Chemical and topographical effects on cell differentiation and matrix elasticity in a corneal stromal layer model

Control and maintenance of the keratocyte phenotype is vital to developing in vitro tissue engineered strategies for corneal repair. In this study the influence of topographical and chemical cues on the mechanical, phenotypical and genotypical behaviour of adult human derived corneal stromal (AHDCS) cells in three dimensional (3D) multi-layered organised constructs is examined. Topographical cues are provided via multiple aligned electrospun nanofiber meshes, which are arranged orthogonally throughout the constructs and are capable of aligning individual cells and permitting cell migration between the layers. The influence of chemical cues is examined using different supplements in culture media. A non-destructive indentation technique and optical coherence tomography are used to determine the matrix elasiticity (elastic modulus) and dimensional changes, respectively. These measurements were indicative of changes in cell phenotype from contractile fibroblasts to quiescent keratocytes over the duration of the experiment and corroborated by qPCR. Constructs containing nanofibers have a higher initial modulus, reduced contraction and organised cell orientation compared to those without nanofibers. Cell-seeded constructs cultured in serum-containing media increased in modulus throughout the culture period and underwent significantly more contraction than constructs cultured in serum-free and insulin-containing media. This implies that the growth factors present in serum promote a fibroblast-like phenotype; qPCR data further validates these observations. These results indicate that the synergistic effect of nanofibers and serum-free media plus insulin supplementation provide the most suitable topographical and chemical environment for reverting corneal fibroblasts to a keratocyte phenotype in a 3D construct

Influence of cell number and collagen concentration on the mechanical behaviour of collagen hydrogel constructs [Abstracts]

Influence of cell number and collagen concentration on the mechanical behaviour of collagen hydrogel constructs [Abstracts

Controlling corneal stromal cell phenotype via chemical cues [Abstract]

Controlling corneal stromal cell phenotype via chemical cues [Abstract

Quotations related to theme 3: Cognitive aspects of fatigue.

<p>Quotations related to theme 3: Cognitive aspects of fatigue.</p

Quotations related to theme 2: Ocular fatigue and fatigue related to ocular complaints.

<p>Quotations related to theme 2: Ocular fatigue and fatigue related to ocular complaints.</p

Quotations related to theme 1: The physical experience of fatigue.

<p>Quotations related to theme 1: The physical experience of fatigue.</p

Ocular surface glucocorticoid bioavailability in health and disease.

<p>Evaluation of tear film glucocorticoid profiles as surrogate readouts of net ocular surface glucocorticoid bioavailability, defines cortisol depletion (reduced cortisol∶cortisone (F∶E ratio, mean±SE) during active untreated pseudomonas keratitis (PSK) but not herpes simplex keratitis (HSK), and amplification during chronic clinically quiescent immune-mediated disease (MMP, mucous membrane pemphigoid; SJS/TEN, Stevens-Johnson Syndrome/Toxic-epidermal Necrolysis). Statistical analysis performed using t-test with two-tailed Mann Whitney post -test. * = P<0.05.</p

Putative interaction of TLR signaling and local regulation of cortisol in the human cornea.

<p>(A) Under physiological conditions, autocrine synthesis of cortisol in corneal epithelial cells contributes to the immunoprotection of the ocular surface mucosa. During induction of ocular surface TLRs cytokines are released in a ligand and cell specific manner. (B) On TLR3 ligation such as chronic immune -mediated disease e.g. SJS-TEN, synthesis of a diverse spectrum of cytokines primarily from the corneal epithelial cells, induces <u>weak</u> monocyte chemotaxis and differentiation to M1 macrophages. These cytokines <u>potently attenuate</u> M1 cortisol biosynthesis leading to a net reduction of ocular surface cortisol levels, promoting recruitment of inflammatory cells necessary for resolving the initial trigger. By contrast, on TLR4 ligation (C) activation of keratocytes to a fibroblast phenotype, form the first line of defense producing chemokines that <u>potently</u> induce monocyte migration to the site of infection for rapid eradication of bacterial invasion. Attenuation of M1 cortisol production is less pronounced and this facilitates resolution of the inflammatory response, limiting tissue damage thereby preserving optical clarity (and sight).</p

Macrophage infiltration in human keratitis.

<p>(A) Immunohistochemistry of the normal human central corneal epithelium showed no evidence of CD68 positive resident macrophages or basal TLR4 expression, although there was some basal TLR3 expression. Corneal stromal infiltration of CD68 positive cells is seen in both herpetic and gram negative keratitis associated with increased TLR3 and TLR4 expression in the corneal epithelium, respectively. (B–E) Migration assay showing culture supernatants of corneal cells having chemotactic potential on monocytes. Cell supernatants from PHCEC/PHKF stimulated with TLR3 (poly I∶C) or TLR4 (LPS) ligands for 16 h were tested for the ability to induce monocyte migration. ‘S’ denotes culture supernatants from PHCEC/PHKF cultures generated from 3 corneal donors, tested on a single allogenic PBMC donor. ‘D’ denotes 3 different PBMC donors subjected to culture supernatant from a single donor derived PHCEC/PHKF treated with TLR3 and TLR4 ligands. Data show that both LPS and Poly I∶C stimulation of corneal cells induce monocyte migration but LPS stimulation of PHKF has the greatest chemotactic potential. (Panels D and E). (F) Culture supernatants from experiments A–D (TLR3 and TLR4 induction of PHCEC/PHKF for 16 h) downregulates M1 macrophage 11β-HSD1 activity. Statistical analysis was carried using one-way ANOVA and comparisons were drawn with untreated control cells vs. TLR3/TLR4 treated cells.</p

Regulation of cytokine production with Cortisol/Dexamethasone on TLR3 or TLR4 stimulated Primary Human Corneal Fibroblasts (PHKF).

<p>Both cortisol (▪, black square) and dexamethasone (▪, gray square) reduced cytokines: VEGF, CCL5, IFN-γ, CXCL-10, IL-8 and GCSF (B) after either or both TLR 3 and 4 stimulation of PHKF. (Values =  Mean+SE, normalising to No Cortisol/Dexamethasone (□, white square) for each treatment n = 3; Statistical analysis 2-way ANOVA with Bonferroni post-test; *p<0.05, **p<0.01, ***p<0.001).</p