Surface Topography and Mechanical Strain Promote Keratocyte Phenotype and Extracellular Matrix Formation in a Biomimetic 3D Corneal Model

Abstract

The optimal functionality of the native corneal stroma is mainly dependent on the well-ordered arrangement of extracellular matrix (ECM) and the pressurized structure. In order to develop an in vitro corneal model, it is crucial to mimic the in vivo microenvironment of the cornea. In this study, the influence of surface topography and mechanical strain on keratocyte phenotype and ECM formation within a biomimetic 3D corneal model is studied. By modifying the surface topography of materials, it is found that patterned silk fibroin film with 600 grooves mm -1 optimally supports cell alignment and ECM arrangement. Furthermore, treatment with 3% dome-shaped mechanical strain, which resembles the shape and mechanics of native cornea, significantly enhances the expression of keratocyte markers as compared to flat-shaped strain. Accordingly, a biomimetic 3D corneal model, in the form of a collagen-modified, silk fibroin-patterned construct subjected to 3% domeshaped strain, is created. Compared to traditional 2D cultures, it supports a significantly higher expression of keratocyte and ECM markers, and in conclusion better maintains keratocyte phenotype, alignment, and fusiform cell shape. Therefore, the novel biomimetic 3D corneal model developed in this study serves as a useful in vitro 3D culture model to improve current 2D cultures for corneal studies.The outermost epithelium, Bowman's layer, the stroma, Descemet's membrane, and the innermost endothelium. [2] The stroma, which constitutes up to 90% of the corneal thickness, is composed of well-organized collagen fibrils and quiescent stromal cells called keratocytes. [3,4] The structure of the stroma is important for the transparency and consequently the vision. The properties of the stroma are based on the combination of the orthogonal lamellar arrangement of aligned collagen fibrils (mainly collagen I and V) [5][6][7][8] and the spacing of these fibrils and the regulation of collagen diameter achieved by proteoglycans (such as lumican and keratocan). [9][10][11][12] It has been shown that keratocytes during in vitro cell culture conditions easily differentiate, as shown by reduced expression of the typical keratocyte markers, including keratocan, CD34, and ALDH3A1. [13,14] However, most of the in vitro corneal studies are conducted on 2D monolayers. [15][16][17] To overcome the limitations of 2D monolayer cell cultures, several 3D corneal in vitro models have been developed using tissue-engineered strategies and a number of biomaterials, such as colla gen, silk, chitosan, and other synthetic polymers. [14,18] Among the various materials, collagen is the most commonly used, as it is the main constituent of native cornea, and collagen-based corneal 3D models have shown promising results as substrates for the culture of corneal cells. [19][20][21] However, few of the 3D corneal in vitro models can replicate the in vivo microenvironment of the native cornea, due to the complexity of the corneal structure and the uniqueness of the mechanical environment created by the ...

Influence of micropatterned substrates on keratocyte phenotype

“…Despite these differences, many of the cells other characteristics were similar on both topographies compared to unpatterned substrates including increases in keratocyte specific gene expression, sGAG release and cell stiffness. While several studies have previously examined the effect of parallel microgrooves or nanogrooves on keratocytes [24][25][26]28 this is the first time that orthogonal substrates have been used with this cell type. It is also pertinent to highlight that this study did not design the orthogonal lattice to mimic the collagen fibril structure in human stroma.…”

“…Then et al (2011), have also previously shown that specific genes expressed by keratocytes could be modulated by culturing the cells on microchannels 46 , although the genes examined were different to those examined in our work. Zhang et al (2017), examined the effect of nanochannels on the expression of lumican, keratocan, collagen I and collagen V and while there were some differences to cells cultured on flat substrates, these were not found to be statistically significant 25 . In our study, keratocytes cultured on microchannels did up-regulate these specific genes.…”

“…Upon the removal of serum, a partial return to a native keratocyte phenotype is achievable [21][22][23] . In this study, we wanted to investigate if topographical cues could influence the restoration of a keratocyte phenotype in serum free media after the cells were initially expanded on culture plastic in serum-supplemented media.Several studies have reported that phenotype and morphology of keratocytes are notably influenced by surface geometry and topography such as parallel linear grooves [24][25][26][27] or concentric circles 13 . In many of these studies, keratocytes stretch and aligned in the groove direction.…”

“…Several studies have reported that phenotype and morphology of keratocytes are notably influenced by surface geometry and topography such as parallel linear grooves [24][25][26][27] or concentric circles 13 . In many of these studies, keratocytes stretch and aligned in the groove direction.…”

3D Miniaturization of Human Organs for Drug Discovery

“…For example, cellular components can be integrated by co-culturing multiple cell types in defined physical arrangements, 3D organization can be mimicked with biomaterial scaffolds and microfluidic channels, mechanical cues can be presented by biomaterials and fluid flow, and soluble stimuli can be delivered via perfusion. [35][36][37][38] Top-down approaches include organ-on-a-chip models, which aim to generate key aspects of an organ structure and function in a microfluidic device. Bottom-up approaches rely on the emergent behavior of biological systems to generate complex tissue-and organ-like constructs.…”