Widyaningrum Rifa, Wu Yu-Wen, Delila Liling, Lee Deng-Yao, Wang Tsung-Jen, Burnouf Thierry
International PhD Program in Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.
Department of Ophthalmology, Faculty of Medicine Public Health and Nursing, Universitas Gadjah Mada-Dr Sardjito General Hospital, Yogyakarta, Indonesia.
Platelets. 2022 Nov 17;33(8):1237-1250. doi: 10.1080/09537104.2022.2105829. Epub 2022 Aug 10.
Corneal endothelial cells (CECs) slowly decrease in number with increasing age, which is a clinical issue as these cells have very limited regenerative ability. Therapeutic platelet biomaterials are increasingly used in regenerative medicine and cell therapy because of their safety, cost-effective manufacture, and global availability from collected platelet concentrates (PCs). Platelet extracellular vesicles (PEVs) are a complex mixture of potent bioactive vesicles rich in molecules believed to be instrumental in tissue repair and regeneration. In this study we investigated the feasibility of using a PEVs preparation as an innovative regenerative biotherapy for corneal endothelial dysfunction. The PEVs were isolated from clinical-grade human PC supernatants by 20,000 × ultracentrifugation and resuspension. PEVs exhibited a regular, fairly rounded shape, with an average size of <200 nm and were present at a concentration of approximately 10 /mL. PEVs expressed cluster of differentiation 41 (CD41) and CD61, characteristic platelets membrane markers, and CD9 and CD63. ELISA and LC-MS/MS proteomic analyses revealed that the PEVs contained mixtures of growth factors and multiple other trophic factors, as well as proteins related to extracellular exosomes with functional activities associated with cell cadherin and adherens pathways. CECs treated with PEVs showed increased viability, an enhanced wound-healing rate, stronger proliferation markers, and an improved adhesion rate. PEVs did not exert cellular toxicity as evidenced by the maintenance of cellular morphology and preservation of corneal endothelial proteins. These findings clearly support further investigations of PEV biomaterials in animal models for translation as a new CEC regeneration biotherapy.
角膜内皮细胞（CECs）的数量会随着年龄的增长而缓慢减少，由于这些细胞的再生能力非常有限，这成为了一个临床问题。治疗性血小板生物材料因其安全性、经济高效的制造以及可从采集的血小板浓缩物（PCs）中全球获取，而越来越多地用于再生医学和细胞治疗。血小板细胞外囊泡（PEVs）是一种复杂的强效生物活性囊泡混合物，富含被认为对组织修复和再生有重要作用的分子。在本研究中，我们调查了使用PEVs制剂作为角膜内皮功能障碍的创新再生生物疗法的可行性。通过20,000×超速离心和重悬从临床级人PC上清液中分离出PEVs。PEVs呈现规则的、相当圆润的形状，平均尺寸<200 nm，浓度约为10 /mL。PEVs表达分化簇41（CD41）和CD61，这是血小板特征性膜标记物，以及CD9和CD63。酶联免疫吸附测定（ELISA）和液相色谱 - 串联质谱（LC - MS/MS）蛋白质组学分析表明，PEVs含有生长因子和多种其他营养因子的混合物，以及与细胞外泌体相关的蛋白质，这些蛋白质具有与细胞钙黏蛋白和黏附途径相关的功能活性。用PEVs处理的CECs显示出活力增加、伤口愈合率提高、增殖标志物更强以及黏附率改善。PEVs未表现出细胞毒性，这通过细胞形态的维持和角膜内皮蛋白的保存得以证明。这些发现明确支持在动物模型中进一步研究PEV生物材料，以便转化为一种新的CEC再生生物疗法。
