Coelho-Sampaio Tatiana, Tenchov Boris, Nascimento Marcos A, Hochman-Mendez Camila, Morandi Verônica, Caarls Michelle Botelho, Altankov George
Institute of Biomedical Sciences, Program of Bioengineering, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
Medical University of Sofia, Department of Medical Physics and Biophysics, Sofia, Bulgaria.
Acta Biomater. 2020 Jul 15;111:242-253. doi: 10.1016/j.actbio.2020.05.021. Epub 2020 May 22.
Tissue engineering demands the development of scaffolds that mimic natural extracellular matrices (ECM). Despite the success in obtaining synthetic interstitial ECM, the production of an artificial basement membrane (BM), the specialized thin sheet of ECM that is pivotal for the functional organization of most tissues and internal organs, is still not achieved. With the long-term aim of developing a flat BM-like structure here we investigated the behavior of acid-soluble Col IV during simultaneous assembly with laminin (LM) in acidic conditions. The underlying rationale was the previously observed phenomenon of acid-triggered LM polymerization, giving rise to biomimetic polylaminin (polyLM) that can be adsorbed on the substrate. Unexpectedly, we found that Col IV (that does not polymerize in acidic conditions) readily incorporated into the polyLM layer, forming a network that mimics to a great extent the characteristic polygonal morphology of single polyLM observable at micrometric scale. Scanning calorimetry and light scattering measurements supported the notion that polyLM and Col IV could directly interact. The biological properties of the proposed artificial BM-like structure were characterized using human keratinocytes (HACAT) and umbilical vein endothelial cells (HUVEC). HACAT formed stratified cell layers on the hybrid polyLM/Col IV layer, but not on Matrigel, nor on LM or Col IV alone, while HUVEC improved cortical F-actin and tight juctions organization on polyLM/Col IV. Thus, the proposed artificial BM reproduces not only morphological but also some functional properties of the natural BM. STATEMENT OF SIGNIFICANCE: Basement membranes (BMs) are flat biological matrices separating tissue compartments in the body. Their peculiar sheet-like structure is thought to result from the association of two independent protein networks of laminin and collagen IV. While pursuing the development of an artificial BM, we found that, when mixed with acid-induced polymerized laminin, collagen IV immediately conformed to the laminin shape. This implies that the protein networks may not be independently assembled as believed so far, but instead that laminin may command the assembly of collagen IV. Our hybrid matrix was structurally more stable than the commercial BM extract Matrigel and, unlike the latter, supported in vitro formation of a stratified layer of keratinocytes that approximated the organization of the natural epidermis.
组织工程需要开发能够模拟天然细胞外基质（ECM）的支架。尽管在获得合成间质ECM方面取得了成功，但人工基底膜（BM）的生产仍未实现，BM是一种特殊的薄片状ECM，对大多数组织和内脏器官的功能组织至关重要。为了长期开发一种扁平的BM样结构，我们在此研究了酸性条件下酸溶性IV型胶原（Col IV）与层粘连蛋白（LM）同时组装时的行为。其基本原理是先前观察到的酸触发LM聚合现象，产生可吸附在底物上的仿生聚层粘连蛋白（polyLM）。出乎意料的是，我们发现Col IV（在酸性条件下不会聚合）很容易融入polyLM层，形成一个在很大程度上模仿在微米尺度上可观察到的单个polyLM特征多边形形态的网络。扫描量热法和光散射测量支持了polyLM和Col IV可以直接相互作用的观点。使用人角质形成细胞（HACAT）和脐静脉内皮细胞（HUVEC）对所提出的人工BM样结构的生物学特性进行了表征。HACAT在polyLM/Col IV混合层上形成分层细胞层，但在基质胶上、单独的LM或Col IV上均未形成，而HUVEC在polyLM/Col IV上改善了皮质F-肌动蛋白和紧密连接的组织。因此，所提出的人工BM不仅再现了天然BM的形态，还再现了其一些功能特性。重要性声明：基底膜（BMs）是分隔体内组织隔室的扁平生物基质。它们独特的片状结构被认为是由层粘连蛋白和IV型胶原这两个独立的蛋白质网络的关联所导致。在追求人工BM的开发过程中，我们发现，当与酸诱导聚合的层粘连蛋白混合时，IV型胶原立即符合层粘连蛋白的形状。这意味着蛋白质网络可能不像迄今为止所认为的那样独立组装，而是层粘连蛋白可能指挥IV型胶原的组装。我们的混合基质在结构上比商业BM提取物基质胶更稳定，并且与后者不同，它支持体外形成近似天然表皮组织的角质形成细胞分层。
