To develop and test a simple enzymatic procedure for isolating chondrons, which consist of the chondrocytes and their surrounding pericellular microenvironment.
Chondrons were obtained by digesting adult human articular cartilage with a mixture of dispase and collagenase. Chondrons and chondrocytes were cultured in alginate beads, immunofluorescence labeled and examined by confocal microscopy.
Comparison of freshly isolated chondrons with cryostat sections of cartilage revealed that type VI collagen, type II collagen and aggrecan were retained, but fibronectin and a unique chondroitin sulfate epitope recognized by the antibody, 7D4, were lost. Comparison of enzymatic and mechanical homogenization methods revealed subtle changes in chondron morphology and retention of fibronectin in mechanically isolated chondrons. Average yield of enzyme-isolated chondrons was slightly lower than that of chondrocytes isolated by pronase and collagenase digestion, but was much greater than that reported for mechanically isolated chondrons. Enzyme-isolated chondron viability was greater than 80% 1 day after isolation, and continued to be above 80% through 7 weeks of alginate bead culture. Viability of isolated chondrocytes was initially greater than 80% but fell to 60-80% with time in culture. Chondrons and isolated chondrocytes had a similar division rate except osteoarthritic chondrons were significantly slower after 2 weeks in culture. Cell division was more rapid for nonosteoarthritic chondrons than for osteoarthritic ones.
Enzymatic isolation of chondrons is relatively simple, gives better yield and viability than mechanical isolation, but comparable yield and viability of traditional chondrocyte isolation. Enzymatic chondron isolation allows the effect of the in vivo-formed pericellular matrix on chondrocyte metabolism to be studied in vitro.