van Osch G J, van den Berg W B, Hunziker E B, Häuselmann H J
Laboratory of Experimental Rheumatology, University Hospital Nijmegen, The Netherlands.
Osteoarthritis Cartilage. 1998 May;6(3):187-95. doi: 10.1053/joca.1998.0111.
Knowledge of matrix assembly is necessary to understand the pathogenesis of disease processes and to find solutions for repair of articular cartilage lesions. The influence of growth factors on matrix assembly is largely unknown. We investigated whether, and to what degree, insulin-like growth factor (IGF-1) and transforming growth factor beta-2 (TGF beta-2) influence proteoglycan synthesis and accumulation in the cell-associated matrix compartment (consisting of pericellular and territorial matrix) compared to the further-removed matrix compartment (consisting of the interterritorial matrix).
Bovine articular chondrocytes were cultured in alginate beads for day 13. The effect of addition of 25 ng/ml IGF-1 or 25 ng/ml TGF beta-2 during the last 7 days in culture was determined. Cell-associated and further-removed matrix compartments were separated by centrifugation after sodium citrate/EDTA treatment. The amount of DNA, the total amount of proteoglycans and the amount of newly synthesized proteoglycans were analyzed biochemically. Morphometric analysis on electron micrographs was used to calculate the volumes of the cell-associated and further-removed matrix components.
It was demonstrated in control beads that 25 +/- 8% of the proteoglycans were laid down in the cell-associated matrix compartment compared with 75 +/- 8% in the further-removed matrix compartment. The cell-associated matrix compartment in intact beads could be recognized in electron microscopy by a delineation of dense amorph material. Morphometric evaluation showed a relative volume of the cell-associated matrix compartment of 5.2 +/- 0.6% compared with 91.3 +/- 0.8% of the further-removed matrix compartment and 3.5 +/- 0.3% of the area occupied by cells. Combination of biochemical and morphometric results showed that the concentration of proteoglycans in the cell-associated matrix compartment was 3.63 +/- 0.32 mg/ml. By adding IGF-1 or TGF beta-2, the amount of both total accumulated proteoglycans and newly synthesized [35S]proteoglycans at day 13 in culture increased. In addition to an overall rise in proteoglycan content, IGF-1 significantly increased (24%) the percentage of proteoglycans laid down in the cell-associated matrix compartment while not changing the relative volume of this compartment (5.2 +/- 0.8%). This leads to a 82% (P < 0.05) increase in the proteoglycan concentration in the cell-associated matrix compartment compared to control beads. In contrast, TGF beta-2 significantly decreased (24%) the relative amount of proteoglycans in the cell-associated matrix compartment which was paralleled by a reduction of the relative volume from 5.2 +/- 0.6 to 3.6 +/- 1.4%. This leads to a significant increase of 87% of the proteoglycan concentration in the cell-associated matrix compartment.
This study demonstrates that both IGF-1 and TGF beta-2 significantly but differently influence proteoglycan synthesis and accumulation in the cell-associated matrix compartment of cultured bovine chondrocytes in alginate. Both growth factors increase the concentration of proteoglycans in the cell-associated matrix compartment. However, addition of TGF beta-2 to bovine articular chondrocytes cultured in alginate beads for 13 days results in a significant reduction of the relative volume of the pericellular matrix compartment compared to controls, indicating differences in assembly of the matrix.
了解基质组装对于理解疾病过程的发病机制以及寻找关节软骨损伤修复方案至关重要。生长因子对基质组装的影响在很大程度上尚不清楚。我们研究了胰岛素样生长因子（IGF-1）和转化生长因子β-2（TGFβ-2）与距离较远的基质区室（由区域间基质组成）相比，是否以及在何种程度上影响细胞相关基质区室（由细胞周和区域基质组成）中蛋白聚糖的合成和积累。
将牛关节软骨细胞在藻酸盐珠中培养13天。测定在培养的最后7天添加25 ng/ml IGF-1或25 ng/ml TGFβ-2的效果。在柠檬酸钠/乙二胺四乙酸（EDTA）处理后通过离心分离细胞相关和距离较远的基质区室。对DNA量、蛋白聚糖总量和新合成的蛋白聚糖量进行生化分析。利用电子显微镜照片的形态计量分析来计算细胞相关和距离较远的基质成分的体积。
在对照珠中证实，25±8%的蛋白聚糖沉积在细胞相关基质区室，而在距离较远的基质区室中为75±8%。完整珠中的细胞相关基质区室在电子显微镜下可通过密集无定形物质的轮廓识别。形态计量评估显示细胞相关基质区室的相对体积为5.2±0.6%，而距离较远的基质区室为91.3±0.8%，细胞所占面积为3.5±0.3%。生化和形态计量结果相结合表明，细胞相关基质区室中蛋白聚糖的浓度为3.63±0.32 mg/ml。通过添加IGF-1或TGFβ-2，培养第13天总积累蛋白聚糖和新合成的[35S]蛋白聚糖的量均增加。除了蛋白聚糖含量总体上升外，IGF-1显著增加（24%）了沉积在细胞相关基质区室中的蛋白聚糖百分比，同时该区域室的相对体积未改变（5.2±0.8%）。这导致与对照珠相比，细胞相关基质区室中蛋白聚糖浓度增加82%（P<0.05）。相比之下，TGFβ-2显著降低（24%）了细胞相关基质区室中蛋白聚糖的相对量，同时相对体积从5.2±0.6%减少到3.6±1.4%。这导致细胞相关基质区室中蛋白聚糖浓度显著增加87%。
本研究表明，IGF-1和TGFβ-2均显著但以不同方式影响藻酸盐中培养的牛软骨细胞的细胞相关基质区室中蛋白聚糖的合成和积累。两种生长因子均增加细胞相关基质区室中蛋白聚糖的浓度。然而，在藻酸盐珠中培养13天的牛关节软骨细胞中添加TGFβ-2导致与对照相比，细胞周基质区室的相对体积显著减少，表明基质组装存在差异。
