RESUMO
OBJECTIVE: Regeneration of the zonal organization of articular cartilage may be an important advancement for cartilage tissue engineering. The first goal of this study was to validate our surgical technique as a method to selectively isolate chondrocytes from different zones of bovine articular cartilage. The second goal was to confirm that chondrocytes from different zones would have different proliferative and metabolic activities in two-dimensional (2-D) and 3-D cultures. Finally, to regenerate the zonal organization, we sought to make multi-layered constructs by encapsulating chondrocytes from different zones of articular cartilage. DESIGN: Cartilage slices were removed from three (upper, middle, and lower) zones of articular cartilage of young bovine legs. Histology and biochemical composition of the cartilage slices were analyzed to confirm that they had been obtained from the proper zone. Growth kinetics and gene expression in monolayer culture and matrix formation in photopolymerizing hydrogels were evaluated. Multi-layered photopolymerizing hydrogels were constructed with chondrocytes from each zone of native cartilage encapsulated. Cell viability and maintenance of the cells in the respective layer were evaluated using the Live/Dead Viability kit and cell tracking protocols, respectively. After 3 weeks, the multi-layered constructs were harvested for histologic examination including immunohistochemistry for type II collagen. RESULTS: Analysis of histology and biochemical composition confirmed that the cartilage slices had been obtained from the specific zone. Chondrocytes from different zones differed in growth kinetics and gene expression in monolayer and in matrix synthesis in 3-D culture. Cells encapsulated in each of the three layers of the hydrogel remained viable and remained in the respective layer in which they were encapsulated. After 3-week culture, each zone of multi-layered constructs had similar histologic findings to that of native articular cartilage. CONCLUSION: We present this as an experimental model to regenerate zonal organization of articular cartilage by encapsulating chondrocytes from different layers in multi-layered photopolymerizing gels.
