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

Abstract

Hyaluronic acid (HA) is a linear polysaccharide naturally found in the eye and therefore is one of the most promising biomaterials for corneal endothelial regenerative medicine. This study reports, for the first time, the development of overrun-processed porous HA hydrogels for corneal endothelial cell (CEC) sheet transplantation and tissue engineering applications. The hydrogel carriers were characterized to examine their structures and functions. Evaluations of carbodiimide cross-linked air-dried and freeze-dried HA samples were conducted simultaneously for comparison. The results indicated that during the fabrication of freeze-dried HA discs, a technique of introducing gas bubbles in the aqueous biopolymer solutions can be used to enlarge pore structure and prevent dense surface skin formation. Among all the groups studied, the overrun-processed porous HA carriers show the greatest biological stability, the highest freezable water content and glucose permeability, and the minimized adverse effects on ionic pump function of rabbit CECs. After transfer and attachment of bioengineered CEC sheets to the overrun-processed HA hydrogel carriers, the therapeutic efficacy of cell/biopolymer constructs was tested using a rabbit model with corneal endothelial dysfunction. Clinical observations including slit-lamp biomicroscopy, specular microscopy, and corneal thickness measurements showed that the construct implants can regenerate corneal endothelium and restore corneal transparency at 4 weeks postoperatively. Our findings suggest that cell sheet transplantation using overrun-processed porous HA hydrogels offers a new way to reconstruct the posterior corneal surface and improve endothelial tissue function.

Fig 1

(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 (n = 4). *P < 0.05 vs all groups.

Fig 2. Time course of in vitro degradability of various HA carriers after incubation at 34°C in BSS containing hyaluronidase.

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

Fig 3

(a) Freezable water content (W _fH/W _s) of various HA carriers. Values are mean ± SD (n = 5). *P < 0.05 vs all groups. (b) Concentration of glucose permeated through various HA carriers at 34°C. Values are mean ± SD (n = 6). *P < 0.05 vs all groups.

Fig 4. 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.

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.

Fig 5

(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 (*P < 0.05; n = 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.

Fig 6. Fabrication of HA-CEC sheet constructs using thermo-responsive culture supports and porous delivery carriers.

(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.

Fig 7. Representative slit-lamp biomicroscopic images of rabbit eyes 4 weeks after surgical treatment of corneal endothelial dysfunction.

(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.

Fig 8. Representative specular microscopic images of rabbit eyes 4 weeks after surgical treatment of corneal endothelial dysfunction.

(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.

Fig 9. Measurements of central corneal thickness 4 weeks after surgical treatment of corneal endothelial dysfunction.

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 (*P < 0.05; n = 6) as compared to the Wound groups.