Eleswarapu Sriram V, Leipzig Nic D, Athanasiou Kyriacos A
Department of Bioengineering, MS-142, Rice University, P.O. Box 1892, Houston, TX 77251, USA.
Cell Tissue Res. 2007 Jan;327(1):43-54. doi: 10.1007/s00441-006-0258-5. Epub 2006 Aug 31.
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.
尽管组织工程领域先前的研究提供了有关关节软骨的重要信息，但其结论是基于总体平均值得出的，并未考虑细胞亚群的差异。为了精确了解软骨细胞，我们研究了软骨区域和接种持续时间对单个软骨细胞基因表达的影响，以选择组织工程的最佳区域（第一阶段），随后评估生长因子暴露对第一阶段所选区域的影响（第二阶段）。在第一阶段，将浅层和中/深层牛关节软骨细胞接种成单层，培养3小时或18小时。在第二阶段，中/深层软骨细胞（在第一阶段选择）接受100 ng/ml胰岛素样生长因子-I（IGF-I）处理3小时。采用实时逆转录/聚合酶链反应定量D-甘油醛-3-磷酸脱氢酶（GAPDH）的丰度以及聚集蛋白聚糖、I型和II型胶原蛋白、软骨寡聚基质蛋白（COMP）、基质金属蛋白酶-1（MMP-1）和金属蛋白酶组织抑制剂-1（TIMP-1）的相对丰度。GAPDH在不同区域存在差异，但时间和IGF-I对其均无影响，这表明GAPDH是每个区域内进行比较的合适管家基因，但不适用于跨区域比较。IGF-I增加了接种18小时的中/深层软骨细胞中聚集蛋白聚糖和II型胶原蛋白的表达。在对照细胞中，TIMP-1表达随时间增加，这表明软骨细胞接种后进入基质保护状态。IGF-I减弱了这种作用，表明用IGF-I处理可使软骨细胞将重点重新放在基质产生上，而不是防止金属蛋白酶的作用。与TIMP-1增加同时，在浅层细胞中18小时可检测到MMP-1，这进一步证明了随着时间推移存在基质降解的趋势。在所有细胞中均未检测到I型胶原蛋白，COMP也未观察到差异，证实未发生去分化或骨关节炎变化。综上所述，这些结果建立了对单个软骨细胞行为的独特认识。
