Supportive properties of basement membrane layer of human amniotic membrane enable development of tissue engineering applications

Abstract

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.

Case Report: Wound Closure Acceleration in a Patient With Toxic Epidermal Necrolysis Using a Lyophilised Amniotic Membrane

“…AM has been shown to: promote epithelialization, to reduce inflammation and fibrosis, to promote neovascularization, painkilling effect and to provide a substrate for skin cell growth, and functions as a biological bandage (Sippel et al, 2001;Cirman et al, 2014). AM also contains some immunoregulatory factors, such as HLA-G and Fas ligand and well-documented re-epithelialization effects, non-tumorigenic, anti-microbial and anti-inflammatory properties (Koizumi et al, 2000;Kubo et al, 2001;Iranpour et al, 2018). The damage to the mucosa of the frontal ocular segment is quite common in acute stage TEN patients and usually manifests as an ocular inflammation with epithelial defects manifesting on the corneal and/or conjunctival epithelium (Figure 1A).…”

Successful Regenerative Endodontic Procedure of a Nonvital Immature Permanent Central Incisor Using Amniotic Membrane as a Novel Scaffold

“…The clinical success encountered with HAM as a scaffold for healing has been mainly attributed to its thick basement membrane and the abundant growth factors present within the membrane. The matrix of basement membrane of HAM contains fibronectin, collagen fibers and other proteoglycans, which provides important biochemical cues for cell adhesion and proliferation, and thus favors tissue reconstruction and remodeling [ 20 ]. Although preservation procedures like freeze-drying and irradiation partially destroyed the amniotic epithelium, the continuity of the basement membrane remains intact [ 21 ], retaining most of the characteristics similar to that of fresh HAM.…”

Applications of Human Amniotic Membrane for Tissue Engineering

“…Recently, adipose-derived MSC and a human immortalized keratinocyte cell line (HaCaT) were seeded on the three different layers of the hAM and cultured for 3 weeks. Cell attachment and viability and the mechanical strengths of the basement membrane were assessed before and after cell culture [101]. All three layers supported the attachment and proliferation of cells with no visible cytotoxic effects.…”