Template Curvature Influences Cell Alignment to Create Improved Human Corneal Tissue Equivalents

Abstract

To accurately create corneal stromal equivalents with native‐like structure and composition, a new biofunctionalized, curved template is developed that allows the precise orientation of cells and of their extracellular matrix. This template is the first demonstration that curvature alone is sufficient to induce the alignment of human corneal stromal cells, which in turn are able to biofabricate stromal tissue equivalents with cornea‐like shape and composition. Specifically, tissues self‐released from curved templates show a highly organized nanostructure, comprised of aligned collagen fibrils, significantly higher expression of corneal stroma‐characteristic markers keratocan, lumican, decorin, ALDH3, and CHST6 (p = 0.012, 0.033, 0.029, 0.003, and 0.02, respectively), as well as significantly higher elastic modulus (p = 0.0001) compared with their planar counterparts. Moreover, curved tissues are shown to support the growth, stratification, and differentiation of human corneal epithelial cells in vitro, while maintaining their structural integrity and shape without any supporting carriers, scaffolds, or crosslinking agents. Together, these results demonstrate that corneal stromal cells can align and create highly organized, purposeful tissues by the influence of substrate curvature alone, and without the need of additional topographical cues. These findings can be important to further understand the mechanisms of corneal biosynthesis both in vitro and in vivo.

3D bioprinting of a corneal stroma equivalent

“…The future success of corneal 3D bioprinting will ultimately depend on the ability of encapsulated cells to mediate ECM remodelling in order to establish tissue functionality. A distinctive feature of corneal fibroblasts presents itself when they are seeded at the base of a curved surface where they have recently been shown to migrate in lattice formation and align collagen in a way that closely resembles its arrangement in the cornea ( Gouveia et al, 2017 ). A significant advantage proffered by the present work is therefore the ability to reproduce curved corneal geometry which is now known to directly influence cell migration and collagen alignment.…”

4D Corneal Tissue Engineering: Achieving Time‐Dependent Tissue Self‐Curvature through Localized Control of Cell Actuators

“…The diverse mechanical properties between center and periphery of self‐curved composite gels (softer in +PA and stiffer in ‐PA regions) led us to investigate how these features impacted the growth of human limbal epithelial stem cells. Previous studies have shown that differences in tissue topography and/or compliance can regulate the phenotype of these cells, with more organized, stiffer collagen‐based substrates promoting epithelial cells differentiation, probably through mechanotransduction signaling pathways . Since the human corneal epithelium requires the support of a well‐ordered native stroma in order to maintain its homeostasis, our experiment aimed at demonstrating the suitability of self‐curved gels to serve as substrate for the adhesion, growth, and differentiation of the epithelial cells.…”

Mechanobiology of the cell–matrix interplay: Catching a glimpse of complexity via minimalistic models

“…Alongside its contribution to understanding the fundamental principles of cell and tissue (patho)physiology, mechanobiology offers a unique approach for regenerative medicine, by exploiting the physical and mechanical properties of the cell and ECM for directing their biological function. Such an approach is already starting to pay dividends for in vitro functional tissue regeneration [98], and we expect it to also help us move towards controlling in situ regeneration [99]. Although many currently available synthetic materials lack various characteristics of natural ECM networks, as noted earlier, there is a growing effort to recapitulate ECM mechanical properties, such as nonlinear behavior [100], for potential biological use.…”