouse articular cartilage was improved in hypoxic culture, with a markedly more fibroblastic appearance seen after greater than 2 passages in normoxic conditions. Micromass cultures maintained in hypoxic conditions demonstrated stronger staining with alcian blue, indicating stronger expression of cartilage-associated glycosaminoglycans. Expansions of human chondrocytes under hypoxic conditions led to an ~ 2-fold increase in the expression of ACAN in comparison to cells in normoxic conditions. Differentiation of passage 2 chondrocytes under hypoxic conditions also improved the expression of ACAN when compared to culturing under normoxia. Ten day hypoxic cultures exhibited an ~ 5-fold increase in ACAN expression in comparison to normoxic cultures. Interestingly, ACAN expression normoxic-cultured cells could be increased by > 4-fold by transfer to hypoxic conditions.

Conclusions: In vivo, the chondrocytes are adapted to an avascular hypoxic environment. Accordingly, applying 5% O2 in the expansion phase in the course of cell-based cartilage repair strategies may more closely mimic the normal chondrocyte microenvironment and may result in a repair tissue with higher quality by increasing the content of glycosaminoglycans.