Allen Claire L, Clare Gerry, Stewart Elizabeth A, Branch Matthew J, McIntosh Owen D, Dadhwal Megha, Dua Harminder S, Hopkinson Andrew
Ophthalmology, Division of Clinical Neuroscience, University of Nottingham, Nottingham, United Kingdom.
PLoS One. 2013 Oct 30;8(10):e78441. doi: 10.1371/journal.pone.0078441. eCollection 2013.
Dried amniotic membrane (AM) can be a useful therapeutic adjunct in ophthalmic surgery and possesses logistical advantages over cryopreserved AM. Differences in preservation techniques can significantly influence the biochemical composition and physical properties of AM, potentially affecting clinical efficacy. This study was established to investigate the biochemical and structural effects of drying AM in the absence and presence of saccharide lyoprotectants and its biocompatibility compared to cryopreserved material.
AM was cryopreserved or dried with and without pre-treatment with trehalose or raffinose and the antioxidant epigallocatechin (EGCG). Structural and visual comparisons were assessed using electron microscopy. Localisation, expression and release of AM biological factors were determined using immunoassays and immunofluorescence. The biocompatibility of the AM preparations co-cultured with corneal epithelial cell (CEC) or keratocyte monolayers were assessed using cell proliferation, cytotoxicity, apoptosis and migration assays.
Drying devitalised AM epithelium, but less than cryopreservation and cellular damage was reduced in dried AM pre-treated with trehalose or raffinose. Dried AM alone, and with trehalose or raffinose showed greater factor retention efficiencies and bioavailability compared to cryopreserved AM and demonstrated a more sustained biochemical factor time release in vitro. Cellular health assays showed that dried AM with trehalose or raffinose are compatible and superior substrates compared to cryopreserved AM for primary CEC expansion, with increased proliferation and reduced LDH and caspase-3 levels. This concept was supported by improved wound healing in an immortalised human CEC line (hiCEC) co-cultured with dried and trehalose or raffinose membranes, compared to cryopreserved and fresh AM.
Our modified preservation process and our resultant optimised dried AM has enhanced structural properties and biochemical stability and is a superior substrate to conventional cryopreserved AM. In addition this product is stable and easily transportable allowing it to be globally wide reaching for use in clinical and military sectors.
干燥羊膜（AM）可作为眼科手术中一种有用的治疗辅助材料，与冷冻保存的AM相比具有后勤方面的优势。保存技术的差异会显著影响AM的生化组成和物理性质，可能影响临床疗效。本研究旨在探讨在有无糖类冻干保护剂的情况下干燥AM的生化和结构效应，以及与冷冻保存材料相比其生物相容性。
AM分别进行冷冻保存，或在有或无海藻糖、棉子糖预处理以及抗氧化剂表没食子儿茶素（EGCG）的情况下进行干燥处理。使用电子显微镜进行结构和视觉比较评估。通过免疫测定和免疫荧光确定AM生物因子的定位、表达和释放。使用细胞增殖、细胞毒性、凋亡和迁移测定评估与角膜上皮细胞（CEC）或角膜细胞单层共培养的AM制剂的生物相容性。
干燥使AM上皮失活，但程度小于冷冻保存，用海藻糖或棉子糖预处理的干燥AM细胞损伤减少。与冷冻保存的AM相比，单独干燥的AM以及添加海藻糖或棉子糖的AM显示出更高的因子保留效率和生物利用度，并在体外表现出更持续的生化因子时间释放。细胞健康测定表明，与冷冻保存的AM相比，添加海藻糖或棉子糖的干燥AM是用于原代CEC扩增的相容且优质的底物，具有更高的增殖率以及更低的乳酸脱氢酶（LDH）和半胱天冬酶-3水平。与冷冻保存和新鲜的AM相比，与干燥的以及添加海藻糖或棉子糖的膜共培养的永生化人CEC系（hiCEC）伤口愈合改善，支持了这一概念。
我们改进的保存工艺以及由此得到的优化干燥AM具有增强的结构特性和生化稳定性，是优于传统冷冻保存AM的底物。此外，该产品稳定且易于运输，使其能够广泛应用于临床和军事领域。
