Influence of micropatterned substrates on keratocyte phenotype

Abstract

Substrate topographic patterning is a powerful tool that can be used to manipulate cell shape and orientation. To gain a better understanding of the relationship between surface topography and keratocyte behavior, surface patterns consisting of linear aligned or orthogonally aligned microchannels were used. Photolithography and polymer molding techniques were used to fabricate micropatterns on the surface of polydimethylsiloxane (PDMS). Cells on linear aligned substrates were elongated and aligned in the channel direction, while cells on orthogonal substrates had a more spread morphology. Both linear and orthogonal topographies induced chromatin condensation and resulted in higher expressions of keratocyte specific genes and sulfated glycosaminoglycans (sGAG), compared with non-patterned substrates. However, despite differences in cell morphology and focal adhesions, many genes associated with a native keratocyte phenotype, such as keratocan and ALDH3A1, remain unchanged on the different patterned substrates. This information could be used to optimize substrates for keratocyte culture and to develop scaffolds for corneal regeneration.Located at the front of the eye, the cornea is the transparent outer component of the eye, responsible for focusing light into the eye and protecting the internal structure from external irritations 1 . The cornea comprises of five main layers: epithelium, Bowman's layer, stroma, Descemet's membrane and endothelium 2,3 . The stroma is the major structural and functional unit of cornea constituting 90% of the total corneal thickness. The collagen fibril structure in the stroma allows it to be transparent which is vital for preserving vision 4 .Aligned collagen nanofibrils in stromal extracellular matrix (ECM) (mainly collagen I and V 5-8 ) form layers arranged orthogonally to one another. Keratocytes, the major cellular component of stroma, are scattered within these layers. Corneal transparency is attributed to the highly ordered orthogonal distribution of fibril layers, spacing between collagen fibrils, modulation of the diameter of collagen fibril attained by proteoglycans (i.e. keratocan and lumican 9-12 ) and crystallin proteins located within the cells. Keratocytes are dendritic in nature with expanded cellular network and compact cell body, enabling them to construct a three-dimensional network of interconnected cells 13,14 . Upon injury the cells lose their dendritic shape and change phenotype becoming fibroblastic or myofibroblastic.Globally around 10 million suffer from vision impairment resulting from corneal injury 13 . Annually around 30,000-40,000 corneal graft replacements are performed within USA and Europe. While rejection rates are low (under 10%) 13 , for high-risk patients the rejection rate can increase to approximately 49% and continues to increase over the patient's life. The limited availability of transplantable donated corneas exacerbates this problem. Synthetic keratoprostheses have been used as an alternative of allografts, however there is a relatively hig...

Designing Scaffolds for Corneal Regeneration

“…Crosslinking silk fibroin films using riboflavin and UVA light has been shown improve their adhesion to the ocular surface 106g. Bioactive molecules, such as retinoic acid, have also been incorporated into silk fibroin to regulate the phenotype of corneal stromal cells 119…”

Polystyrene Pocket Lithography: Sculpting Plastic with Light

“…Surface topographical cues, including parallel microgrooves, have been shown to influence cell orientation, morphology, migration, and differentiation in multiple cell types. [3,[31][32][33][34][35][36][37] We investigated whether we could fabricate microgrooves of different dimensions and control single-cell behavior. The microgrooves (characterized in Figures S10-S12, Supporting Information) were fabricated using three different CQ mask patterns: pattern A with 150 µm-wide transparent line separated by 50 µm spacing, pattern B with 20 µm-wide transparent lines separated by 100 µm spacing, and pattern C with 5 µm transparent lines separated by 45 µm spacing; all dimensions edge to edge.…”

Mechanical Properties of Bioengineered Corneal Stroma

“…The interactions between corneal keratocytes and the ECM is highlighted by various research studies demonstrating that the ECM modulates proliferation, adhesion, differentiation, function and preservation of the morphology of keratocytes, key players for the healing and remodeling of compromised stromal tissue. [43][44][45][46][47] Keratocytes also exhibit durotaxis and mechanosensing, i.e., the migration to stiffer regions of materials. [48,49] Alteration in ECM stiffness can change, for instance, actin stress fiber production and focal adhesions, as well as alter keratocytes' phenotype.…”