Iranpour Sonia, Mahdavi-Shahri Nasser, Miri Raheleh, Hasanzadeh Halimeh, Bidkhori Hamid Reza, Naderi-Meshkin Hojjat, Zahabi Ehsan, Matin Maryam M
Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran.
Research Center for HIV/AIDS, HTLV and Viral Hepatitis, ACECR-Khorasan Razavi Branch, Mashhad, Iran.
Cell Tissue Bank. 2018 Sep;19(3):357-371. doi: 10.1007/s10561-017-9680-z. Epub 2018 Jan 8.
Human amniotic membrane (HAM) has been widely used as a natural scaffold in tissue engineering due to many of its unique biological properties such as providing growth factors, cytokines and tissue inhibitors of metalloproteinases. This study aimed at finding the most suitable and supportive layer of HAM as a delivery system for autologous or allogeneic cell transplantation. Three different layers of HAM were examined including basement membrane, epithelial and stromal layers. In order to prepare the basement membrane, de-epithelialization was performed using 0.5 M NaOH and its efficiency was investigated by histological stainings, DNA quantification, biomechanical testing and electron microscopy. Adipose-derived stromal cells (ASCs) and a human immortalized keratinocyte cell line (HaCaT) were seeded on the three different layers of HAM and cultured for 3 weeks. The potential of the three different layers of HAM to support the attachment and viability of cells were then monitored by histology, electron microscopy and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Moreover, mechanical strengths of the basement membrane were assessed before and after cell culture. The results indicated that the integrity of extra cellular matrix (ECM) components was preserved after de-epithelialization and resulted in producing an intact basement amniotic membrane (BAM). Moreover, all three layers of HAM could support the attachment and proliferation of cells with no visible cytotoxic effects. However, the growth and viability of both cell types on the BAM were significantly higher than the other two layers. We conclude that growth stimulating effectors of BAM and its increased mechanical strength after culturing of ASCs, besides lack of immunogenicity make it an ideal model for delivering allogeneic cells and tissue engineering applications.
人羊膜（HAM）因其许多独特的生物学特性，如提供生长因子、细胞因子和金属蛋白酶组织抑制剂等，已在组织工程中广泛用作天然支架。本研究旨在寻找人羊膜最适合且具支持性的层，作为自体或异体细胞移植的递送系统。研究了人羊膜的三层不同结构，包括基底膜、上皮层和基质层。为制备基底膜，使用0.5 M NaOH进行去上皮处理，并通过组织学染色、DNA定量、生物力学测试和电子显微镜检查来研究其效果。将脂肪来源的基质细胞（ASC）和人永生化角质形成细胞系（HaCaT）接种在人羊膜的三层不同结构上，并培养3周。然后通过组织学、电子显微镜和3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法监测人羊膜的三层不同结构支持细胞附着和活力的潜力。此外，在细胞培养前后评估基底膜的机械强度。结果表明，去上皮处理后细胞外基质（ECM）成分的完整性得以保留，并产生了完整的基底羊膜（BAM）。此外，人羊膜的所有三层结构都能支持细胞的附着和增殖，且无明显细胞毒性作用。然而，两种细胞类型在基底羊膜上的生长和活力均显著高于其他两层。我们得出结论，基底羊膜的生长刺激因子及其在接种脂肪来源的基质细胞后增强的机械强度，加上缺乏免疫原性，使其成为递送异体细胞和组织工程应用的理想模型。
