Poly-ε-lysine based hydrogels as synthetic substrates for the expansion of corneal endothelial cells for transplantation
Poly-ε-lysine based hydrogels as synthetic substrates for the expansion of corneal endothelial cells for transplantation
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DOI：
10.1007/s10856-019-6303-1
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发表时间：
2019-09-01
影响因子：
3.7
通讯作者：
Levis, Hannah J.
中科院分区：
文献类型：
--
作者：
Kennedy, Stephnie;Lace, Rebecca;Levis, Hannah J.
进行性细胞丧失导致的角膜内皮（CE）功能障碍会导致角膜水肿和严重的视力障碍。目前的治疗依赖于供体同种异体组织来替代受损的CE。供体角膜的短缺需要开发生物材料，使角膜内皮细胞（CEC）的体外扩增成为可能。本研究调查了使用与辛二酸交联的聚-β-赖氨酸（p β-赖氨酸）的合成肽水凝胶作为CEC扩增和CE移植物的潜在底物的用途。优化了PINK水凝胶性质以产生薄的、透明的、多孔的和坚固的基底。人角膜内皮细胞系（HCEC-12）在7天后作为汇合的单层附着并生长在pERK水凝胶上，而原代猪CEC（pCEC）从pERK水凝胶上脱离。胶原蛋白I、胶原蛋白IV和纤连蛋白预吸附到pCEC水凝胶增加pCEC粘附在24小时和汇合的单层形成在7天。用预吸附的层粘连蛋白、硫酸软骨素或商业FNC涂层混合物（纤连蛋白、胶原蛋白和白蛋白）观察到最小的细胞粘附。用合成的细胞结合肽H-Gly-Gly-Arg-Gly-Asp-Gly-Gly-OH（RGD）或α 2 β 1整联蛋白识别序列H-Asp-Gly-Glu-Ala-OH（DGEA）官能化pCEC水凝胶导致仅与RGD肽的pCEC粘附增强。在RGD p epsilon K水凝胶上培养5周后，pCEC显示紧密连接的闭合小带1染色和钠钾腺苷三磷酸酶的表达，表明具有功能性CE。这些结果表明，pERK水凝胶可以通过共价结合RGD来定制，以提供用于CEC附着和生长的表面。因此，提供了具有用于扩增同种异体CEC和替换受损CE的治疗应用的合成底物。[图形]。
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-epsilon-lysine (p epsilon K), cross-linked with octanedioic-acid as a potential substrate for CECs expansion and CE grafts. P epsilon K 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 p epsilon K hydrogels as confluent monolayers after 7 days, whereas primary porcine CECs (pCECs) detached from the p epsilon K hydrogel. Pre-adsorption of collagen I, collagen IV and fibronectin to the p epsilon K 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 p epsilon K hydrogel with synthetic cell binding peptide H-Gly-Gly-Arg-Gly-Asp-Gly-Gly-OH (RGD) or alpha 2 beta 1 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 p epsilon K hydrogels showed zonula occludins 1 staining for tight junctions and expression of sodium-potassium adenosine triphosphase, suggesting a functional CE. These results demonstrate the p epsilon K 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.[GRAPHICS].