Lareu Ricky R, Arsianti Irma, Subramhanya Harve Karthik, Yanxian Peng, Raghunath Michael
Tissue Modulation Laboratory, Division of Bioengineering, Faculty of Engineering, National University of Singapore, Singapore.
Tissue Eng. 2007 Feb;13(2):385-91. doi: 10.1089/ten.2006.0224.
The construction of stable engineered tissue depends on the formation of a functional connective tissue produced by cells locally. A major component of connective tissue is collagen. Its deposition into a stable matrix depends on the enzymatic extracellular conversion of procollagen to collagen. This step is very slow in vitro and we hypothesized that this is due to a lack of crowdedness and insufficient excluded volume effect (EVE) in culture media. We used neutral (670 kDa) and negatively charged dextran sulfate (DxS, 500 kDa) to create EVE in cell cultures and to enhance in vitro matrix formation by accelerating procollagen conversion. Biochemical analyses in 2 human fibroblast lines revealed mostly unprocessed procollagen in uncrowded culture medium, whereas in the presence of DxS, procollagen conversion occurred and most of the collagen was associated with the cell layer. Immunocytochemistry confirmed DxS-related collagen deposition that colocalized with fibronectin. The large neutral dextran showed, in identical concentration ranges, no effects that correlated well with its smaller hydrodynamic radius as determined by dynamic light scattering. This predicted a 10 times bigger crowding power of DxS and benchmarks it as a potentially promising crowding agent facilitating the formation of extracellular matrix in vitro.
稳定工程组织的构建依赖于局部细胞产生的功能性结缔组织的形成。结缔组织的主要成分是胶原蛋白。其沉积到稳定基质中依赖于原胶原蛋白在细胞外酶促转化为胶原蛋白。这一步在体外非常缓慢，我们推测这是由于培养基中缺乏拥挤效应和排除体积效应（EVE）不足所致。我们使用中性（670 kDa）和带负电荷的硫酸葡聚糖（DxS，500 kDa）在细胞培养中产生EVE，并通过加速原胶原蛋白转化来增强体外基质形成。对2种人类成纤维细胞系的生化分析表明，在未拥挤的培养基中大多是未加工的原胶原蛋白，而在存在DxS的情况下，发生了原胶原蛋白转化，并且大部分胶原蛋白与细胞层相关。免疫细胞化学证实了与DxS相关的胶原蛋白沉积，其与纤连蛋白共定位。在相同浓度范围内，大的中性葡聚糖没有显示出与其通过动态光散射测定的较小流体力学半径相关的效应。这预测DxS的拥挤能力大10倍，并将其作为一种潜在有前景的拥挤剂，有助于体外细胞外基质的形成。
