Dimensionality Matters: Exploiting UV-Photopatterned 2D and Two-Photon-Printed 2.5D Contact Guidance Cues to Control Corneal Fibroblast Behavior and Collagen Deposition

Cas van der Putten, Gozde Sahin, Rhiannon Grant, Mirko D'Urso, Stefan Giselbrecht, Carlijn V.C. Bouten, Nicholas A. Kurniawan (Corresponding author)

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Abstract

In the event of disease or injury, restoration of the native organization of cells and extracellular matrix is crucial for regaining tissue functionality. In the cornea, a highly organized collagenous tissue, keratocytes can align along the anisotropy of the physical microenvironment, providing a blueprint for guiding the organization of the collagenous matrix. Inspired by this physiological process, anisotropic contact guidance cues have been employed to steer the alignment of keratocytes as a first step to engineer in vitro cornea-like tissues. Despite promising results, two major hurdles must still be overcome to advance the field. First, there is an enormous design space to be explored in optimizing cellular contact guidance in three dimensions. Second, the role of contact guidance cues in directing the long-term deposition and organization of extracellular matrix proteins remains unknown. To address these challenges, here we combined two microengineering strategies-UV-based protein patterning (2D) and two-photon polymerization of topographies (2.5D)-to create a library of anisotropic contact guidance cues with systematically varying height (H, 0 µm ≤ H ≤ 20 µm) and width (W, 5 µm ≤ W ≤ 100 µm). With this unique approach, we found that, in the short term (24 h), the orientation and morphology of primary human fibroblastic keratocytes were critically determined not only by the pattern width, but also by the height of the contact guidance cues. Upon extended 7-day cultures, keratocytes were shown to produce a dense, fibrous collagen network along the direction of the contact guidance cues. Moreover, increasing the heights also increased the aligned fraction of deposited collagen and the contact guidance response of cells, all whilst the cells maintained the fibroblastic keratocyte phenotype. Our study thus reveals the importance of dimensionality of the physical microenvironment in steering both cellular organization and the formation of aligned, collagenous tissues.

Original languageEnglish
Article number402
Number of pages18
JournalBioengineering
Volume11
Issue number4
DOIs
Publication statusPublished - Apr 2024

Funding

This research was funded by the European Research Council (grant 851960), the Ministry of Education, and Science for the Gravitation Program 024.003.013 \u201CMaterials Driven Regeneration\u201D, and the Chemelot InSciTe (Project BM3.02).

FundersFunder number
European Research Council851960
Ministry of Education and ScienceBM3.02

    Keywords

    • UV-based protein patterning
    • collagen alignment
    • contact guidance response
    • cornea
    • corneal fibroblasts
    • two-photon polymerization of topographies

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