Abstract
Dysfunction of the corneal endothelium (CE) resulting from progressive cell loss leads to corneal oedema and significant visual impairment. Current treatments rely upon donor allogeneic tissue to replace the damaged CE. A donor cornea shortage necessitates the development of biomaterials, enabling in vitro expansion of corneal endothelial cells (CECs). This study investigated the use of a synthetic peptide hydrogel using poly-ε-lysine (pεK), cross-linked with octanedioic-acid as a potential substrate for CECs expansion and CE grafts. PεK hydrogel properties were optimised to produce a substrate which was thin, transparent, porous and robust. A human corneal endothelial cell line (HCEC-12) attached and grew on pεK hydrogels as confluent monolayers after 7 days, whereas primary porcine CECs (pCECs) detached from the pεK hydrogel. Pre-adsorption of collagen I, collagen IV and fibronectin to the pεK hydrogel increased pCEC adhesion at 24 h and confluent monolayers formed at 7 days. Minimal cell adhesion was observed with pre-adsorbed laminin, chondroitin sulphate or commercial FNC coating mix (fibronectin, collagen and albumin). Functionalisation of the pεK hydrogel with synthetic cell binding peptide H-Gly-Gly-Arg-Gly-Asp-Gly-Gly-OH (RGD) or α2β1 integrin recognition sequence H-Asp-Gly-Glu-Ala-OH (DGEA) resulted in enhanced pCEC adhesion with the RGD peptide only. pCECs grown in culture at 5 weeks on RGD pεK hydrogels showed zonula occludins 1 staining for tight junctions and expression of sodium-potassium adenosine triphosphase, suggesting a functional CE. These results demonstrate the pεK hydrogel can be tailored through covalent binding of RGD to provide a surface for CEC attachment and growth. Thus, providing a synthetic substrate with a therapeutic application for the expansion of allogenic CECs and replacement of damaged CE.
Highlights
The corneal endothelium (CE) is the inner most layer of the cornea and is composed of a single monolayer of tightly packed, non-replicative endothelial cells on a thickened basement membrane (Descemet’s membrane (DM))
Transparent pεK hydrogel films (Fig. 1a) were fabricated with an open porous structure demonstrated by atomic force microscope (AFM) (Fig. 1b), an average of 118 μm (SD 16 μm) thickness was observed for gels synthesised for cell culture (Table 1)
Representative images of porcine CECs (pCECs) seeded at 24 h on f tissue culture polystyrene (TCPS) and g pεK hydrogel or at 7 days on h TCPS and i pεK hydrogel. j Quantification of cell density at 24 h and 7 days post seeding of pCECs on hydrogels
Summary
The corneal endothelium (CE) is the inner most layer of the cornea and is composed of a single monolayer of tightly packed, non-replicative endothelial cells on a thickened basement membrane (Descemet’s membrane (DM)). The only therapeutic treatment for corneal endothelial dysfunction is corneal transplantation using donor tissue This treatment involves the replacement of the CE with donor CECs on their native DM, using most commonly, partial thickness grafts such as Descemet’s stripping automated endothelial keratoplasty (DSAEK) or Descemet’s membrane endothelial keratoplasty (DMEK). These procedures are not without complications as there is the risk of graft failure (due to rejection or gradual cell loss) [5, 6] and graft survival rate is only 70% at 5 years [7]. At present the ratio of donor tissue to recipient is 1:1 and there is a global shortage of corneas for transplantation, alternative therapeutic methods using expanded CECs are being developed as they offer the advantage of production of several endothelial grafts from one donor to treat multiple recipients [5, 8]
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