Karl David Brown

Ultrathin chitosan-poly(ethylene glycol) hydrogel films for corneal tissue engineering

Due to the high demand for donor corneas and their low supply, autologous corneal endothelial cell (CEC) culture and transplantation for treatment of corneal endothelial dysfunction would be highly desirable. Many studies have shown the possibility of culturing CECs in vitro, but lack potential robust substrates for transplantation into the cornea. In this study, we investigate the properties of novel ultrathin chitosan-poly(ethylene glycol) (PEG) hydrogel films (CPHFs) for corneal tissue engineering applications. Cross-linking of chitosan films with diepoxy-PEG and cystamine was employed to prepare ~50 μm (hydrated) hydrogel films. Through variation of the PEG content (1.5-5.9 wt.%) it was possible to tailor the CPHFs to have tensile strains and ultimate stresses identical to or greater than those of human corneal tissue while retaining similar tensile moduli. Light transmission measurements in the visible spectrum (400-700 nm) revealed that the films were >95% optically transparent, above that of the human cornea (maximum ~90%), whilst in vitro degradation studies with lysozyme revealed that the CPHFs maintained the biodegradable characteristics of chitosan. Cell culture studies demonstrated the ability of the CPHFs to support the attachment and proliferation of sheep CECs. Ex vivo surgical trials on ovine eyes demonstrated that the CPHFs displayed excellent characteristics for physical manipulation and implantation purposes. The ultrathin CPHFs display desirable mechanical, optical and degradation properties whilst allowing attachment and proliferation of ovine CECs, and as such are attractive candidates for the regeneration and transplantation of CECs, as well as other corneal tissue engineering applications.

Biodegradable and biocompatible poly(ethylene glycol)-based hydrogel films for the regeneration of corneal endothelium.

Corneal endothelial cells (CECs) are responsible for maintaining the transparency of the human cornea. Loss of CECs results in blindness, requiring corneal transplantation. In this study, fabrication of biocompatible and biodegradable poly(ethylene glycol) (PEG)-based hydrogel films (PHFs) for the regeneration and transplantation of CECs is described. The 50-μm thin hydrogel films have similar or greater tensile strengths to human corneal tissue. Light transmission studies reveal that the films are >98% optically transparent, while in vitro degradation studies demonstrate their biodegradation characteristics. Cell culture studies demonstrate the regeneration of sheep corneal endothelium on the PHFs. Although sheep CECs do not regenerate in vivo, these cells proliferate on the films with natural morphology and become 100% confluent within 7 d. Implantation of the PHFs into live sheep corneas demonstrates the robustness of the films for surgical purposes. Regular slit lamp examinations and histology of the cornea after 28 d following surgery reveal minimal inflammatory responses and no toxicity, indicating that the films are benign. The results of this study suggest that PHFs are excellent candidates as platforms for the regeneration and transplantation of CECs as a result of their favorable biocompatibility, degradability, mechanical, and optical properties.

Current status and future prospects for cultured limbal tissue transplants in Australia and New Zealand.

Cultured limbal tissue transplants have become widely used over the last decade as a treatment for limbal stem cell deficiency (LSCD). While the number of patients afflicted with LSCD in Australia and New Zealand is considered to be relatively low, the impact of this disease on quality of life is so severe that the potential efficacy of cultured transplants has necessitated investigation. We presently review the basic biology and experimental strategies associated with the use of cultured limbal tissue transplants in Australia and New Zealand. In doing so, we aim to encourage informed discussion on the issues required to advance the use of cultured limbal transplants in Australia and New Zealand. Moreover, we propose that a collaborative network could be established to maintain access to the technology in conjunction with a number of other existing and emerging treatments for eye diseases.

Transforming growth factor β1-induced NADPH Oxidase-4 expression and fibrotic response in conjunctival fibroblasts

Purpose Fibrotic scarring after ocular surgeries and chemical burn injuries can impede clarity of the cornea and cause vision impairment. Transforming growth factor β (TGFβ) signaling pathway is known to mediate fibrotic scarring, and NADPH oxidase-derived reactive oxygen species has been shown to be an effector molecule that facilitates TGFβ1-mediated responses. The present study explores the expression profile and functional importance of NADPH oxidase (Nox) in conjunctival fibroblasts. In addition, the effect of curcumin on the TGFβ1-induced NADPH oxidase expression and collagen synthesis was also investigated. Methods The mRNA expression of Nox isoforms in rabbit conjunctival fibroblasts was measured by real-time PCR. The production of hydrogen peroxide (H2O2) and total collagen by these cells was measured by Amplex Red assay and Picro-Sirius red assay, respectively. Nox4 was knocked down by adenovirus-mediated siRNA targeting Nox4 (Adv-Nox4i). Results We describe for the first time that conjunctival fibroblasts express mRNA encoding for Nox2, Nox3, Nox4, and Nox5. TGFβ1 was found to induce Nox4 mRNA expression and total collagen release by these cells (P < 0.05; n = 4), and both responses are blocked by Smad3 inhibitor SIS3. Suppressing Nox4 gene transcription with Adv-Nox4i completely attenuated TGFβ1-stimulated H2O2 release and collagen production by conjunctival fibroblasts (P < 0.05; n = 4-6). Similarly, curcumin also inhibited TGFβ1-induced Smad3 phosphorylation, Nox4-derived H2O2 production, and total collagen synthesis by conjunctival fibroblasts (P < 0.05; n = 4-6). Conclusions The present study suggests that TGFβ1-mediated production of collagen by conjunctival fibroblasts involves Nox4-derived H2O2 pathway and this effect of Nox4 is abrogated by curcumin. This mechanism might be exploited to prevent fibrotic scarring after surgeries and chemical burn injuries in the eye.