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Journal of Histochemistry and Cytochemistry, Vol. 49, 1211-1220, October 2001, Copyright © 2001, The Histochemical Society, Inc.

ARTICLE

Gene Expression by Human Articular Chondrocytes Cultured in Alginate Beads

Susan Chubinskayaa,c, Klaus Hucha, Monika Schulzea, Lori Ottena, Margaret B. Aydelottea, and Ada A. Colea,b
a Departments of Biochemistry, Rush Medical College at Rush-Presbyterian–St Luke's Medical Center, Chicago, Illinois
b Anatomy, Rush Medical College at Rush-Presbyterian–St Luke's Medical Center, Chicago, Illinois
c Section of Rheumatology, Rush Medical College at Rush-Presbyterian–St Luke's Medical Center, Chicago, Illinois

Correspondence to: Susan Chubinskaya, Dept. of Biochemistry, Rush-Presbyterian–St Luke's Medical Center, 1653 W. Congress Parkway, Chicago, IL 60612. E-mail: schubins@rush.edu

as 0A5395; 1A5554

Culture of articular chondrocytes in alginate beads offers several advantages over culture in monolayer; cells retain their phenotype for 8 months or longer. Earlier studies of chondrocytes cultured in alginate concentrated on collagen and proteoglycan synthesis. However, gene expression by in situ hybridization (ISH) has not been investigated. The purposes of the present study on human chondrocytes were (a) to modify the ISH procedure for the alginate beads to examine the mRNA expression of {alpha}1 (II) procollagen, aggrecan, and two matrix metalloproteinases (MMP-3 and MMP-8) thought to be involved in cartilage matrix degradation, and (b) to compare expression in cultured chondrocytes with that in chondrocytes of intact human cartilage. The modifications made for ISH include the presence of CaCl2 and BaCl2 in the fixation and washing steps and exclusion of cetyl pyridinium chloride. By ISH we show that aggrecan, MMP-3, and MMP-8 are continuously expressed during 8 months of culture. The {alpha}1 (II) procollagen gene is expressed only during the first 2 months of culture and after 3 months its expression is undetectable, which is consistent with its absence in adult articular cartilage. By Western blotting, Type II collagen protein had been synthesized and deposited in both the cell-associated and further-removed matrix compartments at 7 and 14 days of culture. These data indicate that chondrocytes cultured in alginate beads could be preserved for immunohistochemistry and ISH and that culture of human chondrocytes in alginate beads may serve as a good model for studying cartilage-specific phenotype as well as factors that influence cartilage matrix turnover. (J Histochem Cytocem 49:1211–1219, 2001)

Key Words: human articular chondrocytes, alginate beads, in situ hybridization, type II collagen, aggrecan, MMP-3, MMP-8


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