Department of Materials Science and Engineering, University of Delaware, Newark, Delaware 19716, United States.
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, United States.
ACS Appl Mater Interfaces. 2022 Nov 23;14(46):51669-51682. doi: 10.1021/acsami.2c13852. Epub 2022 Nov 11.
Repeated mechanical and chemical insults cause an irreversible alteration of extracellular matrix (ECM) composition and properties, giving rise to vocal fold scarring that is refractory to treatment. Although it is well known that fibroblast activation to myofibroblast is the key to the development of the pathology, the lack of a physiologically relevant model of vocal folds impedes mechanistic investigations on how ECM cues promote myofibroblast differentiation. Herein, we describe a bio-orthogonally cross-linked hydrogel platform that recapitulates the alteration of matrix adhesiveness due to enhanced fibronectin deposition when vocal fold wound healing is initiated. The synthetic ECM (sECM) was established via the cycloaddition reaction of tetrazine (Tz) with slow (norbornene, Nb)- and fast (-cyclooctene, TCO)-reacting dienophiles. The relatively slow Tz-Nb ligation allowed the establishment of the covalent hydrogel network for 3D cell encapsulation, while the rapid and efficient Tz-TCO reaction enabled precise conjugation of the cell-adhesive RGDSP peptide in the hydrogel network. To mimic the dynamic changes of ECM composition during wound healing, RGDSP was conjugated to cell-laden hydrogel constructs via a diffusion-controlled bioorthognal ligation method 3 days post encapsulation. At a low RGDSP concentration (0.2 mM), fibroblasts residing in the hydrogel remained quiescent when maintained in transforming growth factor beta 1 (TGF-β1)-conditioned media. However, at a high concentration (2 mM), RGDSP potentiated TGF-β1-induced myofibroblast differentiation, as evidenced by the formation of an actin cytoskeleton network, including F-actin and alpha-smooth muscle actin. The RGDSP-driven fibroblast activation to myofibroblast was accompanied with an increase in the expression of wound healing-related genes, the secretion of profibrotic cytokines, and matrix contraction required for tissue remodeling. This work represents the first step toward the establishment of a 3D hydrogel-based cellular model for studying myofibroblast differentiation in a defined niche associated with vocal fold scarring.
反复的机械和化学刺激会导致细胞外基质 (ECM) 组成和性质的不可逆转改变，从而导致声带瘢痕形成，且这种瘢痕对治疗具有抗性。尽管众所周知，成纤维细胞向肌成纤维细胞的激活是病理学发展的关键，但缺乏生理相关的声带模型会阻碍对 ECM 线索如何促进肌成纤维细胞分化的机制研究。在此，我们描述了一种生物正交交联水凝胶平台，该平台可以模拟由于启动声带伤口愈合时纤维连接蛋白沉积增加而导致的基质粘附性改变。合成 ECM（sECM）是通过四嗪 (Tz) 与慢反应（降冰片烯，Nb）和快反应（环辛烯，TCO）二烯试剂的环加成反应建立的。相对较慢的 Tz-Nb 键合允许建立用于 3D 细胞包封的共价水凝胶网络，而快速且高效的 Tz-TCO 反应使在水凝胶网络中精确缀合细胞黏附性 RGDSP 肽成为可能。为了模拟伤口愈合过程中 ECM 组成的动态变化，在包封后 3 天，通过扩散控制的生物正交连接方法将 RGDSP 缀接到细胞负载的水凝胶构建体上。在低浓度（0.2 mM）RGDSP 下，当在转化生长因子 β1 (TGF-β1) 条件培养基中维持时，驻留在水凝胶中的成纤维细胞保持静止。然而，在高浓度（2 mM）下，RGDSP 增强了 TGF-β1 诱导的肌成纤维细胞分化，这表现为形成肌动蛋白细胞骨架网络，包括 F-肌动蛋白和α-平滑肌肌动蛋白。RGDSP 驱动的成纤维细胞向肌成纤维细胞的激活伴随着与伤口愈合相关的基因表达增加、促纤维化细胞因子的分泌以及组织重塑所需的基质收缩。这项工作代表了建立用于研究与声带瘢痕相关的特定龛位中的肌成纤维细胞分化的基于 3D 水凝胶的细胞模型的第一步。
