The effects of isolation on chondrocyte gene expression.

Tissue engineering of articular cartilage usually requires the isolation and culture of chondrocytes. Previous studies have suggested that enzymatic isolation may alter the metabolic activity and growth rate of chondrocytes. This study examined the effects of 4 common isolation protocols on chondrocyte gene expression, morphology, and total cell yield immediately following the digest (t = 0) and after 2 culture periods (24 h and 1 week). Cartilage explants were digested using 1 of 4 protocols: (1) 6-h collagenase digest, (2) 22-h collagenase digest, (3) 45-min trypsin digest followed by a 3-h collagenase digest, or (4) 1.5-h pronase digest followed by a 3-h collagenase digest. Gene expression levels for glyceraldehyde-3-phosphate dehydrogenase, type I collagen, type II collagen, aggrecan, superficial zone protein, matrix metalloproteinase- 1, and tissue inhibitor of metalloproteinase-1 were measured at t = 0 h, 24 h, and 1 week using quantitative reverse transcriptase-polymerase chain reaction. In this study, cell yield was greatest for the 22-h collagenase and pronase-collagenase digests. However, the data indicate that a 6-h collagenase digest has the fewest gene expression changes compared to native cells. For tissue engineering, data from this study suggest that when cell yield is critical, a 22-h collagenase digest is preferable, but when obtaining cells closest to native chondrocytes is more desired, the 6-h collagenase digest is more beneficial.