M1 - De novo architectural control of extracellular matrix self-assembly on 2D hydrogels

Introduction: : Alterations in tissue extracellular matrix (ECM) composition, architecture, and mechanics have been characterized in multiple biological processes including development, aging, and disease. For example, architectural changes in the form of increased collagen I fiber endpoints have been observed in the aging cardiac ECM, accompanied by tissue stiffening (from ~10 kPa to ~40 kPa) and increased fibronectin [1]. Cells are well known to respond to ECM cues via both biochemical and mechano- signaling pathways, but little is known about the role of ECM architecture. Recent studies using engineered biomaterial systems (e.g., fibrous hydrogels and electrospun surfaces) have found that cells can perceive architectural information including fiber directionality (isotropic vs. anisotropic) and network density (coarse vs. fine mesh) via mechanosensitive YAP signaling. However, providing synergistic biomimetic ECM cues that are present in tissues (composition, architecture, and mechanics) to cells in vitro, while allowing for independent ECM parameter tunability, remains a challenge. Thus, we developed a method to control the de novo self-assembly of decellularized ECM (dECM) coated on a stiffness tunable 2D hydrogel, enabling a fully decoupled yet physiological-relevant system that enables the identification of ECM parameter-driven cellular function.

Materials and

Methods: : Porcine hearts were perfused with PBS and left ventricles were dissected. Tissues were then decellularized with sodium deoxycholate with constant stirring, followed by snap freezing and lyophilization. Lyophilized dECM was then pulverized, solubilized, and neutralized. Homogenized dECM solutions were quantified for protein concentration using a BCA assay kit, diluted to consistent concentrations among biological replicates, and cryopreserved pending usage. Polyacrylamide hydrogels were fabricated on hydrophilic coverslips and activated with sulfo-SANPAH under UV illumination. Thawed dECM solutions were treated with a crosslinking agent of varying concentrations and incubated on sulfo-SANPAH treated hydrogels to facilitate ECM self-assembly and attachment. Confocal microscopy of immunostained samples (collagen I and laminin) was used to characterize the self-assembled dECM on hydrogel surfaces and TWOMBLI plugin was used to characterize architectural differences of the dECM networks. Nanoindentation was performed to characterize the stiffness of hydrogels coated with dECM. Primary cardiac fibroblasts (CFs) were seeded on these samples followed by immunofluorescence (IF) staining of alpha-smooth muscle actin (a-SMA) and yes-associated protein 1 (YAP1) to investigate fibroblast activation and function.

Results, Conclusions, and Discussions:: Prior to pulverization, immunohistochemistry of left ventricle tissues confirmed full decellularization (no DAPI/actin signal) while preserving key ECM components (collagen and laminin). Solubilized dECM was shown to self-assemble on top of polyacrylamide hydrogels, forming full ECM networks in 2D. This ~5 um thick ECM network allows cells to sense both the biochemical and architectural cues from the ECM, as well as the mechanical cues from the underlying substrate. By altering the concentration of the crosslinking agent, the fiber network was adjusted from a coarse to a fine mesh, as visualized in phase contrast and confocal microscopy (Figure 1. A-C). Confocal imaging followed by TWOMBLI analysis characterized the architectural differences in de novo self-assembled dECM regulated by crosslinker concentration. These architectural readouts were compared with previous data on ECM architecture in young versus aged murine hearts to provide biological relevance to the network structure [1]. Nanoindentation confirmed substrate stiffness mimicking young (~10 kPa) versus aged (~40 kPa) murine hearts was independent of dECM architecture. When seeded with primary CFs, IF of YAP and aSMA showed architecture-specific activation phenotypes. Future work aims to provide a mechanistic understanding of how ECM architecture regulates CF fate. Overall, we describe a method to control ECM architecture on 2D hydrogels with physiologically-relevant tunable stiffness to understand how cells integrate combinatorial ECM cues.

Acknowledgements and/or References (Optional):: [1] Sun, A.R., Ramli, M.F.H., Shen, X., Ramakanth, K.K., Chen, D., Hu, Y., Vidyasekar, P., Foo, R.S., Long, Y., Zhu, J., Ackers-Johnson, M., Young, J.L. (2025): Hybrid hydrogel-extracellular matrix scaffolds identify biochemical and mechanical signatures in cardiac aging. Nature Materials (in press).