Qi Wen-ning, Scully Sean P
Division of Orthopaedic Surgery, Duke University Medical Center, Durham, NC 27710, USA.
J Orthop Res. 2003 Mar;21(2):282-9. doi: 10.1016/S0736-0266(02)00136-5.
The articular cartilage extracellular matrix (ECM) interfaces with chondrocytes and influences many biological processes important to cartilage homeostasis and repair. The alginate bead culture system can be viewed as a model of cartilage repair in which the chondrocyte attempts to recreate the pericellular matrix while maintaining a differentiated phenotype. The purpose of this study was to evaluate the alteration in epitopes of proteoglycan and tenascin synthesized by chondrocytes in the presence of exogenous extracellular type II collagen. We evaluated the effects on four biomarkers associated with the creation of the denovo matrix using ELISA and immunohistochemistry: keratan sulfate epitope (5D4), 3B3(-) neoepitope of chondroitin-6- sulfate, 3B3(+) chondroitinase-generated epitope of chondroitin-6-sulfate, and tenascin-C expression. TGF-beta1 stimulated the production of 3B3(+), 5D4, and tenascin-C in a dose-dependent manner and decreased 3B3(-) levels. Following the addition of exogenous type II collagen, 3B3(-) increased and tenascin-C decreased but did not change the direction of TGF-beta1 effects. In contrast, 5D4 expression decreased in the presence of collagen II as TGF-beta1 increased to 10 ng/ml. Interestingly, the amount of 3B3(+) epitope was not affected by the incorporation of type II collagen. Immunohistochemistry found there was no significant difference in distribution of these biomarkers in the presence and absence of extracellular type II collagen incorporation. These results elucidate the subtle biochemical differences in ECM synthesized by chondrocytes in the presence of type II collagen and further characterize the role played by ECM in the TGF-beta1 regulation of the articular cartilage physiology.
关节软骨细胞外基质（ECM）与软骨细胞相互作用，并影响许多对软骨稳态和修复至关重要的生物学过程。藻酸盐珠培养系统可被视为软骨修复模型，其中软骨细胞试图在维持分化表型的同时重建细胞周围基质。本研究的目的是评估在存在外源性细胞外II型胶原蛋白的情况下，软骨细胞合成的蛋白聚糖和腱生蛋白表位的变化。我们使用酶联免疫吸附测定（ELISA）和免疫组织化学评估了对与新生基质形成相关的四种生物标志物的影响：硫酸角质素表位（5D4）、硫酸软骨素-6-硫酸酯的3B3(-)新表位、硫酸软骨素-6-硫酸酯的3B3(+)软骨素酶产生的表位以及腱生蛋白-C表达。转化生长因子-β1（TGF-β1）以剂量依赖性方式刺激3B3(+)、5D4和腱生蛋白-C的产生，并降低3B3(-)水平。添加外源性II型胶原蛋白后，3B3(-)增加，腱生蛋白-C减少，但未改变TGF-β1的作用方向。相比之下，当TGF-β1增加到10 ng/ml时，在存在II型胶原蛋白的情况下5D4表达下降。有趣的是，3B3(+)表位的量不受II型胶原蛋白掺入的影响。免疫组织化学发现，在存在和不存在细胞外II型胶原蛋白掺入的情况下，这些生物标志物的分布没有显著差异。这些结果阐明了在存在II型胶原蛋白的情况下软骨细胞合成的ECM中的细微生化差异，并进一步表征了ECM在TGF-β1调节关节软骨生理学中所起的作用。
