Gene expression of single articular chondrocytes

Abstract

Although previous studies in the field of tissue engineering have provided important information about articular cartilage, their conclusions are based on population averages and do not account for variations in cell subpopulations. To obtain a precise understanding of chondrocytes, we investigated the effects of cartilage zone and seeding duration on single chondrocyte gene expression to select an optimal zone for tissue engineering (Phase I), followed by an evaluation of growth factor exposure on the zone selected in Phase I (Phase II). In Phase I, superficial and middle/deep bovine articular chondrocytes were seeded in monolayers for 3 or 18 h. In Phase II, middle/deep chondrocytes (selected in Phase I) received 100 ng/ml insulin-like growth factor-I (IGF-I) for 3 h. Real-time reverse transcription/polymerase chain reaction was used to quantify the abundance of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the relative abundances of aggrecan, collagens I and II, cartilage oligomeric matrix protein (COMP), matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of metalloproteinase-1 (TIMP-1). GAPDH varied zonally, but neither time nor IGF-I had an effect on it, suggesting that GAPDH is a suitable housekeeping gene for comparisons within each zone, but not across zones. IGF-I increased the expression of aggrecan and collagen II in middle/deep chondrocytes seeded for 18 h. TIMP-1 expression increased with time in control cells, suggesting that chondrocytes enter a matrix protective state after seeding. IGF-I diminished this effect, suggesting that treatment with IGF-I refocuses chondrocytes on matrix production rather than on protection from metalloproteinases. Concomitant to increasing TIMP-1, MMP-1 was detectable by 18 h in superficial cells, providing further evidence of a trend toward matrix degradation with time. Collagen I was undetected in all cells, and no differences were observed for COMP, confirming that no dedifferentiation or osteoarthritic changes occurred. Taken together, these results establish a unique understanding of individual chondrocyte behavior.

Differences in matrix accumulation and hypertrophy in superficial and deep zone chondrocytes are controlled by bone morphogenetic protein

“…Previous studies including our own have reported several differences between SZC and DZC Darling and Athanasiou, 2005a;Darling and Athanasiou, 2005b;Darling et al, 2004;Dowthwaite et al, 2004;Eleswarapu et al, 2006;Marles et al, 1991;Siczkowski and Watt, 1990;Stephens et al, 1992;Waldman et al, 2003;Zanetti et al, 1985). In the current study, we have identified BMP as an important factor in the differential capacities of SZC and DZC to accumulate matrix and to hypertrophy.…”

“…The distinction is important as the use of BMPs and/or other growth factors in cell-based strategies for cartilage tissue engineering or regeneration cannot be optimized without specific knowledge about the potentially distinct effects of such factors on SZC versus DZC. Recent reports on the effects of other growth factors such as transforming growth factor β and insulin-like growth factor 1 indicate that chondrocyte sub-populations have zonespecific responses (Darling and Athanasiou, 2005a;Eleswarapu et al, 2006). In this study we compared morphology, gene expression, matrix accumulation and quality by SZC versus DZC in a scaffold-free in vitro model of cartilage-like tissue formation.…”

Regional Effects of Enzymatic Digestion on Knee Meniscus Cell Yield and Phenotype for Tissue Engineering

“…Specific primers for each gene were identified from the literature [37][38][39][40] and are listed in Table 1. Gene expression data were analyzed using a two-way analysis of variance (ANOVA), with a significance level of p < 0.05.…”

Chondrocyte-associated antigen and matrix components in a 2- and 3-dimensional culture of rat chondrocytes

“…Moreover, human chondrocytes with high chondrogenic capacity were found to express markers associated with cell-cell or cell-matrix interactions, while a low capacity for matrix production was associated with higher expression of IGF-1 and catabolic genes, e.g., matrix metalloproteinases (37). In relation to the findings presented above, it is also important to note that articular cartilage chondrocytes differ in response to IGF-1 depending on the zone from which they were isolated (38).…”