Institute for Tissue Engineering and Regenerative Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong,Hong Kong Special Administrative Region; School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, Hong Kong Special Administrative Region.
ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, and the Department of Chemical Engineering, The University of Melbourne, Parkville, Victoria, 3010, Australia.
Mater Sci Eng C Mater Biol Appl. 2020 Jan;106:110280. doi: 10.1016/j.msec.2019.110280. Epub 2019 Oct 7.
A faithful reconstruction of the native cellular microenvironment is instrumental for tissue engineering. Macromolecular crowding (MMC) empowers cells to deposit their own extracellular matrix (ECM) in greater amounts, and thus contributes to building tissue-specific complex microenvironments in vitro. Dextran sulfate (DxS, 500 kDa), a semi-synthetic sulfated polyglucose, was shown previously at a fractional volume occupancy (FVO) of 5.2% (v/v; 100 μg/ml) to act as a potent molecular crowding agent in vitro. When added to human mesenchymal stromal cell (MSC) cultures, DxS enhanced fibronectin and collagen I deposition several-fold also at concentrations with negligible FVO (<1% v/v). In a cell-free system, incubation of culture media supplemented with fetal bovine serum (FBS), purified fibronectin or collagen I with DxS led to a co-deposition of respective components, exhibiting a similar granular pattern as observed in cell culture. Aggregation of FBS components, fibronectin or collagen I with DxS was confirmed by dynamic light scattering, where an increase in hydrodynamic radius in the respective mixtures was observed. FBS- and fibronectin aggregates could be dissociated with increasing salt concentrations, indicating electrostatic forces to be responsible for the aggregation. Conversely, collagen I-DxS aggregates increased in size with increasing ion concentration, likely caused by charge screening of collagen I, which is net negatively charged at neutral pH, thus permitting weaker intermolecular interactions to occur. The incorporation of DxS into the ECM resulted in altered ECM topography and stiffness. DxS-supplemented cultures exhibited potentiated bioactivity, such as enhanced adipogenic and especially osteogenic differentiation under inductive conditions. We propose an alternative mechanism by which DxS drives ECM deposition via aggregation, and in an independent manner from MMC. A deeper understanding of the underlying mechanism will enable optimized engineering approaches for ECM-rich tissue constructs.
在组织工程中，对天然细胞微环境进行忠实重建至关重要。大分子拥挤（MMC）使细胞能够沉积更多的细胞外基质（ECM），从而有助于在体外构建具有组织特异性的复杂微环境。硫酸葡聚糖（DxS，500kDa）是一种半合成的硫酸多糖，先前已证明在体积分数占有率（FVO）为 5.2%（v/v；100μg/ml）时，它在体外是一种有效的分子拥挤剂。当添加到人骨髓间充质基质细胞（MSC）培养物中时，DxS 在浓度具有可忽略的 FVO（<1%v/v）时也能使纤连蛋白和 I 型胶原的沉积增加数倍。在无细胞体系中，在含有胎牛血清（FBS）、纯化的纤连蛋白或 I 型胶原的培养基中孵育 DxS 会导致各成分的共沉积，表现出与细胞培养中观察到的相似的颗粒状模式。通过动态光散射证实了 DxS 与 FBS 成分、纤连蛋白或胶原 I 的聚集，在各自的混合物中观察到水动力半径增加。随着盐浓度的增加，FBS 和纤连蛋白的聚集物可以解离，表明静电作用力是聚集的原因。相反，随着离子浓度的增加，胶原 I-DxS 聚集物的尺寸增加，这可能是由于胶原 I 的电荷屏蔽，在中性 pH 下胶原 I 带负电荷，从而允许较弱的分子间相互作用发生。DxS 掺入 ECM 会导致 ECM 形貌和刚度发生改变。在诱导条件下，补充 DxS 的培养物表现出增强的生物活性，例如增强的脂肪生成和特别是成骨分化。我们提出了一种替代机制，即 DxS 通过聚集来驱动 ECM 沉积，并且与 MMC 独立。对潜在机制的更深入了解将使优化富含 ECM 的组织构建体的工程方法成为可能。
