Poly-ε-lysine based hydrogels as synthetic substrates for the expansion of corneal endothelial cells for transplantation

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.

Poly-Epsilon-Lysine Hydrogels with Dynamic Crosslinking Facilitates Cell Proliferation

“…Despite this unexpected difference in mechanical properties with the incorporation of RGD in the hydrogel formulation, HG-PεK constructs displayed good cytocompatibility with higher metabolic activity/viability detected in HG-PεK with 0 and 0.2 mM of RGD leading to three-fold increase in cell number after 48 h cell culture. Cell adhesion and metabolic activity have been reported in chemically static poly-ε-lysine hydrogels with enhanced stiffness (Young modulus of 0.11 MPa) [ 51 ]. Here, these investigations demonstrated that the dynamic crosslinked HG-PεK with molar ratio of 1:2 (storage moduli of 0.02 MPa) formulated with low RGD concentrations provided enough stability and structural support, like chemically static poly-ε-lysine hydrogels, to afford cellular adhesion and proliferation.…”

“…Herein, the naturally occurring poly-ε-lysine, without further functionalization, was exploited in the design of arrays of dynamic hydrogels that were crosslinked via reversible Schiff-base bond formation with a 4-armed PEG-aldehydes. There are a limited number of studies with hydrogels including poly-ε-lysine [ 51 ], or modified versions of poly-ε-lysine [ 52 ], to form conventional chemically crosslinked hydrogels, for example via amide bond crosslinking. To the best of our knowledge, however, no designed dynamic poly-ε-lysine hydrogels have been proposed as ECM mimics [ 53 ].…”

Advances and trends of hydrogel therapy platform in localized tumor treatment: A review

“…For tissue engineering part, the support for histogenesis process needs to be emphasized. Functional groups of hydrogels can provide combination site for cells, such as natural adhesive proteins laminin and fibronectin, 179,180 which are essential for cell attachment. And OH and NH 2 surfaces have been proved to facilitate cell bioactivity such as ALP expression, 181 sign of osteogenic differentiation.…”

Corneal endothelium tissue engineering: An evolution of signaling molecules, cells, and scaffolds toward 3D bioprinting and cell sheets

“…Due to biocompatibility, degradability, and mechanical and optical properties, this scaffold is considered an appropriate candidate for the regeneration and transplantation of HCECs (Ozcelik et al, 2014). Kennedy et al (2019) used synthetic hydrogel poly‐ε‐lysine cross‐linking with octanedioic‐acid, which produced a thin, transparent, porous, and robust substrate for the growing of CECs. The functionalization of this hydrogel with arginine‐glycine‐aspartic acid (RGD) provides a suitable surface for the CEC attachment and facilitates the generation of confluent monolayers with functional cell surface proteins (Kennedy et al, 2019).…”