Chen Ya-Nan, Guo Rui-Qin, Liang Bo-Yu, Ke Hong-Qin, Dong Meng-Jie, He Ming-Fang, Yang Ji, Liu Hai
Department of Ophthalmology, The Eye Disease Clinical Medical Research Center of Yunnan Province, The Eye Disease Clinical Medical Center of Yunnan Province, Second People's Hospital of Yunnan Province, The Affiliated Hospital of Yunnan University, Kunming, China.
Front Med (Lausanne). 2025 Jul 1;12:1592123. doi: 10.3389/fmed.2025.1592123. eCollection 2025.
This study aimed to develop a human acellular amniotic membrane (HAAM) scaffold suitable for corneal endothelial transplantation. The HAAM was engineered using sequential chemical treatments and physical agitation to remove cellular components while preserving the extracellular matrix structure. The study sought to evaluate the biocompatibility and functional properties of the HAAM when seeded with immortalized human corneal endothelial cells (HCECs), with the ultimate goal of providing a potential therapeutic option for corneal endothelial dysfunction.
The HAAM was fabricated through a series of chemical treatments involving trypsin/EDTA, Triton X-100, sodium deoxycholate, and peracetic acid/ethanol, combined with physical agitation. Following lyophilization, the HAAM was sterilized and coated with fibronectin and chondroitin sulfate (FNC) to enhance cell adhesion. HCECs were then seeded onto the HAAM scaffold. Biocompatibility was assessed by evaluating cell adhesion using microscopy, cell viability using CCK-8 and EdU assays, and cell proliferation. Functional validation included immunofluorescence detection of tight junction proteins (ZO-1), transcriptome sequencing (RNA-seq), and quantitative PCR (qPCR) to analyze the expression of genes regulating barrier function, ion transport, and extracellular matrix synthesis. Additionally, the expression of key genes critical for endothelial function was assessed to validate the functionality of the HAAM-based corneal endothelial transplantation membrane.
The HAAM was successfully prepared, maintaining an intact collagen fiber structure. HCECs adhered closely to the HAAM scaffold, forming a continuous monolayer. The HAAM promoted cell viability and proliferation, as evidenced by positive expression of tight junction proteins and upregulation of key functional genes. Transcriptome analysis identified genes involved in proliferation and matrix synthesis, further supporting the biocompatibility and functional properties of the HAAM.
The HAAM scaffold demonstrated excellent transparency, mechanical properties, and biocompatibility, making it suitable for the attachment and proliferation of HCECs. The effective maintenance of key functional gene expression levels suggests that the HAAM functionally mimics the characteristics of the natural corneal endothelial layer. These findings provide experimental evidence for the potential clinical application of the HAAM in corneal endothelial transplantation, offering a promising therapeutic option for patients with corneal endothelial dysfunction. Further studies are warranted to explore the long-term efficacy and safety of the HAAM in preclinical and clinical settings.
本研究旨在开发一种适用于角膜内皮移植的人脱细胞羊膜（HAAM）支架。通过序贯化学处理和物理搅拌对HAAM进行工程化处理，以去除细胞成分，同时保留细胞外基质结构。该研究旨在评估接种永生化人角膜内皮细胞（HCEC）时HAAM的生物相容性和功能特性，最终目标是为角膜内皮功能障碍提供一种潜在的治疗选择。
通过一系列化学处理制备HAAM，这些处理包括胰蛋白酶/乙二胺四乙酸（EDTA）、曲拉通X-100、脱氧胆酸钠和过氧乙酸/乙醇，并结合物理搅拌。冻干后，对HAAM进行灭菌，并用纤连蛋白和硫酸软骨素（FNC）包被以增强细胞黏附。然后将HCEC接种到HAAM支架上。通过显微镜评估细胞黏附、使用CCK-8和EdU检测法评估细胞活力以及细胞增殖来评估生物相容性。功能验证包括紧密连接蛋白（ZO-1）的免疫荧光检测、转录组测序（RNA-seq）和定量聚合酶链反应（qPCR），以分析调节屏障功能、离子转运和细胞外基质合成的基因表达。此外，评估对内皮功能至关重要的关键基因的表达，以验证基于HAAM的角膜内皮移植膜的功能。
成功制备了HAAM，其胶原纤维结构保持完整。HCEC紧密黏附于HAAM支架，形成连续的单层。HAAM促进了细胞活力和增殖，紧密连接蛋白的阳性表达和关键功能基因的上调证明了这一点。转录组分析确定了参与增殖和基质合成的基因，进一步支持了HAAM的生物相容性和功能特性。
HAAM支架表现出优异的透明度、机械性能和生物相容性，使其适合HCEC的黏附和增殖。关键功能基因表达水平的有效维持表明HAAM在功能上模拟了天然角膜内皮层的特征。这些发现为HAAM在角膜内皮移植中的潜在临床应用提供了实验证据，为角膜内皮功能障碍患者提供了一种有前景的治疗选择。有必要进一步研究探索HAAM在临床前和临床环境中的长期疗效和安全性
