Representative slit-lamp biomicroscopic images of rabbit eyes 4 weeks after surgical treatment of corneal endothelial dysfunction.

<p>(a) Wound group: cornea denuded of endothelium, (b) OFHA group: endothelial scrape-wounded cornea implanted with HA carriers, and (c) OFHA+CEC group: endothelial scrape-wounded cornea implanted with HA carriers and bioengineered CEC sheet. Scale bars: 5 mm.</p

Representative specular microscopic images of rabbit eyes 4 weeks after surgical treatment of corneal endothelial dysfunction.

<p>(a) Wound group: cornea denuded of endothelium, (b) OFHA group: endothelial scrape-wounded cornea implanted with HA carriers, and (c) OFHA+CEC group: endothelial scrape-wounded cornea implanted with HA carriers and bioengineered CEC sheet.</p

Cell viability of rabbit CEC cultures was determined by staining with Live/Dead Viability/Cytotoxicity Kit in which the live cells fluoresce green and dead cells fluoresce red.

<p>Fluorescence images of cells in (a) controls (without test materials) after 8 h of direct contact with different types of HA samples (b) AHA, (c) FHA, and (d) OHFA. Scale bars: 50 μm.</p

Measurements of central corneal thickness 4 weeks after surgical treatment of corneal endothelial dysfunction.

<p>Wound group: cornea denuded of endothelium, OFHA group: endothelial scrape-wounded cornea implanted with HA carriers, and OFHA+CEC group: endothelial scrape-wounded cornea implanted with HA carriers and bioengineered CEC sheet. The dash line represents the preoperative corneal thickness. An asterisk indicates statistically significant differences (*<i>P</i> < 0.05; <i>n</i> = 6) as compared to the Wound groups.</p

Time course of in vitro degradability of various HA carriers after incubation at 34°C in BSS containing hyaluronidase.

<p>An asterisk indicates statistically significant differences (*<i>P</i> < 0.05; <i>n</i> = 5) for the mean value of degradability compared with the value at the previous time point. ^#<i>P</i> < 0.05 vs all groups (compared only within each time point group).</p

Investigation of Overrun-Processed Porous Hyaluronic Acid Carriers in Corneal Endothelial Tissue Engineering - Fig 5

<p>(a) Gene expression level of ATP1A1 in rabbit CECs after 8 h of direct contact with various HA carriers, measured by real-time reverse transcription polymerase chain reaction. Normalization was done by using GAPDH. Data in the experimental groups are percentages relative to that of control groups (cells cultured in the absence of HA materials). An asterisk indicates statistically significant differences (*<i>P</i> < 0.05; <i>n</i> = 4) as compared to the control groups. (b) Western blot analysis of ATP1A1 expression in the rabbit CECs after 8 h of direct contact with HA carriers. Lane 1: control (without HA materials), Lane 2: AHA, Lane 3: FHA, and Lane 4: OFHA groups.</p

Investigation of Overrun-Processed Porous Hyaluronic Acid Carriers in Corneal Endothelial Tissue Engineering - Fig 1

<p>(a) Cross-section and (b) surface images obtained by scanning electron microscopy of the HA carriers. Scale bars: 100 μm. (c) Pore size and (d) porosity of various HA carriers. Values are mean ± SD (<i>n</i> = 4). *<i>P</i> < 0.05 vs all groups.</p

Fabrication of HA-CEC sheet constructs using thermo-responsive culture supports and porous delivery carriers.

<p>(a) Picture of bioengineered cell sheet graft (asterisk) attached to the porous hydrogel carrier (arrow) from OFHA group. Scale bars: 5 mm. (b) Light micrograph of cross-section of the construct stained with Hoechst 33258. Large arrow: larger pore on the interior of the carrier; Fine arrow: smaller pore on the surface of the carrier; Asterisk: cell sheet. Scale bars: 200 μm.</p

Characterization of Cross-Linked Porous Gelatin Carriers and Their Interaction with Corneal Endothelium: Biopolymer Concentration Effect

<div><p>Cell sheet-mediated tissue regeneration is a promising approach for corneal reconstruction. However, the fragility of bioengineered corneal endothelial cell (CEC) monolayers allows us to take advantage of cross-linked porous gelatin hydrogels as cell sheet carriers for intraocular delivery. The aim of this study was to further investigate the effects of biopolymer concentrations (5–15 wt%) on the characteristic and safety of hydrogel discs fabricated by a simple stirring process combined with freeze-drying method. Results of scanning electron microscopy, porosity measurements, and ninhydrin assays showed that, with increasing solid content, the pore size, porosity, and cross-linking index of carbodiimide treated samples significantly decreased from 508±30 to 292±42 µm, 59.8±1.1 to 33.2±1.9%, and 56.2±1.6 to 34.3±1.8%, respectively. The variation in biopolymer concentrations and degrees of cross-linking greatly affects the Young’s modulus and swelling ratio of the gelatin carriers. Differential scanning calorimetry measurements and glucose permeation studies indicated that for the samples with a highest solid content, the highest pore wall thickness and the lowest fraction of mobile water may inhibit solute transport. When the biopolymer concentration is in the range of 5–10 wt%, the hydrogels have high freezable water content (0.89–0.93) and concentration of permeated glucose (591.3–615.5 µg/ml). These features are beneficial to the in vitro cultivation of CECs without limiting proliferation and changing expression of ion channel and pump genes such as ATP1A1, VDAC2, and AQP1. In vivo studies by analyzing the rabbit CEC morphology and count also demonstrate that the implanted gelatin discs with the highest solid content may cause unfavorable tissue-material interactions. It is concluded that the characteristics of cross-linked porous gelatin hydrogel carriers and their triggered biological responses are in relation to biopolymer concentration effects.</p></div

Quantitative real-time reverse transcription polymerase chain reaction and Western blot analyses.

<p>(a) Gene expression level of ATP1A1, VDAC2, and AQP1 in rabbit corneal endothelial cells after 12 h of direct contact with various gelatin samples, measured by real-time RT-PCR. Normalization was done by using GAPDH. Data in the experimental groups are percentages relative to that of control groups (cells cultured in the absence of gelatin materials). An asterisk indicates statistically significant differences (*<i>P</i><0.05; <i>n</i> = 3) as compared with the control groups. (b) Western blot analysis of Na⁺,K⁺-ATPase expression in the rabbit corneal endothelial cells after 12 h of direct contact with gelatin samples. Lane 1: control (without gelatin materials), Lane 2: G5, Lane 3: G10, and Lane 4: G15 groups.</p