Determine whether the structure of the type VI collagen component of the chondrocyte pericellular matrix (PCM) generated by agarose-embedded chondrocytes in culture is similar to that found in native articular cartilage.
Confocal microscopy, quick-freeze deep-etch electron microscopy, and real-time polymerase chain reaction (PCR) were used to investigate temporal and spatial patterns of type VI collagen protein deposition and gene expression by bovine chondrocytes during 4 weeks of culture within a 2% agarose hydrogel. Similar analyses were performed on chondrocytes within samples of intact cartilage obtained from the same joint surfaces as those used for cell isolation for comparison.
Type VI collagen accumulated uniformly around cells embedded in agarose, with the rate of deposition slowing after the second week. After 1 week, PCM fibrils were observed to be oriented perpendicular to the cell surface, in contrast with the primarily tangential fibrillar arrangement observed in native articular cartilage. Expression of col6 in agarose-embedded cells was initially much higher ( approximately 400%) than that in chondrocytes within cartilage. Expression of col6 in the cultured chondrocytes declined by approximately 60% after 1 week, and remained stable thereafter.
PCM structure and composition around cells in a hydrogel scaffold may be different than that in native cartilage, with potential implications for mass transport, mechanotransduction, and ultimately, the success of tissue engineering approaches.