Chemical modification of human decellularized extracellular matrix for incorporation into phototunable hybrid-hydrogel models of tissue fibrosis

Tissue fibrosis remains a serious health condition with high morbidity and mortality rates. There is a critical need to engineer model systems that better recapitulate the spatial and temporal changes in the fibrotic extracellular microenvironment and enable study of the cellular and molecular alterations that occur during pathogenesis. Here, we present a process for chemically modifying human decellularized extracellular matrix (dECM) and incorporating it into a dynamically tunable hybrid-hydrogel system containing a poly(ethylene glycol)-alpha methacrylate (PEGαMA) backbone. Following modification and characterization, an off-stoichiometry thiol-ene Michael addition reaction resulted in hybrid-hydrogels with mechanical properties that could be tuned to recapitulate many healthy tissue types. Next, photoinitiated, free-radical homopolymerization of excess α-methacrylates increased crosslinking density and hybrid-hydrogel elastic modulus to mimic a fibrotic microenvironment. The incorporation of dECM into the PEGαMAhydrogel decreased the elastic modulus and, relative to fully synthetic hydrogels, increased the swelling ratio, the average molecular weight between crosslinks, and the mesh size of hybrid-hydrogel networks. These changes were proportional to the amount of dECM incorporated into the network. Dynamic stiffening increased the elastic modulus and decreased the swelling ratio, average molecular weight between crosslinks, and the mesh size of hybrid-hydrogels, as expected. Stiffening also activated human fibroblasts, as measured by increases in average cellular aspect ratio (1.59 ± 0.02 to 2.98 ± 0.20) and expression of α-smooth muscle actin (αSMA). Fibroblasts expressing αSMA increased from 24.4% to 51.8% upon dynamic stiffening, demonstrating that hybrid-hydrogels containing human dECM support investigation of dynamic mechanosensing. These results improve our understanding of the biomolecular networks formed within hybrid-hydrogels: this fully human phototunable hybrid-hydrogel system will enable researchers to control and decouple the biochemical changes that occur during fibrotic pathogenesis from the resulting increases in stiffness to study the dynamic cell-matrix interactions that perpetuate fibrotic diseases.

组织纤维化仍旧是一种严重的健康问题，其发病率和死亡率均较高。迫切需要构建能够更好地模拟纤维化细胞外微环境中空间和时间变化的模型系统，并能够研究发病机制过程中发生的细胞和分子改变。在此，我们介绍了一种化学修饰人源性去细胞外基质（dECM）并将其整合至含有聚乙二醇-α甲基丙烯酸酯（PEGαMA）骨架的动态可调谐混合水凝胶系统的工艺。经过修饰和表征后，非化学计量学的硫醇-烯基迈克尔加成反应导致混合水凝胶的机械性能可调，以模拟多种健康组织类型。随后，通过光引发的自由基均聚化过量的α-甲基丙烯酸酯，增加了交联密度和混合水凝胶的弹性模量，以模拟纤维化微环境。dECM的整合入PEGαMA水凝胶中降低了弹性模量，与全合成水凝胶相比，增加了溶胀率、交联之间的平均分子量和混合水凝胶网络的网孔尺寸。这些变化与整合入网络中的dECM量成正比。动态硬化增加了弹性模量，并降低了溶胀率、交联之间的平均分子量和混合水凝胶的网孔尺寸，如预期。硬化还激活了人成纤维细胞，如通过平均细胞长宽比（1.59 ± 0.02至2.98 ± 0.20）和α-平滑肌肌动蛋白（αSMA）的表达增加所测得。在动态硬化后，表达αSMA的成纤维细胞从24.4%增加到51.8%，表明含有人类dECM的混合水凝胶支持动态机械传感的研究。这些结果加深了我们对于混合水凝胶内形成的生物分子网络的理解：这一全人源光调谐混合水凝胶系统将使研究者能够控制和解耦纤维化发病机制过程中发生的生化变化，以及由此导致的刚度增加，以研究持续纤维化疾病的动态细胞-基质相互作用。