Vasudevan Balamurugan, Chang Qing, Wang Bin, Huang Siyang, Sui Yulong, Zhu Wenjie, Fan Qing, Song Yisheng
Affiliated Hospital of Medical College, Qingdao University, Qingdao, China.
Nanobiomedicine (Rij). 2020 Dec 29;7:1849543520983173. doi: 10.1177/1849543520983173. eCollection 2020 Jan-Dec.
Trehalose is a disaccharide molecule consisting of two molecules of glucose. Industrially, trehalose is derived from corn starch and utilized as a drug. This study aims to examine whether the integration of nanoparticle-encapsulated trehalose to the Ice-Free Cryopreservation (IFC) method for preserving heart valves has better cell viability, benefits to protect the extracellular matrix (ECM), and reduce immune response after storage. For the experiment to be carried out, we obtained materials, and the procedures were carried out in the following manner. The initial step was the preparation of hydroxyapatite nanoparticles, followed by precipitation to acquire Apatite colloidal suspensions. Animals were obtained, and their tissue isolation and grouping were done ethically. All samples were then divided into four groups, Control group, Conventional Frozen Cryopreservation (CFC) group, IFC group, and IFC + T (IFC with the addition of 0.2 M nanoparticle-encapsulated Trehalose) group. Histological analysis was carried out via H&E staining, ECM components were stained with Modified Weigert staining, and the Gomori Ammonia method was used to stain reticular fibers. Alamar Blue assay was utilized to assess cell viability. Hemocompatibility was evaluated, and samples were processed for immunohistochemistry (TNFα and IL-10). Hemocompatibility was quantified using Terminal Complement Complex (TCC) and Neutrophil elastase (NE) as an indicator. The results of the H&E staining revealed less formation of extracellular ice crystals and intracellular vacuoles in the IFC + T group compared with all other groups. The CFC group's cell viability showed better viability than the IFC group, but the highest viability was exhibited in the IFC + T group (70.96 ± 2.53, P < 0.0001, n = 6). In immunohistochemistry, TNFα levels were lowest in both IFC and IFC + T group, and IL-10 expression had significantly reduced in IFC and IFC + T group. The results suggested that the nanoparticle encapsulated trehalose did not show significant hemocompatibility issues on the cryopreserved heart valves.
海藻糖是一种由两个葡萄糖分子组成的二糖分子。在工业上，海藻糖由玉米淀粉衍生而来并用作药物。本研究旨在检验将纳米颗粒包裹的海藻糖整合到用于保存心脏瓣膜的无冰冷冻保存（IFC）方法中是否具有更好的细胞活力、对保护细胞外基质（ECM）有益以及降低储存后的免疫反应。为了进行该实验，我们获取了材料，并按以下方式进行操作。第一步是制备羟基磷灰石纳米颗粒，随后进行沉淀以获得磷灰石胶体悬浮液。获取动物，并在伦理上对其组织进行分离和分组。然后将所有样本分为四组，对照组、传统冷冻保存（CFC）组、IFC组和IFC + T（添加0.2 M纳米颗粒包裹海藻糖的IFC）组。通过苏木精 - 伊红（H&E）染色进行组织学分析，用改良魏格特染色对ECM成分进行染色，并用Gomori氨法对网状纤维进行染色。使用阿拉玛蓝测定法评估细胞活力。评估血液相容性，并对样本进行免疫组织化学处理（检测肿瘤坏死因子α（TNFα）和白细胞介素 - 10（IL - 10））。使用末端补体复合物（TCC）和中性粒细胞弹性蛋白酶（NE）作为指标对血液相容性进行量化。H&E染色结果显示，与所有其他组相比，IFC + T组细胞外冰晶和细胞内空泡的形成较少。CFC组的细胞活力显示出比IFC组更好的活力，但最高活力出现在IFC + T组（70.96 ± 2.53，P < 0.0001，n = 6）。在免疫组织化学中，TNFα水平在IFC组和IFC + T组中最低，并且IL - 10表达在IFC组和IFC + T组中显著降低。结果表明，纳米颗粒包裹的海藻糖在冷冻保存的心脏瓣膜上未显示出明显的血液相容性问题。
