Margaret B. Aydelotte

Differences between sub-populations of cultured bovine articular chondrocytes. I. Morphology and cartilage matrix production

Bovine articular chondrocytes cultured in agarose gel comprise a heterogeneous population when judged by morphological and histochemical criteria. The purpose of the present experiments was to compare, under the same conditions of culture, sub-populations of chondrocytes derived from different depths of articular cartilage. Sub-populations of chondrocytes were cultured separately following their isolation from slices of articular cartilage cut from successive depths of the tissue. Chondrocytes derived from superficial and deep zones differed significantly in morphology, rate of proliferation, and activity in secreting a proteoglycan-rich extracellular matrix. The differences are sufficient to account for the heterogeneity observed in cultures of the entire cell population, and the correlate well with known variations with depth in morphology and histochemistry of intact articular cartilage. These results demonstrate that articular chondrocytes continue in culture to express metabolic differences which reflect their original anatomical location; such differences may have important functional significance.

Differences between sub-populations of cultured bovine articular chondrocytes. II. Proteoglycan metabolism

Sub-populations of bovine articular chondrocytes derived from different depths of the cartilage showed differences in accumulation of proteoglycan-rich extracellular matrix in culture. To extend these morphological studies, the synthesis and catabolism of 35S-labeled proteoglycans have been examined in similar cultures. Chondrocytes from deep zones synthesized significantly more proteoglycans than cells from the superficial zone. While all populations of chondrocytes synthesized predominantly aggregating proteoglycans, a higher proportion of isotope was present in non-aggregating proteoglycans in cultures of superficial chondrocytes, by comparison with those of deep cells. Proteoglycans were degraded more rapidly by superficial cells than by chondrocytes from deeper layers. These results correlate both with previous histochemical studies of similar cultures, and with known depth-related variations in biochemical composition of intact articular cartilage.

Gene Expression by Human Articular Chondrocytes Cultured in Alginate Beads

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 α1 (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 α1 (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.

Influence of interleukin-1 on the morphology and proteoglycan metabolism of cultured bovine articular chondrocytes.

Bovine articular chondrocytes cultured in agarose gel in the presence of serum elaborated a highly organized extracellular matrix rich in proteoglycans and collagens. The cultures were evaluated quantitatively by radiosulfate labeling of proteoglycans, and by densitometry following staining with alcian blue. In addition, immunohistochemical methods were used to demonstrate the presence of several components of cartilage proteoglycan molecules. Treatment with Interleukin-1 (Il-1) or retinol resulted in diminished synthesis and enhanced catabolism of matrix proteoglycans, but the chondrocytes were more sensitive to human recombinant Il-1 alpha than to Il-1 beta. Treatment with Il-1 alpha or retinol resulted in a profound disorganization of the residual matrix around the majority of the chondrocytes, while Il-1 beta caused much less severe changes. Some variation in cellular response to Il-1 alpha may result from the heterogeneity previously reported among articular chondrocytes.

Culture of chondrocytes in alginate gel: variations in conditions of gelation influence the structure of the alginate gel, and the arrangement and morphology of proliferating chondrocytes.

SummarySodium alginate, which gels in the presence of calcium ions, is commonly used for culture of anchorage-independent cells, such as chondrocytes. Normally, the gel appears microscopically homogeneous but, depending on the conditions of gelation, it may contain a varying number of small channels that extend inward from the surface. We have examined the influence of these channels on the morphology of cultured chondrocytes entrapped in alginate beads. Growth-plate or articular chondrocytes cultured in alginate normally proliferate and form rounded cell clusters but, in alginate beads containing numerous channels, many chondrocytes become aligned and form columns similar to those in the growth plate in vivo. As the pattern of cellular growth and morphology in alginate is profoundly influenced by the presence of channels in the gel, further studies were conducted to determine what specific conditions of gelation affect their formation. The channels are especially numerous when both the alginate and the gelling solutions lack sodium ions or other monovalent cations. The channels are cavities in the gel formed by particulate blocking of the rapid diffusion of calcium ions from the gelling solution into the boundary of the calcium alginate solution, and hence they extend inward from cells at the surface of the alginate gel. An understanding of the conditions under which these channels develop makes it possible either to avoid their formation or, alternatively, to enhance the number of channels in order to encourage proliferating cells to grow in radial columns, rather than in a less organized pattern characteristic of most culture systems.

COMBINATION OF DIGITAL IMAGE ANALYSIS AND POLARIZATION MICROSCOPY : THEORETICAL CONSIDERATIONS AND EXPERIMENTAL DATA

The potentialities of polarization microscopy has been greatly increased by using specific stains for selective enhancement of the optical anisotropy of a macromolecular constituent of cells and tissues. Such stainings have proved to be especially useful in exploring the spatial orientation pattern of the extracellular matrix components. The retardation value, which characterizes quantitatively the degree of submicroscopic orientation, can be measured traditionally with a compensator plate. This technique, however, is time‐consuming and greatly dependent on visual judgment. Several attempts have been made to combine digital image analysis and polarization microscopy to improve the measuring technique in unstained structures. In this paper, we summarize theoretical considerations and experimental data to show the advantages and limitations of this methodological approach when using stained and birefringent specimens. The technique we are suggesting is the measurement of the light intensity using a 12 bit cCCD camera attached to a polarized light microscope and digital image analysis system. The theoretical basis is given by the Fresnel equation describing the relationship between light intensity and retardation value. According to this, there is a sin2 function between the light intensity and the retardation value. The same relationship of these two parameters was observed in our experiments on the birefringent extracellular matrix around chondrocytes grown in agarose gel and interterritorial and territorial matrix of canine articular cartilage stained with picrosirius red. Our results suggest that the retardation values can be calculated directly from the light intensity values if the retardation value is lower than λ/2. Microsc. Res. Tech. 43:511–517, 1998.

Evidence of an Eicosanoid Contribution to IL-1 Induction of IL-6 in Human Articular Chondrocytes.

It has been demonstrated previously that interleukin-1 (IL-1) induces articular cartilage explants and chondrocytes in culture to produce elevated levels of inflammatory mediators such as interleukin-6 (IL-6) and prostaglandins. Previous studies have also demonstrated a relationship between IL-6 secretion and the ability of IL-1 to modulate proteoglycan synthesis by chondrocytes. In this study we have utilized an alginate culture system in an effort to investigate a role for eicosanoids in IL-1 induction of IL-6 expression in human articular chondrocytes. IL-1 treatment of chondrocytes cultured in alginate resulted in increased synthesis of IL-6 and prostaglandins, but not leukotrienes. Cyclo-oxygenase inhibitor, indomethacin (5 μg ml-1), was able to inhibit prostaglandin synthesis to below basal levels with no significant effect on the levels of IL-6 released by chondrocytes in response to IL-1. When chondrocytes were treated with 5 μg ml-1 indomethacin and 10 μM of the general lipoxygenase inhibitor, nordihydroguiaretic acid (NDGA), an approximate 50% decrease in IL-1-induced IL-6 expression was observed. Alone, levels of NDGA specific for lipoxygenase inhibition (10 μM) did not affect IL-1-induced IL-6 expression, but higher levels of NDGA (50 μM) which inhibited both prostaglandin and leukotriene biosynthesis reduced IL-1-induced IL-6 expression to the same extent as that observed with 5 μg ml-1 indomethacin and 10 μM NDGA. This inhibition of IL-6 expression by NDGA and indomethacin was dose responsive and also reversible with the addition of exogenous prostaglandin E2 (PGE2) or leukotriene B4 (LTB4). Although IL-1-induced IL-6 expression was only affected when both prostaglandin and leukotriene biosynthesis were inhibited, elevated levels of PGE2 but not leukotriene B4, C4, D4, or E4 were observed in the culture medium of IL-1-treated chondrocytes. These findings may indicate that cyclo-oxygenase products such as PGE2 normally contribute to IL-1 induction of IL-6 expression in chondrocytes, and under conditions when cyclo-oxygenase is inhibited, lipoxygenase products alternatively contribute to this response.

Effects of Misoprostol with Interleukin-1 on Proteoglycan Metabolism of Cultured Articular Chondrocytes.

Bovine and normal human articular chondrocytes in suspension culture were treated with misoprostol (an analog of prostaglandin E1 [PGE1]), alone and in combination with interleukin-1 (IL-1) and/or diclofenac sodium, to study effects on proteoglycan metabolism. At concentrations of 50 ng ml-1 and above, misoprostol suppressed synthesis of proteoglycans but did not affect their rate of catabolism. The mild inhibitory effect of misoprostol on proteoglycan synthesis was additive to that of IL-1, especially in chondrocytes from the superficial zone of bovine articular cartilage. In IL-1-treated cultures, diclofenac Na caused a modest improvement in proteoglycan synthesis, but this beneficial effect was diminished by the simultaneous addition of misoprostol. Cartilage or chondrocyte cultures treated with IL-1, in which proteoglycan synthesis is suppressed, serve as model systems in which to study metabolic responses of chondrocytes to potential therapeutic agents, but in these experiments, no chondroprotective effects of misoprostol were observed in IL-1-activated chondrocytes.