Ultrathin chitosan–poly(ethylene glycol) hydrogel films for corneal tissue engineering Berkay Ozcelik a , Karl D. Brown b , Anton Blencowe a , Mark Daniell b , Geoff W. Stevens a , Greg G. Qiao a,⇑ a Department of Chemical and Biomolecular Engineering, The University of Melbourne, Victoria 3010, Australia b Centre for Eye Research Australia (CERA), Royal Victorian Eye and Ear Hospital, Peter Howson Wing, Victoria 3002, Australia article info Article history: Received 27 August 2012 Received in revised form 16 November 2012 Accepted 17 January 2013 Available online 29 January 2013 Keywords: Corneal endothelium Chitosan Ultrathin film Hydrogel Tissue engineering abstract 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 chito- san–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 lm (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 demon- strated 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 regen- eration and transplantation of CECs, as well as other corneal tissue engineering applications. Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. 1. Introduction Corneal endothelial cells (CECs) are specialized, polygonal- shaped cells that reside on the inner surface of the cornea within the aqueous chamber [1]. CECs are responsible for actively pump- ing fluids across the cornea to maintain corneal transparency [1]. Human CECs do not regenerate in vivo and upon loss of their func- tion corneal transplantation is required to restore vision [1,2]. Var- ious factors can lead to the loss of function of the CEC layer, including ageing, trauma and disease [1,3]. Once the number of these cells reduces below a critical value, the cornea loses its opti- cal clarity due to oedema, which subsequently leads to blindness [1]. Various transplantation methods are available, ranging from the replacement of the whole cornea to the replacement of only the diseased cell layer [4–7]. As a result of the highly invasive nat- ure of full-thickness corneal transplants, less invasive methods have been developed. For example, Descemet’s stripping endothe- lial keratoplasty (DSEK) [8,9] involves removing the Descemet’s membrane, along with the non-functioning CECs, from the cornea via a small incision into the anterior chamber. Subsequently, a thin layer of donor corneal endothelium supported by a section of the stroma is inserted into the anterior chamber to replace the CECs [8]. Since only a thin layer of tissue is replaced via a small incision, the technique is less invasive, healing rates are more rapid and the chances of infection are greatly reduced [9]. As with all types of corneal transplantation techniques utilizing donor tissue, there are also risks of rejection and graft failure with the DSEK procedure [9–11]. As such, development of an autologous transplant for the treatment of corneal endothelial dysfunction would be ideal. A wide variety of substrates have been investigated for the regeneration and potential implantation of CECs [12–19]. In partic- ular, collagen-based materials demonstrate desirable properties for the attachment and proliferation of CECs but generally have tensile strengths inferior to that of the human cornea [12–14]. The effects of harvesting, isolation and purification negatively im- pact upon the mechanical properties of collagen, whereby they are rapidly degraded due to disassembly of the natural structure and cross-links present in vivo [20]. Ultimately, this hinders their appli- cation in surgical procedures such as DSEK, which are physically demanding and require robust substrates for implantation. In addition, since most natural polymers are sourced from animals, 1742-7061/$ - see front matter Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2013.01.020 ⇑ Corresponding author. E-mail address: gregghq@unimelb.edu.au (G.G. Qiao). Acta Biomaterialia 9 (2013) 6594–6605 Contents lists available at SciVerse ScienceDirect Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat