Quantitative analysis of temporal and spatial variations of chondrocyte behavior in engineered cartilage during long-term culture

Abstract

In this work, we present the fact that chondrocyte activity differs in relation to their position in an engineered cartilage construct. Chondrocytes from porcine articular cartilage were cultured in a monolayer. Then the cell/extracellular matrix (ECM) membrane was peeled off and centrifuged into a three-dimensional (3D) pellet-type construct. Cultivated in a static condition, the constructs were harvested at specific time intervals (1, 2, 3, and 5 weeks) and manually cored using a biopsy punch to separate the core from the remaining construct. The resultant parts, core and peripheral remnant were thus obtained and subjected to analysis individually. Cell density (10(6) cells/cm(3)) of the core was significantly higher at 1 week than that of the periphery but this trend was reversed at later time points. Cell viability was remarkably better in the peripheral tissue. Alcian blue staining of glycosaminoglycan (GAG) revealed an intense blue staining from the periphery, exhibiting a steep gradient in distribution of GAG concentration. The gene expression ratio of collagen type II to I appeared to be more altered in the periphery, possibly suggesting cell dedifferentiation, especially at later time points (> 2 weeks). The mRNA levels of matrix metalloproteinase-1 (MMP-1) and MMP-13 remained in the normal range, whereas collagen type X expression was more significantly upregulated at the periphery. This study showed that chondrocyte behavior could be highly variable in the extent of their proliferation, differentiation and dedifferentiation, depending on their physical location within 3D engineered cartilage construct.