How preparation and preservation procedures affect the properties of amniotic membrane? How safe are the procedures?

JBUR 5867 No. of Pages 18

burns xxx (2019) xxx –xxx

Available online at www.sciencedirect.com

ScienceDirect journal homepage: www.elsevier.com/locate/burns

Review

How preparation and preservation procedures affect the properties of amniotic membrane? How safe are the procedures? Mazaher Gholipourmalekabadi a,b,c , Behrouz Farhadihosseinabadi d,e , Mehdi Faraji a,b, Mohammad Reza Nourani f,g, * a

Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran Department of Tissue Engineering & Regenerative Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran c Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medicine Sciences, Tehran, Iran d Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran e Student Research Committee, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran f Chemical Injuries Research Center, Systems Biology and Poisoning Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran g Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran b

article info

abstract

Article history:

Human amniotic membrane (AM) has been widely used for tissue engineering and

Accepted 3 July 2019

regenerative medicine applications. AM has many favorable characteristics such as high

Available online xxx

biocompatibility, antibacterial activity, anti-scarring property, immunomodulatory effects, anti-cancer behavior and contains several growth factors that make it an excellent natural

Keywords: Human amniotic membrane

candidate for wound healing. To date, various methods have been developed to prepare, preserve, cross-link and sterilize the AM. These methods remarkably affect the morphological, physico-chemical and biological properties of AM. Optimization of an effective and safe

Abbreviations: AAM, air-dried amniotic membrane; ANG, angiopoietin; bFGF, basic fibroblast growth factor; BMP, bone morphogenetic protein; CAM, cryopreservation of amniotic membrane; DMSO, dimethyl sulfoxide; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; EGF, epidermal growth factor; FAM, fresh amniotic membrane; GDF, growth differentiation factor; GM-CSF, granulocyte-macrophage colony stimulating factor; HNP, human neutrophil peptides; IGF, insulin-like growth factor; IL, interleukin; MDR, multidrug resistant; MCP, monocyte chemotactic protein; MCSF, macrophage colony stimulating factor; MIP, macrophage inflammatory protein; OPG, osteoprotegerin; PDGF, platelet-derived growth factor; RANTES, regulated on activation, normal T cell expressed and secreted; SLPI, secretory leukocyte proteinase inhibitor; VEGF, endothelial growth factor; AM, human amniotic membrane; BDNF, brain-derived neurotrophic factor; BLC, B-lymphocyte chemoattractant; BPI, bactericidal/permeability-increasing protein; DAM, decellularized amniotic membrane; ECM, extracellular matrix; EDTA, ethylenediaminetetraacetic acid; EMEM, eagles’ minimum essential medium; GA, glutaraldehyde; GH, growth hormone; HGF, hepatocyte growth factor; IFNy, interferon gamma; IGFBP, insulin-like growth factor-binding protein; LAM, lyophilized amniotic membrane; MIF, migration inhibitory factor; MIG, monokine induced by gamma-interferon; MRP8/14, Calprotectin; PBS, phosphate buffered saline; PIGF, placental growth factor; SDS, sodium dodecyl sulphate; TIMP, tissue inhibitors of metalloproteases. * Corresponding author at: Chemical Injuries Research Center, Systems Biology and Poisoning Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran. E-mail addresses: [email protected], [email protected] (M.R. Nourani). https://doi.org/10.1016/j.burns.2019.07.005 0305-4179/© 2019 Elsevier Ltd and ISBI. All rights reserved.

Please cite this article in press as: M. Gholipourmalekabadi, et al., How preparation and preservation procedures affect the properties of amniotic membrane? How safe are the procedures?, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.005

JBUR 5867 No. of Pages 18

2

burns xxx (2019) xxx –xxx

Preparation

method for preparation and preservation of AM for a specific application is critical. In this

Tissue engineering

review, the isolation, different methods of preparation, preservation, cross-linking and

Regenerative medicine

sterilization as well as their effects on properties of AM are well discussed. For each section, at

Preservation

least one effective and safe protocol is described in detail. © 2019 Elsevier Ltd and ISBI. All rights reserved.

Decellularization

Contents 1. 2. 3.

4.

5. 6. 7. 8.

1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Amniotic membrane components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological properties of amniotic membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Wound healing and anti-scarring properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AM as a cell delivery system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. 3.3. Angiogenic property . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antibacterial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Anti-inflammatory property . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Isolation and preparation of AM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Preparation and preservation of amniotic membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. 4.2.1. Preparation: fresh amniotic membrane (FAM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. Preparation: decellularized amniotic membrane (DAM) . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3. Preservation: cryopreservation and tissue banking of amniotic membrane (CAM) . . . 4.2.4. Preservation: air-dried amniotic membrane (AAM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.5. Preservation: lyophilized amniotic membrane (LAM) . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-linking of amniotic membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sterilization of amniotic membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FDA regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction

The use of human amniotic membrane (AM) in treatment of wounds and burns have been described for over a century [1]. In the 1910s, AM was used for the first time as a transplantable material and skin graft substitute at the Johns Hopkins Hospital [2]. In the second half of the 20th century, many scientific groups in all over the world began their clinical investigations focusing on possible therapeutic applications of AM, especially as a natural biological dressing for different types of wounds [3]. Possession of clinically important characteristics, including anti-inflammatory behavior, angiogenic property, anti-microbial, nonimmunigenic, epithelialization induction, as well as the presence of a number of important growth factors, have made AM a potential candidate for healing wounds, particularly burns [4–7]. Already, AM is widely used in various areas of tissue engineering such as skin wound healing and ophthalmology, both in animal and human (Table 1) [8–12]. AM can be used fresh or preserved, intact or decellularized, depending available facility, wound type and required life style. To date, many methods have been developed for preparation, preservation, cross-linking and sterilization of AM. Each of these methods remarkably affect the mechanical, physico-chemical and biological

.... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... ....

.... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... ....

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...

.... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... ....

00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00

behaviors of AM [13,14]. It is very critical to optimize and use an appropriate AM preservation method for a specific clinical applications. In this article, various methods of preparation of preservation of AM, from tissue collection to grafting are reviewed in details. For each step, an effective and viable procedure is described. Effects of different preparation and preservation methods on mechanical and biological properties of AM are well discussed.

2.

Amniotic membrane components

AM is the innermost fetal membrane with average thickness of 0.5 mm. AM is composed of three different histological layers: an epithelial monolayer, a thick basement membrane layer and an underneath layer of avascular mesenchymal tissue. The latter layer can be subdivided into the compact, fibroblast and intermediate or spongy sublayers (Fig. 1) [6,15]. Amniotic epithelial cells contain nearly a small number of intracytoplasmic organelles, microvilli on the apical surface and pinocytic vesicles and also produce several cytokines/ factors known to promote cell proliferation and differentiation [3,16]. Amniotic epithelial cells are possibly involved in the secretory and intra-/trans-cellular transport activities [17]. AM basement membrane is one of the thickest basement

Please cite this article in press as: M. Gholipourmalekabadi, et al., How preparation and preservation procedures affect the properties of amniotic membrane? How safe are the procedures?, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.005

JBUR 5867 No. of Pages 18

3

burns xxx (2019) xxx –xxx

Table 1 – Summary of potential applications of the amniotic membrane based scaffolds in tissue engineering. The AM component

Species

Target cell/tissue

Cryopreserved AM Cryopreserved AM Fresh AM Denuded AM Intact/denuded AM Amniotic epithelial cells Denuded AM Denuded AM AECs/intact AM Cryopreserved AM Cryopreserved AM Nonpreserved AM Denuded AM Denuded AM

Human Human Human Rabbit Rabbit Mouse Rat Rat Mouse – Human Human Mouse Rabbit

Eye Eye Skin Eye Cartilage beta-glucoronidase secretory cell Peripheral nerve Peripheral nerve Hepatocyte Endothelial cell Eye Eye Skin Skin

Reference [175] [176] [54] [177] [178] [179] [180] [181] [182] [183] [184] [76] [185] [24]

Fig. 1 – Histological structure of human amniotic membrane [186].

membrane among human tissues and contains different types of collagen, a-actinin, actin, several cytokeratins, spectrin, vimentin, ezrin, desmoplakin and laminin [16,18,19]. The collagen in the basement membrane provides a strong tensile strength in AM [20]. The inner compact layer in avascular mesenchymal tissue is in contact with the basement membrane. The fibroblast layer contains collagens types I and III that are arranged in parallel bundles to maintain the mechanical integrity of AM. In addition, type V and VI collagens create cross-links between interstitial collagen and epithelial basement membrane. The intermediate layer, also known as spongy layer, is in touch to chorionic membrane and consists of a large number of proteoglycans and glycoproteins with a nonfibrillar network of type III collagen [6,21]. As mentioned earlier, AM is a rich source of various biologically active substances which are involved in tissue regeneration and wound healing. The most important biologically active substances within AM are wound healingpromoting factors such as transforming growth factors alpha and beta (TGF-a and TGF-b), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF). Various cytokines, chemokines and antibacterial substances presented in AM is listed in Table 2. Biological properties of AM are described below in more details.

3.

Biological properties of amniotic membrane

3.1.

Wound healing and anti-scarring properties

AM accelerates wound healing with minimal post wound scar formation. AM inhibits scarring by down-regulation of TGF-b1 which is responsible for induced fibrotic responses through activation of fibroblasts [16]. The primary inhibitor of TGF-b1 within the AM is hyaluronic acid existed in mesenchymal portion [22]. It was found that the low/late reepithelialization and chronic inflammation, essentially caused by infections, are the major inducer factors in scar tissue formation. AM, as a promising biological membrane, also minimize scar formation in skin wounds through secretion of EGF, KGF and HGF which are essential growth factors involved in epithelialization and wound healing [23,24]. AM exerts its anti-inflammatory activity through migration inhibitory factor (MIF) and down-regulation of the pro-inflammatory cytokines expression such as IL-1a, IL-1b and proteinase activity [25,26]. Additionally, AM has a broadspectrum antibacterial activities that enable it to prevent post-wound infections, the most important stimulus of inflammatory responses [27].

Please cite this article in press as: M. Gholipourmalekabadi, et al., How preparation and preservation procedures affect the properties of amniotic membrane? How safe are the procedures?, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.005

JBUR 5867 No. of Pages 18

4

burns xxx (2019) xxx –xxx

Table 2 – Cytokines, chemokines and antibacterial substances presented in AM. Biologically active substances presented in AM Cytokine bFGF EG-VEGF VEGF BMP-5 EGF FGF-4 BDNF GH DPPIV/CD26 HB-EGF GCSF GM-CSF Serpin MCSF GDF-15

IGF-1 IGFBP-1 IGFBP-2 IGFBP-3 IGFBP-4 IGFBP-6 TGF-a TGF-b PDGF-AA PDGF-BB TIMP-1 TIMP-2 TIMP-4 KGF;FGF-7

INFg IL-1a IL-1b IL-4 IL-6 IL-15 IL-17 OPG TNF HGF B-NGF PIGF ANG-2 PAI-1

Chemokine

Antibacterial substances

IL-8 IL-16 Eotaxin-2 MCP-1 BLC I-309 MIG MIP-1a MIP-1b MIP-1d RANTES

Cystatin E b-defensins SLPI Elafin BPI HNP LL-37 Lysozyme b2-macroglobulin MRP8/14 Ubiquitin

Abbreviations: ANG, angiopoietin; BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; BLC, B-lymphocyte chemoattractant; BMP, bone morphogenetic protein; BPI, bactericidal/permeability-increasing protein; EGF, epidermal growth factor; GDF, growth differentiation factor; GH, growth hormone; GM-CSF, granulocyte-macrophage colony stimulating factor; HGF, hepatocyte growth factor; HNP, human neutrophil peptides; IFNy, interferon gamma; IGF, insulin-like growth factor; IGFBP, insulin-like growth factor-binding protein; IL, interleukin; MCP, monocyte chemotactic protein; MCSF, macrophage colony stimulating factor; MIG, monokine induced by gamma-interferon; MIP, macrophage inflammatory Protein; MRP8/14, Calprotectin; OPG, osteoprotegerin; PDGF, platelet-derived growth factor; PIGF, placental growth factor; RANTES, regulated on activation, normal T cell expressed and secreted; SLPI, secretory leukocyte proteinase inhibitor; VEGF, endothelial growth factor.

3.2.

AM as a cell delivery system

Intact AM is a great source of native high-molecular-weight hyaluronic acid and acts as a ligand for CD44 which is an antigen expressed by different cells [28]. Moreover, laminin, fibronectin and different types of collagens and proteoglycans are other components of AM that could act as a ligand for integrin receptors. These promising properties make AM as an appropriate biological membrane support cell adhesion, ingrowth, spreading and eventually tissue regeneration. In our previously studies, we showed that AM is an excellent delivery system with high biocompatibility and no toxicity for both mouse and human stromal cells [29,30]. It has also been shown that AM can be served as a promising delivery system for TGF-b3 expressing bone marrow stromal cells [31].

3.3.

Angiogenic property

Angiogenesis or neovascularization is the formation of new capillaries from preexisting microvessels. Neovascularization plays a critical role in wound healing in physiologic level [32,33]. The effects of AM and AM-derived cells on angiogenesis are poorly understood and controversial. AM showed both angiogenesis and anti-angiogenesis properties [16,34]. The angiogenic factors including vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), angiogenin, interferon-g, interleukin-6 (IL-6), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) are secreted by amniotic mesenchymal cells [35,36]. In contrast, amniotic epithelial cells secret some antiangiogenic factors such as IL-1 receptor antagonist, tissue

inhibitors of metalloproteases (TIMP-1, -2, -3 and -4), thrombospondin-1, endostatin and heparin sulfate proteoglycan [32,34].

3.4.

Antibacterial activity

Intact skin acts as a primary physical barrier against invading pathogenic bacteria and viruses disrupted mainly by acute and chronic wounds [37]. AM grafting not only acts as a biological barrier, but also expresses several anti-bacterial and anti-viral components. Covering the wound surface with AM decreases the risk of infection through prevention of bacterial infiltration and growth [38]. b-defensins, especially b3-defensin, expressed at mucosal surfaces by epithelial cells and leukocytes, are the primary responsible element for antimicrobial features of AM. The b3-defensin is the predominant defensin in the AM and considered as an important part of the innate immune system [39–41]. Moreover, secretory leukocyte proteinase inhibitor (SLPI) and elafin, two low-molecular-mass elastase inhibitors that are mainly expressed by AM-derived cells and at mucosal sites, have strong anti-inflammatory and anti-microbial abilities [39,40]. In addition to AM, amniotic fluid shows a wide-spectrum antibacterial activities. Several antimicrobial peptides and proteins such as bactericidal/permeability-increasing protein (BPI) [42], human neutrophil peptides (HNP) 1, 2 and 3 [43], LL-37 [44], lysozyme [44], b2-macroglobulin [45], calprotectin (MRP8/14) [46] and ubiquitin [47] have been documented in amniotic fluid. Miller et al. [48] demonstrated that amniotic fluid obtained during the first, second and third trimesters have low, intermediate and high inhibitory effects on

Please cite this article in press as: M. Gholipourmalekabadi, et al., How preparation and preservation procedures affect the properties of amniotic membrane? How safe are the procedures?, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.005

JBUR 5867 No. of Pages 18

burns xxx (2019) xxx –xxx

microorganisms, respectively. Antibacterial activity of AM against various bacterial strains has been investigated by several studies [49–51]. For instance, based on the results reported by Robson and Krizek, AM-implanted rat burn wounds showed a decreased rate of infections, compared with untreated burn wounds [52,53]. In another study, Mohammadi et al. [54] treated the patients with chronic burn wounds with AM and they showed that the AM prevented postburn infections in comparison with the un-implanted burn wounds. During the last years, overuse of antibiotics in health setting especially in burn center has led to an elevated prevalence of multidrug resistant (MDR) bacteria. Although many efforts have been carried out to develop an efficient antibacterial agent for prevention or treatment of MDR bacteria, increased incidence of such bacteria has still remained as a major concern in healthcare community.

3.5.

Anti-inflammatory property

Anti-inflammatory effect of AM has been well-documented in the literature [55–58]. This characteristic of AM can be attributed to the cells and matrix of AM. High adhesion characteristic of AM’s matrix for inflammatory cells and driving these cells to apoptosis through pro-apoptotic agents play a critical role in the context [12]. Shimmura et al. [59] showed that the cultured inflammatory cells on AM surface exhibited the characteristic of cells undergoing apoptosis in both morphological and molecular aspects. AM’s matrix also showed positive impacts on the suppression of pro-inflammatory cytokines production by the inflammatory cells. For

5

instance, the seeded human limbal epithelial cells on AM’s matrix secreted lower amount of IL-1b at both protein and mRNA levels compared to control. On the other hand, these cells showed higher expression of anti-inflammatory cytokines such as interleukin-1 receptor antagonist. In addition to the matrix, the cells derived from AM have an undeniable role in its anti-inflammatory characteristic. It was reported that both human amniotic epithelial cells (hAEC) and human amniotic mesenchymal stromal cells (hAMSC) exerted their anti-inflammatory effects via secreting various factors [60]. For example, Kang et al. [61] reported some soluble factors responsible for anti-inflammatory effects of hAMSC including prostaglandin E2 (PGE2), TGF-b, hepatocyte growth factor (HGF), and indoleamine 2, 3 dioxygenase (IDO). They found that co-culture of hAMSC with peripheral blood mononuclear cell (PBMC) could remarkably diminish production of some pro-inflammatory cytokines by PBMCs such as IL-17 and INF-g. PBMCs also showed a higher amount of IL-10, as an anti-inflammatory cytokine, in the presence of hAMSC. In another study, Li et al. [62] investigated the effect of hAECs’s supernatant on the biological behaviors of inflammatory cells. They reported that the soluble factors released by these cells strongly reduced the chemotactic activity of neutrophils and macrophages as well as diminished the proliferation of B and T cells. They also considered some agents including RAIL (tumor necrosis factor-related apoptosis-inducing ligand), Fas ligand (FasL), TNF-a, T, TGF-b, and MIF as the most important factors related to the immunosuppressive activity of hAECS.

Fig. 2 – Isolation, preparation, preservation, cross-linking and sterilization of amniotic membrane (AM). After isolation of AM from placenta, separation from chorion and several washing, AM can be used fresh or decellularized. Fresh or decellularized AM can be preserved by air-drying, freeze drying and cryopreservation. The prepared AM can be cross-link and/or sterilized. Permission for reuse of tissue is obtained [51].

Please cite this article in press as: M. Gholipourmalekabadi, et al., How preparation and preservation procedures affect the properties of amniotic membrane? How safe are the procedures?, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.005

JBUR 5867 No. of Pages 18

6

burns xxx (2019) xxx –xxx

4.

Isolation and preparation of AM

4.1.

Isolation

AM should be taken from placenta of mothers after their cesarean-section deliveries. Protein and growth factors content of AM depends on gestational age and donor age. It was shown that total protein content and HGF, KGF, bFGF and TGFb1 have lower level in AM of mothers with higher donor and gestational ages [63]. Placenta derived from natural vaginal delivery is not a good choice for AM isolation. It was reported that natural placenta delivery may cause structural changes and contaminated with vaginal flora organisms [64]. All the placenta collection, transfer to clean room in transport media, separation of AM and preservation must be carried out under sterile conditions. For isolation of AM, placenta is collected from consenting mothers upon elective cesarean delivery

under sterile condition. Placenta should be transferred to clean room (under the laminar flow hood) in appropriate transfer medium. Various transport medium has been used for this purpose. The Eagles’ minimum essential medium (EMEM) supplemented with 3.3% L-glutamine and antibiotics cocktail (50 mg/ml gentamicin, 100 units/ml penicillin, 200 mg/ml ciprofloxacin) and antifungal (1 mg/ml Amphotericin B) has widely been used as transport medium [29,65,66]. Placenta must be serologically negative for human immunodeficiency virus types I and II, human hepatitis virus types B (HBV) and C (HCV), syphilis, gonorrhea, toxoplasmosis, cytomegalovirus and Treponema pallidum infections [13,29,65,67]. It is recommended to obtain additional consent from mother for subsequent screening of HIV 3–4 month post placenta collection. The placenta must be further tested for bacterialand fungal culture once transport to clean room. All the screening positive samples must be excluded from further operation to avoid risk of transmissible infections [13,65,68]. It is

Fig. 3 – (a) Strong antibacterial activity of both fresh and decellularized AM against S. aureus (ATCC 25,923) and P. aeruginosa (ATCC 27833) [86]. (b) Growth inhibition zone formed around fresh (FAM), cryopreserved (CAM) and freeze-dried AM (LAM) for P. aeruginosa (ATCC 27853), E. coli T3 and E. coli T4; indicating no significant effects of preservation on antibacterial property of the AM [51]. Please cite this article in press as: M. Gholipourmalekabadi, et al., How preparation and preservation procedures affect the properties of amniotic membrane? How safe are the procedures?, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.005

JBUR 5867 No. of Pages 18

burns xxx (2019) xxx –xxx

noted that all the screening test should be carried out by a standard laboratory, and the result records and detailed medical history of donor should be kept for 11 years [65,69]. Placenta should place in a sterile stainless steel containing transport medium. After several washing with sterile phosphate buffered saline (PBS) to remove blood clot, chorion must be completely peeled off from amniotic membrane under clean laminar flow workbench. Chorion has high immunogenicity and provoke host immune system and cause graft rejection. So, chorion and chorion fragments should be completely removed from the AM by a round-ended forcep or cell culture scraper to avoid any risk of inflammatory reaction and rejection [29,66,67,70]. It is noted that transport and work solution should be prepared 1–2 weeks before operation. pH and sterility of the solution should be tested and confirmed one day before use. It is recommended to prepare solution fresh (up to 1– 2 weeks before operation) [65]. All the media preparation should get relevant standard by a competent certifying institution. After this step, AM is ready to preserve in various forms for soft tissue engineering clinical applications. After isolation, AM can be used fresh or preserved and stored until use (Fig. 2).

4.2.

Preparation and preservation of amniotic membrane

AM can be used “fresh”, however in the most countries, because of their legal regulation, the AM must be preserved for 6 month until confirming negative screening result for HIV. Several techniques such as lyophilization and cryopreservation (by glycerol or Dimethyl sulfoxide (DMSO)) have been developed to preserve AM for long storage. Correct preparation and preservation choice deeply depends on AM applications and needed storage time [65,71,72]. It is also reported that handling procedures and method of preservation can significantly affect the AM morphology, transparency, thickness, biochemical composition, protein and growth factors contents [23,73,74]. Herein, various AM preparation and preservation procedures as well as their effects on mechanical and biological properties of AM are discussed.

4.2.1.

Preparation: fresh amniotic membrane (FAM)

Fresh AM (FAM) has widely been used in soft tissue engineering [54,75,76]. Native structure and preserved growth factors and living stem cells make FAM a promising amniotic product for tissue engineering [54,75,76]. A randomized clinical trial study on 38 patients (mean age of 27.18
6.38 and burn in 29.18
7.23 TBSA%) with chronic burn wounds show better graft take, healing and protection against post-burn infections [54]. FAM also show to have a great impact on chronic nonhealing ulcers [75]. It is reported that both fibroblast and epithelial cells are alive after isolation from FAM, while these cells lost their viability after isolation of cryopreserved AM (CAM) [77]. Short storage time and slight inflammatory reaction are the main disadvantages of fresh AM [29,67,75].

4.2.2.

Preparation: decellularized amniotic membrane (DAM)

As discussed earlier in this article, epithelial side of AM contains a monolayer of epithelial cells and mesenchymal side contains various cell types such as fibroblasts and mesenchymal stem cells. Although amniotic membrane-derived cells do not express MHC II and co-stimulatory molecules [78]. Some studies reported

7

a mild immunogenicity of FAM that may cause some inflammatory responses of host, leading delayed healing [29,67,79]. The question that whether FAM or decellularized AM (DAM) is more appropriate for ex vivo and in vivo applications of this membrane still remains unanswered. Some researchers reported that epithelial cells of AM secret some growth factors and cytokines useful for cell proliferation and wound healing [80–82]. On the other hand, several studies demonstrated that DAM can support cell growth and cell adhesion and is better choice for corneal and skin wound healing than FAM [83,84]. Decellularization of AM is the best way to overcome its antigenicity. Nevertheless, decellularization can significantly decrease thickness, mechanical property and immunogenicity of AM, while increase its degradation rate and safety [29,67]. It was demonstrated that the DAM is almost fully degraded after 7 days treatment with lysozyme solution, while FAM show less than 80% weight loss [29]. DAM is a superior substrate for culture of corneal epithelial cells compared to FAM [83]. In addition, both FAM and DAM showed to be a promising delivery system for adipose tissue-derived mesenchymal stem cells [30]. Decellularization did not affect the limbal epithelial cells, but accelerated cell migration compared to FAM [85]. We recently demonstrated that although both FAM and DAM inhibit the growth of some standard strain bacteria, these membranes are not able to protect entirely the burn patients against MDR bacteria [86] (Fig. 3a). Several decellularization agents such as Sodium dodecyl sulphate (SDS), triton X-100, dispase, thermolysin, urea, ammonia, EDTA and trypsin have been used to decellularize AM for soft tissue engineering uses [13,29,67,87–89]. Almost all of the decellularization procedures are detergent-, enzyme- or mechanical-based technique or a combination thereof [13,29,67,90] with various ranges of success. SDS is an ionic detergent. SDS has been widely used in decellularization of organs, as this detergent lyse cells with a minor damage to extracellular matrix (ECM). Tissue damage of SDS is dose dependent. In high concentration, SDS causes excessive tissue damage [91,92]. Triton X-100 is a nonionic detergent that can degrade lipid– lipid and lipid–protein interactions. So, this detergent has fewer damage effects on ECM compared to SDS [93]. Some researchers used a combination of SDS and triton X-100 to achieve better decellularization with intact ECM [94]. Urea is easily available decellularization agent. Urea detaches epithelial cells through solubilizing proteins [95]. Decellularization of AM with urea is very simple and fast. Treatment of AM with 5M ice-cold urea for 5 min showed to completely remove epithelial cells, while maintained ECM components and various growth factors [96]. Ethanol is another easily available decellularization agent. Decellularization with ethanol is very fast and safe. Ethanol is not a potent decellularization agent. So, successful decellularization can be achieved by further aggressive scrapping after ethanol treatment [96]. Decellularization of AM with 20% ethanol for 30 s, followed with aggressive scrapping revealed successful decellularization of AM with maintained ECM composition, intact basement membrane and growth factors expression. Ethanol cannot completely remove epithelial cells, so some epithelial remnants may remain after decellularization [96]. Presence of epithelial remnants may cause

Please cite this article in press as: M. Gholipourmalekabadi, et al., How preparation and preservation procedures affect the properties of amniotic membrane? How safe are the procedures?, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.005

JBUR 5867 No. of Pages 18

8

burns xxx (2019) xxx –xxx

micro- and nano-roughness on surface of DAM and thereby may better support proliferation and adhesion of seeded cells [97,98]. Trypsin is another decellularization agent. This enzyme detaches the cells and denude organs. However, trypsin can further degrade ECM proteins and growth factors in higher concentration or exposure time, as this enzyme is a protease [93]. Trypsin (0.1% to 0.25% at 37  C for 30 min) is often used together with ethylenediaminetetraacetic acid (EDTA). EDTA maintains activity of trypsin and also weaken cell attachment

to ECM by chelating calcium and magnesium [93,99]. 0.25% trypsin–EDTA showed to maintain some ECM components such as collagen types IV and VII, and laminin-5. Higher concentration of trypsin or treatment time can degrade ECM of AM [96]. Treatment of AM with EDTA followed with mechanical scrapping is another decellularization technique to fabricate an inexpensive acellular AM with intact ECM [29,68]. Dispase and thermolysin are two proteases which are produced by Bacillus, a genus of gram-positive bacteria. These two enzymes disrupt cell-ECM interactions and detach cells

Fig. 4 – Decellularization of amniotic membrane by enzyme- (a) and chemical/mechanical- (b) based decellularization protocols. Please cite this article in press as: M. Gholipourmalekabadi, et al., How preparation and preservation procedures affect the properties of amniotic membrane? How safe are the procedures?, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.005

JBUR 5867 No. of Pages 18

burns xxx (2019) xxx –xxx

from cell culture plate and ECM and can be used as decellularization agents [68,100]. Dispase, as a protease, can damage collagen types IV and VII, fibronectin and laminin [13,101]. The rate of damage to ECM directly depends on concentration of dispase and treatment time [102]. In a study conducted by Hopkinson et al., it was revealed that basement membrane can be completely degraded after 10 min incubation and treatment with dispase II [68]. Dispase II may not be a safe decellularization agent, as this enzyme causes irreversible damages to ECM proteins and growth factors [102]. Nevertheless, some AM products decellularized by dispase II (5 min to 2 h treatment with 1.2 U/ml enzyme at 25  C) could get clinical use permission and are already used by surgeons [103,104]. Some successful results obtained from clinical use of AM decellularized by dispase II attracted the attention of researchers [104–106]. These findings may be due to better basement membrane exposure of dispase II treated AM compared to intact AM. Comparison between three decellularization agents (EDTA, dispase II and thermpolysin) alone and followed with mild, increased and most scrapping (using a commercially available Surgical-K sponge, Katena, Denville, NJ) showed various rates of decellularization, basement membrane and ECM damages. They reported that thermolysin effectively denudes AM with minimal destruction of basement membrane and ECM [68]. Saghizadeh et al. also reported a simple procedure for decellularization of AM using EDTA, NaOH and scrapping. They showed fully decellularization of AM with intact ECM [87]. The decellularization of AM can be determined by various examinations, but the most valuable examinations for this aim are staining with hematoxylin & eosin and DNA count [29,67]. Here, we describe two reproducible and well cited decellularization protocols in details (Fig. 4). Example 1: The following enzyme-based decellularization protocol was reported by Wilshaw et al. [67] (Fig. 4a). (1) Treat AM with 10 mM tris buffer (hypotonic) with pH 8.0 containing 0.1% EDTA, protease inhibitors, 10 KIU/mL aprotinin for 16 h at 4  C. (2) Replace tris buffer with new tris buffer containing 0.03% SDS and incubate for 24 h at 25  C. (3) After several washing with TBS (pH 7.6), treat tissue with DNase (50 U/mL) and RNase (1 U/mL) in a reaction buffer (pH 7.5) of 50 mM trishydrochloric acid (HCl), 50 mg/ml bovine serum albumin and 10 mM magnesium chloride for 3 h at 37  C. Agitate tissue gently. (4) Wash the samples with TBS and sterilize with 0.1%v/v peracetic acid in PBS (pH 7.2–7.4) for 3 h at 25  C. (5) Wash again the samples with TBS. The decellularized tissue is now ready for use or further process on preservation. Example 2: The following simple and cost-effective decellularization procedure was reported by Gholipourmalekabadi et al. [29] (Fig. 4b). This is a chemical/mechanical-based decellularization protocol. (1) Treat the isolated AM with 0.2% EDTA for 30 min at 37  C, (2) Treat the tissue with 0.5 M NaOH for 30 s. (3) Place the tissue in 5% ammonium chloride and shake vigorously and scrap to remove the remaining cells from tissue. (4) After final wash with PBS (3), transfer the AM to 20  C for 2 h, then 80  C for 12 h. (5) Freeze dry the decellularized tissue, seal with a double film package and sterile with gamma irradiation (25 kGy). The dehydrated AM can be stored in room temperature for 3 years.

9

4.2.3. Preservation: cryopreservation and tissue banking of amniotic membrane (CAM) Cryopreservation is the most common preservation method for AM. There are several published clinical data that guarantee the safety, effectiveness and advantages of this preservation method [13,107]. Several studies confirmed the potential of cryopreserved AM (CAM) in management of various skin wounds (such as burns, diabetic) and ophthalmology [77,108–110]. Both FAM (mounted on nitrocellulose backing paper stored in CPTES solution containing 2.5% chondroitin sulphate at +4  C) and CAM (in 50% glycerol stored at 80  C) accelerated cornea re-epithelialization and healing [111]. AM can be cryopreserved by glycerol or DMSO at 80  C for up to 12 months [38,65,66,69,111]. Cryopreservation of AM in 10% DMSO or 50% glycerol is a standard method for preservation of AM for tissue engineering and regenerative applications in the European Union [87,112]. It is important to use an additive such as glycerol rather than straight freezing the AM. It was shown that storage methods (straight or with glycerol additive) do not affect the histology structure, tensile strength and Young’s modulus of AM. The viability of the cells showed to significantly decrease in CAM samples compared to FAM, but glycerol CAM showed higher cell viability than straight freezing method. Tensile strength and Young’s modulus increased in longer storage time. Glycerol CAM also maintained bFGF secretion compared to straight frozen samples [113]. Storage time depends on storage temperature, as CAM in 20  C and 80  C can be stored for 4 weeks and 6 months [114]. It may be possible to store AM in 85% glycerol at 80  C for a long time. In this regard, Ravishanker et al. demonstrated that the AM frozen in 85% glycerol in normal saline at 80  C for 2.5 years are effective in treatment of superficial and superficial partial thickness burns [115]. Anticancer activity, angiogenic (mesodermal side) and antiangiogenic (epithelial side) property of AM can be maintained after 6 month cryopreservation of AM in freezing solution (70% PBS, 10% FBS and 10% DMSO in DMEM) at 80  C [116]. AM can maintain its antibacterial activity after cryopreservation at 80  C. Antimicrobial peptides present in AM endow it a strong and broad-spectrum antibacterial activity. Mao et al. [117] indicated that CAM maintains the expression of two antimicrobial peptides (human beta-defensins 2 and 3) after thawing in room temperature for 3–5 min and culture in DMEM supplemented with 10% FBS for 22 h in standard shaker incubator (37  C, 5% CO2 and 95% humidity). Nevertheless, it was reported that growth factors and cytokines lost during preservation of AM in DMSO [118]. However, Cooke et al. [119] showed that the structure and composition of AM is better conserved in cryopreservation method compared to freeze drying method. Both CAM and powdered AM showed higher rate of growth factors and protein release compared to LAM [120]. A viable and safe procedure for AM cryopreservation and banking is described step by step below [65,66,121]. Briefly, (1) Place and spread AM on nitrocellulose membrane (0.22 mm), epithelial side up. (2) Cut AM/nitrocellulose in a desired size and left AM to adhere to nitrocellulose. (3) Add 50 ml of freezing solution (~1 g sample/1 ml freezing solution) and store at 80  C. 50 ml freezing solution contains: 25 ml sterilized glycerol (autoclaved), 25 ml DMEM (or Roswell Park Memorial Institute

Please cite this article in press as: M. Gholipourmalekabadi, et al., How preparation and preservation procedures affect the properties of amniotic membrane? How safe are the procedures?, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.005

JBUR 5867 No. of Pages 18

10

burns xxx (2019) xxx –xxx

medium RPMI [122]) supplemented with phenol red, 3.3% L-glutamine, 100 mg/ml ciprofloxacin, 50 units/ml penicillin, 25 mg/ml gentamicin, and 0.5 mg/ml Amphotericin B. (4) One of the samples is randomly subjected for bacterial and fungal tests. For this purpose, 200 ml of bacteria/fungal medium (100 ml brain heart infusion medium and 100 ml of thioglycolate) is inoculated with 5 ml of samples, left for 21 days in 37  C to check the bacterial and fungal sterility. Discard the batch of AM if you observed any sign of microorganism growth. As described earlier in this article, the screening test for HIV should be repeated 3–4 month after placenta collection. Storage time depends on storage temperature and countryspecific regulations. It is reported that the cryopreserved AM gradually lost its quality (cell viability, growth factor content and structural stability) after 2–3 months [23,123,124]. (5) Cryopreserved AM should be thawed at 4C for 30 min and room temperature for 10 min before grafting and transferred to surgery room on ice.

4.2.4.

Preservation: air-dried amniotic membrane (AAM)

Air-drying is a very simple and fast method for preservation of AM. To prepare an air-dried AM (AAM), spread the isolated AM on a stainless steel (epithelial side down, spongy side up) under a sterile laminar flow (clean room) and left at 37  C for 24 h. Place AM in a double plastic bag and seal. The packaged AAM is now ready for further sterilization, if needed [107,125–127]. Gamma irradiation at 25 kGy is a validated sterilization method for AAM. A sample should be randomly selected for bacterial and fungal cell culture before and after sterilization process [125]. Although AAM was recommended by Singh et al. [126] as a very promising wound dressing with high stability and suitability for storage in various environmental conditions, some studies revealed that CAM has superior properties (such as better retained basement membrane with higher growth factors concentrations) than AAM in ophthalmology applications [107].

4.2.5.

Preservation: lyophilized amniotic membrane (LAM)

Lyophilization (freeze drying) is a reliable preservation method for long-term storage of AM [72,128,129], even at room temperature. Histological structure of lyophilized AM (LAM) remains unchanged, but it was shown that AM lost its total protein and some growth factors concentration during lyophilization compared to FAM and CAM [129–131]. However, no difference in in vivo cell survival was observed between LAM and CAM. In contrast, it was also reported that there is no difference between ECM components in LAM and CAM [128]. LAM has superior properties such as safety, higher graft take, longer shelf life, and is easily handled and transport compared to CAM. Gamma irradiation of LAM makes it a very safe product for soft tissue engineering [72,132,133]. Both CAM and LAM showed to have great potential in reconstruction of rabbit corneal defect [133]. LAM supports various cell growth and adhesion and can be served as a great cell delivery system [30,134]. LAM seeded with autologous epithelium and fibroblasts accelerated the healing of the alkali-injured rabbit ocular surface [134]. Therani et al. [51] revealed that cryopreservation and freeze-drying do not affect the antibacterial activity of the AM in comparison with FAM. According to their results, antibacterial activity of the AM is depending on the

bacterial strains (Fig. 3b). It was detected by Nakamura et al. [128] that LAM sterilized by gamma irradiation retained its morphological, physical and biological characteristics after freeze drying process, and in vivo study in ocular surface reconstruction of rabbit model was comparable to CAM. Trehalose is a disaccharide composed of two molecules of glucose and produced by a wide variety of organisms such as bacteria, fungi, plants and invertebrate animals to serve as an energy source and also protect the cells against dehydration [135]. It was shown that pre-treatment with 10% trehalose before freeze drying can highly protect the AM against the physical, biological, and morphological changes during freeze drying process [136]. Transparency and mechanical properties of AM can be improved by gradually dehydration at 4–8  C and cross-linking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccimide [137]. A simple and reliable lyophilization protocol is described step by step below [29,129]. Briefly, (1) Spread AM on a nitrocellulose membrane (0.22 mm), epithelial side up. (2) Cut the membrane in a desired size. (3) Transfer AM/nitrocellulose membrane to 80  C for 24 h. (4) Freeze dry the membrane for 24 h. (5) Place membrane in a double film pack. (6) Sterilize membrane by gamma irradiation at 25 kGy. (7) Store at room temperature until use. (8) For bacterial and fungal culture test, and second screening test for HIV, please see step#4 of cryopreservation protocol.

5.

Cross-linking of amniotic membrane

Nanofibrous collagen proteins in ECM of AM are stabilized by peptide bridges. Tissue damage cause increased release and activity of tissue collagenase. Tissue collagenase can degrade both collagen and peptide bridges and cause biodegradation of AM [138]. Despite many favorable characteristics of AM in soft tissue engineering, its fast degradation, fast detachment (due to tearing of the tissue at the suture sites) and low mechanical properties remains problematic. FAM, acellular LAM, LAM and CAM are degraded within 1, 1, 2 and 2–3 weeks, respectively [29,139]. Degradation rate of AM can decrease with crosslinking of tissue with an appropriate cross-linking agents. Several cross-linking agents such as glutaraldehyde (GA), Aluminium sulfate (Al2(SO4)3) and EDC are already used for this purpose with various rate of success [140]. Almost all of cross-linking agents have cytotoxicity effects in high concentrations. So, it is important to use a safe and effective cross-linker with optimal crosslinking concentration to guarantee molecular stability of AM and minimal its cytotoxicity effects. The samples should also be well washed to remove all the remained cross-linker agents from tissue [140,141]. EDC, as a zero-length cross-linker, cross-links proteinbased matrices through activation of carboxylic acid groups in amino acids and formation of bridges between proteins [142,143]. EDC does not alter molecular structure of proteins. Therefore, this cross-linking agent has higher biocompatibility compared to GA [144]. The presence of some amino acids in proteins affect the efficacy of crosslinking with EDC. It was reported that the proteins with higher lysine content are better

Please cite this article in press as: M. Gholipourmalekabadi, et al., How preparation and preservation procedures affect the properties of amniotic membrane? How safe are the procedures?, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.005

JBUR 5867 No. of Pages 18

burns xxx (2019) xxx –xxx

cross-linked with EDC and become resistant to degradation by proteases [145]. The AM cross-linked with GA showed to have high resistance to collagenase [139,146]. Despite several reports on use of EDC, this cross-linking agent has limited cross-linking efficacy. The AM cross-linked with EDC showed approximately 20% weight lost after 4 weeks treatment with matrix metalloproteinase at 37  C. The degradation rate of EDC-cross linked AM significantly decreased with the increase of lysine concentration in cross-linking system up to 30 mM [147]. Nevertheless, an alternative cross-linking agent such as GA is needed to achieve a highly stable AM. GA, as a non-zero-length cross-linker, is widely used as an efficient chemical cross-linker. GA directly joints the molecules together by covalent bonds and stabilizes tissue/ scaffolds against enzymatic degradation [141,148]. Cytotoxicity effects of GA depends on GA concentration in reaction system [149]. Although GA is known as a very toxic chemical agent in higher concentration [150], it is already used as crosslinker agent for some commercially available products [151]. Aldehyde groups formed following GA treatment can be capped by glycine [152]. Therefore, embedding GA crosslinked materials in glycine aqueous solutions can reduce the cytotoxicity effects of GA [141,152]. Treatment of AM (4  4 cm piece) with a cross-linking solution containing 0.1% GA/20% dextran for 30 min at 25  C increased biochemical and stability of AM with a degradation time of a couple months and makes it suitable for cornea reconstruction [139]. Hyper-dried AM cross-linked with 0.1% GA showed to stay on conjunctival defect of 74-year-old woman for 24-month [153]. Aluminum sulfate is another agent used for cross-linking of AM. Aluminum sulfate significantly increased biomechanical properties of AM, but the effects of this agent on biodegradation of AM was not reported [140].

6.

Sterilization of amniotic membrane

Sterilization is a critical step to minimal the risk of infection transmission by AM. Gamma irradiation and paracetic acid (PAA) are two commonly used sterilization agents for AM [148,154–157]. Finding a safe and appropriate dose of these agents is critical for successful sterilization and effective antibacterial, antiviral and antifungal activities [13,158]. Combination of freeze-drying and gamma irradiation is an effective preservation and sterilization method that has been widely used for AM [128,159]. It was revealed that gamma irradiation in different doses of 25–50 kGy does not affect the water absorption capacity, chemical and structural properties, and water vapor transmission rate of AM [160]. Another study, however, detected a significant decrease in growth factors concentrations after radiation of AM with higher doses of gamma rays (up to 50 kGy). Based on their results, freeze drying does not affect the growth factor concentration compared to fresh-frozen AM [161]. Djefal et al. [162] validated an appropriate and effective dose of gamma irradiation at 25 kGy for safe and fully sterilization of AM. Gamma irradiation in higher doses can cause destruction and degradation of all three main layers of AM [163]. Gamma irradiation at dose of 15 kGy did not affect the expression of growth factors in irradiated CAM

11

compared to non-irradiated FAM and CAM [164]. It was also demonstrated that gamma irradiation of glycerol preserved AM at doses  at 25 kGy does not affect the morphology and appearance of AM [165]. Mrázová et al. [166] compared the structural changes in frozen porcine skin xenografts, human skin allografts and human amnion tissues after sterilization with different doses of electron beam (15, 25, 50 kGy) and gamma rays (12.5, 25, 35, 50 kGy). They showed that both electron beam and gamma rays similarly changed the cells and extracellular matrix of tissues, significantly damaged the basement membrane. Electron beam radiation at the dose of 15 kGy caused disintegration of the epithelial basement membrane. Both radiation types at the dose of 25 kGy damaged the elastic and collagen fibers in the xenograft dermis at the dose of 25 kGy of both radiation type and elastic and collagen fibers. Almost all of the data obtained from relevant researches and clinical based studies proved and confirmed the safe and effective dose of gamma irradiation at 15 kGy and 25 kGy for CAM and LAM, respectively. It was shown that irradiation at 25 kGy could sterilize the AM and provide the microbiological safety for clinical use [167]. PAA, also known as peroxyacetic acid, is another effective sterilization agent that has been used for sterilization of various biomaterials [13,154]. PAA is an inorganic chemical peroxide with high antibacterial, antifungal, antiviral and anti-spore agent [168,169]. PAA is decomposed to acetic acid and oxygen peroxide and thus is not toxic for human cells [157]. PAA sterilization did not alter the structure and component (elastin, GAG, collagen, laminin and fibronectin) of AM when compared to gamma irradiation [67,163]. This is consistent with the results obtained by Lomas et al. [169], who showed that structure and component of ECM is maintained after sterilization of acellular human skin by PAA. Wilshaw et al. [67] successfully sterilized DAM with 0.1% PAA in PBS (pH 7.2–74, with no calcium and magnesium) for 3 h at 25  C. During the sterilization process, the samples were gently agitated to increase the effectiveness of sterilization. Then, the samples were washed three times to discard PAA from DAM. PAA-sterilized DAM supported the limbal epithelial stem cells growth and adhesion ex vivo [85]. In another study, the effects of supercritical carbon dioxide (SCCO2) alone and in combination with PAA on sterilization and structural damages of AM were evaluated. They showed that combination of SCCO2 with PAA successfully sterilized AM with minimal changes in tissue architecture, extracellular matrix components (type IV collagen, elastin and glycosaminoglycans) the biophysical properties of the tissue. They concluded that SCCO2 can be used as an excellent sterilization agent for preservation of AM, while preserve its biological attributes [170]. The use of PAA for sterilization of AAM and LAM was also recommended by Versen-Höynck et al. [163]. A combination of PAA and ethanol was suggested as an effective sterilization method for AM. Physicochemical and biological properties of AM were maintained after sterilization with PAA and ethanol [127]. Clinical use of PAA/ethanol as a sterilization solution for AM showed a promising result in treatment of corneal and conjunctival lesions [171].

Please cite this article in press as: M. Gholipourmalekabadi, et al., How preparation and preservation procedures affect the properties of amniotic membrane? How safe are the procedures?, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.005

JBUR 5867 No. of Pages 18

12

7.

burns xxx (2019) xxx –xxx

FDA regulation

In the United State, the regulations of cell and tissue based products were released by the Food and Drug Administration (FDA). Many attempts are being made by FDA to modify and revise the existing regulations for defining the best regulatory framework to cover all aspects of the cell and tissue products. This framework is included in the sections 351 and 361 of the Public Health Service (PHS), Act of 1944, and in Title 21 of the Federal Code of Federal Regulations of 2001, Part 1271, also referred to as 21 CFR Part 1271. Sections 351 and 361 of PHS define biological products and give FDA the authority to prevent the transmission of communicable diseases, respectively (FDA, 2017k) [172,173]. 21 CFR Part 1271 provide some outlines for regulations of human cells and tissues products (referred to as HCT/Ps; FDA, 2017d). HCT/Ps are defined as any products based on the cells and tissues such as transplantation, implantation, infusion, and other relevant products (FDA, 2017d); and tiered into three regulation classification systems (FDA, 2001c). i) Low risk cell and tissue based products: these products such as blood transfusion and organ transplant do not need FDA pre-approval (21 CFR Part 1270 (FDA, 2017c) or 21 CFR Part 1271.15 (FDA, 2017d). ii) Middle risk cell and tissue based products: these products, defined also to as 361 HCT/P products, are not subjected to pre-market FDA approval or clearance (21 CFR Part 1271.10; FDA, 2017d). The products are categorized into 361 HCT/P products according to the following criteria: (1) minimally manipulated, (2) homologous applications, (3) no systematic effects and no dependence to the living cells metabolic activity for their primary function, except for autologous applications, allogeneic applications in the first- or second-degree blood relative, or for reproductive goal (FDA, 2017d), and (4) no combination with another article (except for crystalloids, water or a sterilizing, preserving, or storage agents”. All these exemptions must not make clinical safety concerns. iii) High risk cell and tissue based products: these products (referred to as “biological products” or “351 products”) do not meet the criteria stated in middle risk cell and tissue based products, and must follow the regulations of both pre-market and post-market as medical devices, drugs, or biologics (21 CFR Part 1271.20; FDA, 2017d). FDA provides a general definition of “minimal manipulation” and “more than minimal manipulation” in 21 CFR under Part 1271. The definition of “minimal manipulation” depends on whether the relevant cells/tissues are structural tissues, those tissues which serve as a barrier or provide mechanical support within the body, such as skin, bone, blood vessels, AM or adipose tissue, or are considered as cells/nonstructural tissues, those that serve as predominantly metabolic or biochemical roles in the body, such as bone marrow aspirate, cord blood, pancreatic tissue and lymph nodes (FDA, 2014b). Minimal manipulation for structural tissues is defined as “processing that does not alter the original relevant characteristics of the tissue relating to the tissue’s utility for reconstruction, repair, or replacement” ((21 C.F.R. x 1271.3(f)(1)). In

these cases, for example, the changes in shape or size of structural tissues during manipulation with no significant changes in their physical composition and relevant functionality is considered as “minimal manipulation” (FDA, 2017d). Lyophilizing, grinding and packing of AM as powder alter the physical integrity of membrane and affect its relevant function as a membranous barrier, and therefore are considered as “more than minimal manipulation” (FDA, 2017n). Separation of structural tissues into components and cell isolation can be considered as “minimal manipulation”, if the separation process does not alter the original characteristics and function of tissue. For instance, separation of epidermis or connective tissues from skin for preparation of decellularized dermal grafts would be considered as minimally manipulated products, because the process retain the integrity and relevant function of these products (FDA, 2017d). In contrast, isolation of collagen from skin and cross-linking by chemical agents alter the strength and structural integrity and are defined as “more than minimal manipulation”. For non-structural tissues, FDA defined “minimal manipulation” as “processing that does not alter the relevant biological characteristics of cells or tissues” (FDA, 2017d). Metabolic activity, proliferation and differentiation capacity are the main relevant biological characteristics in this definition. It was also defined by FDA that grinding, cutting, shaping, freezing, lyophilization, soaking in antibiotic solution, sterilization by gamma irradiation and demineralization of bone products are considered as minimal manipulation [172–174]. In November 2017, the guidance was finalized and released as “Regulatory Consideration for Human Cells, Tissues, and Cellular and Tissue-Based Products: Minimal Manipulation and Homologous Use” (FDA, 2017m). The finalized guidance provides further definition and discussion to clarify whether some preparation and preservation procedures such as decellularization and cryopreservation for structural tissues would be qualified as “minimal manipulation” or “more than minimal manipulation” (FDA, 2017n). The term “homologous use” is defined by FDA in 21 CFR Part 1271 as “the repair, reconstruction, replacement, or supplementation of a recipient’s cells or tissues with an HCT/P that performs the same basic function or functions in the recipient as in the donor.” (FDA, 2017d). To determine the recognition of HCT/P for homologous uses, FDA examines the statements released by the manufacturer such as labelling, marketing and others. Uses of dermis as dural patch and cartilage in the bladder are some examples of non-homologous uses. The use of AM for treatment of corneal defects is qualified as a structural homologous use, while the application of AM in repair of bone defects is considered as structural nonhomologous use [172–174]. It is very important and would be cost-benefit if manufacturers prove that their product meet the definition of a 361 HCT/P under FDA’s regulations. Manufacturers and sponsors are allowed to self-determine whether their HCT/ Ps are qualified as 361 HCT/Ps. In some cases, it is easy to define the category of the products based on “minimal manipulation” and “homologous use”. But in some other products, the manufactures are recommended to seek advice from FDA agency prior to marketing about the eligibility of their products to be categorized as a 361 HCT/P. As mentioned above, the FDA

Please cite this article in press as: M. Gholipourmalekabadi, et al., How preparation and preservation procedures affect the properties of amniotic membrane? How safe are the procedures?, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.005

JBUR 5867 No. of Pages 18

burns xxx (2019) xxx –xxx

determination depends on the claims and statements released by manufacturers about a specific product. A claim may lead to FDA to conclude non-homologous use of product, but a minor revision of claim may alter the FDA decision and qualify the same product as a 361 HCT/P. For example, the use of dermal product to cover/protect a tendon would be qualified as a homologous use, while, the use of the same dermal graft to replace/repair a tendon would be considered as a nonhomologous use. Therefore, a manufacturer should be very careful about the precise claims made for each product and is recommended to get FDA’s advice for each product before going to market.

8.

Conclusion

AM has many interesting biological characteristic that make it an excellent candidate for tissue engineering and regenerative medicine. This membrane is used in various forms of fresh, decellularize, air-dried, freeze dried, cryopreserved with or without sterilization and crosslinking. As we described in this article, preparation, preservation, sterilization and crosslinking can significantly affect the physico-chemical biological properties of AM. It is very important to set up an optimal preparation and preservation methods with minimal destruction of ECM and growth factors lost. Although the AM prepared with almost all of these procedures got permission in clinical uses, but there is still no a completely safe method to produce an intact preserved AM for a long time. Recent advances in tissue engineering and regenerative medicine may able us to develop an ideal procedure for this purpose.

Conflict of interest The authors have no conflict of interest to disclose.

REFERENCES

[1] Ilic D, Vicovac L, Nikolic M, Ilic EL. Human amniotic membrane grafts in therapy of chronic non-healing wounds. Br Med Bull 2016;ldv053. [2] Davis JS. Skin transplantation. Johns Hopkins Hospital Reports. 1910;15:307–96. [3] Litwiniuk M, Grzela T. Amniotic membrane: new concepts for an old dressing. Wound Repair Regen 2014;22:451–6. [4] Schulze U, Hampel U, Sel S, Goecke TW, Thäle V, Garreis F, et al. Fresh and cryopreserved amniotic membrane secrete the trefoil factor family peptide 3 that is well known to promote wound healing. Histochem Cell Biol 2012;138:243–50. [5] Garfias Y, Zaga-Clavellina V, Vadillo-Ortega F, Osorio M, Jimenez-Martinez MC. Amniotic membrane is an immunosuppressor of peripheral blood mononuclear cells. Immunol Invest 2011;40:183–96. [6] Niknejad H, Peirovi H, Jorjani M, Ahmadiani A, Ghanavi J, Seifalian AM. Properties of the amniotic membrane for potential use in tissue engineering. Eur Cells Mater 2008;15:88–99. [7] John T. Human amniotic membrane transplantation. Ophthalmol Clin North Am 2003;16:43–65.

13

[8] Costa E, Murta JN. Amniotic membrane in ophthalmology. Amniotic membrane. Springer; 2015. p. 105–22. [9] Gholipourmalekabadi M, Samadikuchaksaraei A, Seifalian AM, Urbanska AM, Ghanbarian H, Hardy JG, et al. Silk fibroin/ amniotic membrane 3D bi-layered artificial skin. Biomed Mater 2018;13:035003. [10] Gholipourmalekabadi M, Seifalian AM, Urbanska AM, Omrani MD, Hardy JG, Madjd Z, et al. 3D protein-based bilayer artificial skin for the guided scarless healing of third-degree burn wounds in vivo. Biomacromolecules 2018;19:2409–22. [11] Liu J, Sheha H, Fu Y, Liang L, Tseng SC. Update on amniotic membrane transplantation. Expert Rev Ophthalmol 2010;5:645–61. [12] Gholipourmalekabadi M, Chauhan NPS, Farhadihosseinabad B, Samadikuchaksaraei A. Human amniotic membrane as a biological source for regenerative medicine. Perinatal tissuederived stem cells. Springer; 2016. p. 81–105. [13] Riau AK, Beuerman RW, Lim LS, Mehta JS. Preservation, sterilization and de-epithelialization of human amniotic membrane for use in ocular surface reconstruction. Biomaterials 2010;31:216–25. [14] Hermans MH. Preservation methods of allografts and their (lack of) influence on clinical results in partial thickness burns. Burns 2011;37:873–81. [15] Bourne GL. The microscopic anatomy of the human amnion and chorion. Am J Obstet Gynecol 1960;79:1070–3. [16] Mamede A, Carvalho M, Abrantes A, Laranjo M, Maia C, Botelho M. Amniotic membrane: from structure and functions to clinical applications. Cell Tissue Res 2012;349:447–58. [17] Pollard SM, Aye N, Symonds E. Scanning electron microscope appearances of normal human amnion and umbilical cord at term. BJOG Int J Obstet Gynaecol 1976;83:470–7. [18] King BF. Distribution and characterization of anionic sites in the basal lamina of developing human amniotic epithelium. Anat Rec 1985;212:57–62. [19] Akashi T, Miyagi T, Ando N, Suzuki Y, Nemoto T, Eishi Y, et al. Synthesis of basement membrane by gastrointestinal cancer cell lines. J Pathol 1999;187:223–8. [20] Diaz-Prado S, Muiños-Lopez E, Fuentes-Boquete I, de Toro FJ, Garcia FJB. Human amniotic membrane: a potential tissue and cell source for cell therapy and regenerative medicine. Emerging trends in cell and gene therapy. Springer; 2013. p. 55–78. [21] Epstein FH, Parry S, Strauss JF. Premature rupture of the fetal membranes. N Engl J Med 1998;338:663–70. [22] Fairbairn N, Randolph M, Redmond R. The clinical applications of human amnion in plastic surgery. J Plast Reconstr Aesthetic Surg 2014;67:662–75. [23] Koizumi N, Inatomi T, Sotozono C, Fullwood NJ, Quantock AJ, Kinoshita S. Growth factor mRNA and protein in preserved human amniotic membrane. Curr Eye Res 2000;20:173–7. [24] Gholipourmalekabadi M, Khosravimelal S, Nokhbedehghan Z, Sameni M, Jajarmi V, Urbanska AM, et al. Modulation of hypertrophic scar formation using amniotic membrane/ electrospun silk fibroin bilayer membrane in a rabbit ear model. ACS Biomater Sci Eng 2019;5:1487–96. [25] Solomon A, Rosenblatt M, Monroy D, Ji Z, Pflugfelder SC, Tseng SC. Suppression of interleukin 1a and interleukin 1b in human limbal epithelial cells cultured on the amniotic membrane stromal matrix. Br J Ophthalmol 2001;85:444–9. [26] Olive V, Cuzin F. The spermatogonial stem cell: from basic knowledge to transgenic technology. Int J Biochem Cell Biol 2005;37:246–50. [27] Gholipourmalekabadi M, Bandehpour M, Mozafari M, Hashemi A, Ghanbarian H, Sameni M, et al. Decellularized human amniotic membrane: more is needed for an efficient dressing for protection of burns against antibiotic-resistant bacteria isolated from burn patients. Burns 2015;41:1488–97.

Please cite this article in press as: M. Gholipourmalekabadi, et al., How preparation and preservation procedures affect the properties of amniotic membrane? How safe are the procedures?, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.005

JBUR 5867 No. of Pages 18

14

burns xxx (2019) xxx –xxx

[28] Higa K, Shimmura S, Shimazaki J, Tsubota K. Hyaluronic acid-CD44 interaction mediates the adhesion of lymphocytes by amniotic membrane stroma. Cornea 2005;24:206–12. [29] Gholipourmalekabadi M, Mozafari M, Salehi M, Seifalian A, Bandehpour M, Ghanbarian H, et al. Development of a costeffective and simple protocol for decellularization and preservation of human amniotic membrane as a soft tissue replacement and delivery system for bone marrow stromal cells. Adv Healthc Mater 2015;4:918–26. [30] Gholipourmalekabadi M, Sameni M, Radenkovic D, Mozafari M, Mossahebi-Mohammadi M, Seifalian A. Decellularized human amniotic membrane: how viable is it as a delivery system for human adipose tissue-derived stromal cells? Cell Prolif 2016;49:115–21. [31] Samadikuchaksaraei A, Mehdipour A, Habibi Roudkenar M, Verdi J, Joghataei MT, As’ adi K, et al. A dermal equivalent engineered with TGF-b3 expressing bone marrow stromal cells and amniotic membrane: cosmetic healing of full-thickness skin wounds in rats. Artif Organs 201640:. [32] Niknejad H, Paeini-Vayghan G, Tehrani F, Khayat-Khoei M, Peirovi H. Side dependent effects of the human amnion on angiogenesis. Placenta 2013;34:340–5. [33] Milan PB, Lotfibakhshaiesh N, Joghataie M, Ai J, Pazouki A, Kaplan D, et al. Accelerated wound healing in a diabetic rat model using decellularized dermal matrix and human umbilical cord perivascular cells. Acta Biomater 2016;45:234–46. [34] Hao Y, Ma DH-K, Hwang DG, Kim W-S, Zhang F. Identification of antiangiogenic and antiinflammatory proteins in human amniotic membrane. Cornea 2000;19:348–52. [35] Burgos H. Angiogenic factor from human term placenta. Purification and partial characterization. Eur J Clin Invest 1986;16:486–93. [36] Wolbank S, Hildner F, Redl H, Van Griensven M, Gabriel C, Hennerbichler S. Impact of human amniotic membrane preparation on release of angiogenic factors. J Tissue Eng Regen Med 2009;3:651–4. [37] Thomas-Virnig CL, Centanni JM, Johnston CE, He L-K, Schlosser SJ, Van Winkle KF, et al. Inhibition of multidrugresistant Acinetobacter baumannii by nonviral expression of hCAP-18 in a bioengineered human skin tissue. Mol Ther 2009;17:562–9. [38] Baradaran-Rafii A, Aghayan H-R, Arjmand B, Javadi M-A. Amniotic membrane transplantation. J Ophthalmic Vis Res 20072:. [39] Buhimschi IA, Jabr M, Buhimschi CS, Petkova AP, Weiner CP, Saed GM. The novel antimicrobial peptide b3-defensin is produced by the amnion: a possible role of the fetal membranes in innate immunity of the amniotic cavity. Am J Obstet Gynecol 2004;191:1678–87. [40] King A, Paltoo A, Kelly R, Sallenave J-M, Bocking A, Challis J. Expression of natural antimicrobials by human placenta and fetal membranes. Placenta 2007;28:161–9. [41] Krisanaprakornkit S, Weinberg A, Perez CN, Dale BA. Expression of the peptide antibiotic human b-defensin 1 in cultured gingival epithelial cells and gingival tissue. Infect Immun 1998;66:4222–8. [42] Elsbach P, Weiss J. The bactericidal/permeability-increasing protein (BPI), a potent element in host-defense against gramnegative bacteria and lipopolysaccharide. Immunobiology 1993;187:417–29. [43] Spitznagel JK. Antibiotic proteins of human neutrophils. J Clin Invest 1990;86:1381. [44] Yoshio H, Tollin M, Gudmundsson GH, Lagercrantz H, Jörnvall H, Marchini G, et al. Antimicrobial polypeptides of human vernix caseosa and amniotic fluid: implications for newborn innate defense. Pediatr Res 2003;53:211–6.

[45] Kim J-Y, Park S-C, Lee J-K, Choi SJ, Hahm K-S, Park Y. Novel antibacterial activity of b 2-microglobulin in human amniotic fluid. PLoS One 2012;7:e47642. [46] Lehrer RI. Holocrine secretion of calprotectin: a neutrophilmediated defense against Candida albicans? J Lab Clin Med 1993;121:193. [47] Kim J-Y, Lee SY, Park S-C, Shin SY, Choi SJ, Park Y, et al. Purification and antimicrobial activity studies of the Nterminal fragment of ubiquitin from human amniotic fluid. Biochim Biophys Acta 2007;1774:1221–6. [48] Miller J, Michel J, Bercovici B, Argaman M, Sacks T. Studies on the antimicrobial activity of amniotic fluid. Am J Obstet Gynecol 1976;125:212–4. [49] Inge E, Talmi YP, Sigler L, Finkelstein Y, Zohar Y. Antibacterial properties of human amniotic membranes. Placenta 1991;12:285–8. [50] Lo V, Pope E. Amniotic membrane use in dermatology. Int J Dermatol 2009;48:935–40. [51] Tehrani FA, Ahmadiani A, Niknejad H. The effects of preservation procedures on antibacterial property of amniotic membrane. Cryobiology 2013;67:293–8. [52] Robson MC, Krizek TJ. The effect of human amniotic membranes on the bacteria population of infected rat burns. Ann Surg 1973;177:144. [53] Robson M, Krizek T. Clinical experiences with amniotic membranes as a temporary biologic dressing. Conn Med 1974;38:449. [54] Mohammadi AA, Jafari SMS, Kiasat M, Tavakkolian AR, Imani MT, Ayaz M, et al. Effect of fresh human amniotic membrane dressing on graft take in patients with chronic burn wounds compared with conventional methods. Burns 2013;39:349–53. [55] Magatti M, Cargnoni A, Silini AR, Parolini O. Epithelial and mesenchymal stromal cells from the amniotic membrane: both potent immunomodulators. Perinatal stem cells. Elsevier; 2018. p. 147–55. [56] Navas A, Magaña-Guerrero FS, Domínguez-López A, ChávezGarcía C, Partido , Graue-Hernández EO, et al. Antiinflammatory and anti-fibrotic effects of human amniotic membrane mesenchymal stem cells and their potential in corneal repair. Stem Cells Transl Med 2018;7:906–17. [57] Wu Z, Liu X, Yuan D, Zhao J. Human acellular amniotic membrane is adopted to treat venous ulcers. Exp Ther Med 2018;16:1285–9. [58] Farhadihosseinabadi B, Farahani M, Tayebi T, Jafari A, Biniazan F, Modaresifar K, et al. Amniotic membrane and its epithelial and mesenchymal stem cells as an appropriate source for skin tissue engineering and regenerative medicine. Artif Cells Nanomed Biotechnol 2018;46:431–40. [59] Shimmura S, Shimazaki J, Ohashi Y, Tsubota K. Antiinflammatory effects of amniotic membrane transplantation in ocular surface disorders. Cornea 2001;20:408–13. [60] Manuelpillai U, Moodley Y, Borlongan C, Parolini O. Amniotic membrane and amniotic cells: potential therapeutic tools to combat tissue inflammation and fibrosis? Placenta 2011;32: S320–5. [61] Kang JW, Koo HC, Hwang SY, Kang SK, Ra JC, Lee MH, et al. Immunomodulatory effects of human amniotic membranederived mesenchymal stem cells. J Vet Sci 2012;13:23–31. [62] Li H, Niederkorn JY, Neelam S, Mayhew E, Word RA, McCulley JP, et al. Immunosuppressive factors secreted by human amniotic epithelial cells. Invest Ophthalmol Vis Sci 2005;46:900–7. [63] López-Valladares MJ, Teresa Rodríguez-Ares M, Touriño , Gude F, Teresa Silva M, Couceiro J. Donor age and gestational age influence on growth factor levels in human amniotic membrane. Acta Ophthalmol 201088:. Aghayan HR, Goodarzi P, Baradaran-Rafii A, Larijani B, Moradabadi L, Rahim F, et al. Bacterial contamination of

Please cite this article in press as: M. Gholipourmalekabadi, et al., How preparation and preservation procedures affect the properties of amniotic membrane? How safe are the procedures?, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.005

JBUR 5867 No. of Pages 18

burns xxx (2019) xxx –xxx

[64] amniotic membrane in a tissue bank from Iran. Cell Tissue Bank 2013;14:401–6. [65] Madhavan HN, Priya K, Malathi J, Joseph PR. Preparation of amniotic membrane for ocular surface reconstruction. Indian J Ophthalmol 2002;50:227.  N. Preparation and preservation of amniotic [66] Dekaris I, Gabric membrane. Eye Banking: Karger Publishers; 2009. p. 97–104. [67] Wilshaw S-P, Kearney JN, Fisher J, Ingham E. Production of an acellular amniotic membrane matrix for use in tissue engineering. Tissue Eng 2006;12:2117–29. [68] Hopkinson A, Shanmuganathan VA, Gray T, Yeung AM, Lowe J, James DK, et al. Optimization of amniotic membrane (AM) denuding for tissue engineering. Tissue Eng C Methods 2008;14:371–81. [69] Dua HS. Amniotic membrane transplantation. Br J Ophthalmol 1999;83:748–52. [70] Mohan S, Budhiraja I, Saxena A, Khan P, Sachan S. Role of multilayered amniotic membrane transplantation for the treatment of resistant corneal ulcers in North India. Int Ophthalmol 2014;34:485–91. [71] Hanada K, Shimazaki J, Shimmura S, Tsubota K. Multilayered amniotic membrane transplantation for severe ulceration of the cornea and sclera. Am J Ophthalmol 2001;131:324–31. [72] Nakamura T, Inatomi T, Sekiyama E, Ang LP, Yokoi N, Kinoshita S. Novel clinical application of sterilized, freezedried amniotic membrane to treat patients with pterygium. Acta Ophthalmol 2006;84:401–5. [73] Connon CJ, Doutch J, Chen B, Hopkinson A, Mehta JS, Nakamura T, et al. The variation in transparency of amniotic membrane used in ocular surface regeneration. Br J Ophthalmol 2010;94:1057–61. [74] Hopkinson A, McIntosh RS, Tighe PJ, James DK, Dua HS. Amniotic membrane for ocular surface reconstruction: donor variations and the effect of handling on TGF-b content. Invest Ophthalmol Vis Sci 2006;47:4316–22. [75] Sabo M, Moore S, Yaakov R, Doner B, Patel K, Serena TE. Fresh hypothermically stored amniotic allograft in the treatment of chronic nonhealing ulcers: a prospective case series. Chronic Wound Care Manag Res 2018;5:1–4. [76] Gündüz K, Uçakhan ÖÖ, Kanpolat A, Günalp I. Nonpreserved human amniotic membrane transplantation for conjunctival reconstruction after excision of extensive ocular surface neoplasia. Eye 2006;20:351. [77] Kruse F, Joussen A, Rohrschneider K, You L, Sinn B, Baumann J, et al. Cryopreserved human amniotic membrane for ocular surface reconstruction. Graefes Arch Clin Exp Ophthalmol 2000;238:68–75. [78] Cananzi M, Atala A, De Coppi P. Stem cells derived from amniotic fluid: new potentials in regenerative medicine. Reprod Biomed Online 2009;18:17–27. [79] Magatti M, Vertua E, Cargnoni A, Silini A, Parolini O. The immunomodulatory properties of amniotic cells: the two sides of the coin. Cell Transplant 2018;27:31–44. [80] Uchida S, Inanaga Y, Kobayashi M, Hurukawa S, Araie M, Sakuragawa N. Neurotrophic function of conditioned medium from human amniotic epithelial cells. J Neurosci Res 2000;62:585–90. [81] Touhami A, Grueterich M, Tseng SC. The role of NGF signaling in human limbal epithelium expanded by amniotic membrane culture. Invest Ophthalmol Vis Sci 2002;43:987–94. [82] Shao C, Sima J, Zhang SX, Jin J, Reinach P, Wang Z, et al. Suppression of corneal neovascularization by PEDF release from human amniotic membranes. Invest Ophthalmol Vis Sci 2004;45:1758–62. [83] Koizumi N, Fullwood NJ, Bairaktaris G, Inatomi T, Kinoshita S, Quantock AJ. Cultivation of corneal epithelial cells on intact and denuded human amniotic membrane. Invest Ophthalmol Vis Sci 2000;41:2506–13.

15

[84] Koizumi N, Rigby H, Fullwood NJ, Kawasaki S, Tanioka H, Koizumi K, et al. Comparison of intact and denuded amniotic membrane as a substrate for cell-suspension culture of human limbal epithelial cells. Graefes Arch Clin Exp Ophthalmol 2007;245:123–34. [85] Shortt AJ, Secker GA, Lomas RJ, Wilshaw SP, Kearney JN, Tuft SJ, et al. The effect of amniotic membrane preparation method on its ability to serve as a substrate for the ex-vivo expansion of limbal epithelial cells. Biomaterials 2009;30:1056–65. [86] Gholipourmalekabadi M, Sameni M, Hashemi A, Zamani F, Rostami A, Mozafari M. Silver-and fluoride-containing mesoporous bioactive glasses versus commonly used antibiotics: activity against multidrug-resistant bacterial strains isolated from patients with burns. Burns 2016; 42:131–40. [87] Saghizadeh M, Winkler MA, Kramerov AA, Hemmati DM, Ghiam CA, Dimitrijevich SD, et al. A simple alkaline method for decellularizing human amniotic membrane for cell culture. PLoS One 2013;8:e79632. [88] Taghiabadi E, Nasri S, Shafieyan S, Firoozinezhad SJ, Aghdami N. Fabrication and characterization of spongy denuded amniotic membrane based scaffold for tissue engineering. Cell Journal (Yakhteh) 2015;16:476. [89] Zhang T, Yam GH-F, Riau AK, Poh R, Allen JC, Peh GS, et al. The effect of amniotic membrane de-epithelialization method on its biological properties and ability to promote limbal epithelial cell culture. Invest Ophthalmol Vis Sci 2013;54:3072–81. [90] He Q, Chen B, Wang Z, Li Q. The experimental study of culture in vitro of fibroblasts seeded onto human amnion extracellular matrix (HA-ECM). Zhonghua Zheng Xing Wai Ke Za Zhi. 2002;18:229–31. [91] Ott HC, Matthiesen TS, Goh S-K, Black LD, Kren SM, Netoff TI, et al. Perfusion-decellularized matrix: using nature’s platform to engineer a bioartificial heart. Nat Med 2008;14:213. [92] Guyette JP, Gilpin SE, Charest JM, Tapias LF, Ren X, Ott HC. Perfusion decellularization of whole organs. Nat Protoc 2014;9:1451. [93] Gilbert TW, Sellaro TL, Badylak SF. Decellularization of tissues and organs. Biomaterials 2006;27:3675–83. [94] Reing JE, Brown BN, Daly KA, Freund JM, Gilbert TW, Hsiong SX, et al. The effects of processing methods upon mechanical and biologic properties of porcine dermal extracellular matrix scaffolds. Biomaterials 2010;31:8626–33. [95] Bennion BJ, Daggett V. The molecular basis for the chemical denaturation of proteins by urea. Proc Natl Acad Sci 2003;100:5142–7. [96] Mehta J, Beuerman R, Thein Z, Tan D. Modification of human amniotic membrane as a carrier for stem cell transplantation. Invest Ophthalmol Vis Sci 2007;48:453. [97] Dolatshahi-Pirouz A, Pennisi CP, Skeldal S, Foss M, Chevallier J, Zachar V, et al. The influence of glancing angle deposited nano-rough platinum surfaces on the adsorption of fibrinogen and the proliferation of primary human fibroblasts. Nanotechnology 2009;20:095101. [98] Reich U, Mueller PP, Fadeeva E, Chichkov BN, Stoever T, Fabian T, et al. Differential fine-tuning of cochlear implant material–cell interactions by femtosecond laser microstructuring. J Biomed Mater Res B Appl Biomater 2008;87:146–53. [99] Tudorache I, Cebotari S, Sturz G, Kirsch L, Hurschler C, Hilfiker A, et al. Tissue engineering of heart valves: biomechanical and morphological properties of decellularized heart valves. J Heart Valve Dis 2007;16:567–73 discussion 74. [100] Lynch AP, Ahearne M. Strategies for developing decellularized corneal scaffolds. Exp Eye Res 2013;108:42–7.

Please cite this article in press as: M. Gholipourmalekabadi, et al., How preparation and preservation procedures affect the properties of amniotic membrane? How safe are the procedures?, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.005

JBUR 5867 No. of Pages 18

16

burns xxx (2019) xxx –xxx

[101] Espana EM, Romano AC, Kawakita T, Di Pascuale M, Smiddy R, Tseng SC. Novel enzymatic isolation of an entire viable human limbal epithelial sheet. Invest Ophthalmol Vis Sci 2003;44:4275–81. [102] Lim LS, Riau A, Poh R, Tan D, Beuerman R, Mehta J. Effect of dispase denudation on amniotic membrane. Mol Vis 2009;15:1962. [103] Ang LP, Tan DT, Cajucom-Uy H, Beuerman RW. Autologous cultivated conjunctival transplantation for pterygium surgery. Am J Ophthalmol 2005;139:611–9. [104] Ti S-E, Anderson D, Touhami A, Kim C, Tseng SC. Factors affecting outcome following transplantation of ex vivo expanded limbal epithelium on amniotic membrane for total limbal deficiency in rabbits. Invest Ophthalmol Vis Sci 2002;43:2584–92. [105] Ohno-Matsui K, Mori K, Ichinose S, Sato T, Wang J, Shimada N, et al. In vitro and in vivo characterization of iris pigment epithelial cells cultured on amniotic membranes. Mol Vis 2006;12:1022–32. [106] Yam H-F, Pang C-P, Fan DS-P, Fan B-J, Yu EY-W, Lam DS-C. Growth factor changes in ex vivo expansion of human limbal epithelial cells on human amniotic membrane. Cornea 2002;21:101–5. [107] Thomasen H, Pauklin M, Steuhl K-P, Meller D. Comparison of cryopreserved and air-dried human amniotic membrane for ophthalmologic applications. Graefes Arch Clin Exp Ophthalmol 2009;247:1691–700. [108] Werber B, Martin E. A prospective study of 20 foot and ankle wounds treated with cryopreserved amniotic membrane and fluid allograft. J Foot Ankle Surg 2013;52:615–21. [109] Georgiadis NS, Ziakas NG, Boboridis KG, Terzidou C, Mikropoulos DG. Cryopreserved amniotic membrane transplantation for the management of symptomatic bullous keratopathy. Clin Exp Ophthalmol 2008;36:130–5. [110] Kesting MR, Loeffelbein DJ, Steinstraesser L, Muecke T, Demtroeder C, Sommerer F, et al. Cryopreserved human amniotic membrane for soft tissue repair in rats. Ann Plast Surg 2008;60:684–91. [111] Adds PJ, Hunt CJ, Dart JK. Amniotic membrane grafts,“fresh” or frozen? A clinical and in vitro comparison. Br J Ophthalmol 2001;85:905–7. [112] Yazdanpanah G, Paeini-Vayghan G, Asadi S, Niknejad H. The effects of cryopreservation on angiogenesis modulation activity of human amniotic membrane. Cryobiology 2015;71:413–8. [113] Wagner M, Walter P, Salla S, Johnen S, Plange N, Rütten S, et al. Cryopreservation of amniotic membrane with and without glycerol additive. Graefes Arch Clin Exp Ophthalmol 2018;1–10. [114] Ashraf NN, Siyal NA, Sultan S, Adhi MI. Comparison of efficacy of storage of amniotic membrane at-20 and80 degrees centigrade. J Coll Physicians Surg Pak 2015;25:264–7. [115] Ravishanker R, Bath A, Roy R. “Amnion Bank”—the use of long term glycerol preserved amniotic membranes in the management of superficial and superficial partial thickness burns. Burns 2003;29:369–74. [116] Modaresifar K, Azizian S, Zolghadr M, Moravvej H, Ahmadiani A, Niknejad H. The effect of cryopreservation on anti-cancer activity of human amniotic membrane. Cryobiology 2017;74:61–7. [117] Mao Y, Hoffman T, Singh-Varma A, Duan-Arnold Y, Moorman M, Danilkovitch A, et al. Antimicrobial peptides secreted from human cryopreserved viable amniotic membrane contribute to its antibacterial activity. Sci Rep 2017;7:13722. [118] Hennerbichler S, Reichl B, Pleiner D, Gabriel C, Eibl J, Redl H. The influence of various storage conditions on cell viability in amniotic membrane. Cell Tissue Bank 2007;8:1–8.

[119] Cooke M, Tan E, Mandrycky C, He H, O’Connell J, Tseng S. Comparison of cryopreserved amniotic membrane and umbilical cord tissue with dehydrated amniotic membrane/ chorion tissue. J Wound Care 2014;23:465–76. [120] Russo A, Bonci P, Bonci P. The effects of different preservation processes on the total protein and growth factor content in a new biological product developed from human amniotic membrane. Cell Tissue Bank 2012;13:353–61. [121] Qureshi IZ, Fareeha A, Khan WA. Technique for processing and preservation of human amniotic membrane for ocular surface reconstruction. World Acad Sci Eng Technol 2010;69:763–6. [122] Laurent R, Nallet A, Obert L, Nicod L, Gindraux F. Storage and qualification of viable intact human amniotic graft and technology transfer to a tissue bank. Cell Tissue Bank 2014;15:267–75. [123] Kubo M, Sonoda Y, Muramatsu R, Usui M. Immunogenicity of human amniotic membrane in experimental xenotransplantation. Invest Ophthalmol Vis Sci 2001;42:1539–46. [124] Sippel KC, Ma JJ, Foster CS. Amniotic membrane surgery. Curr Opin Ophthalmol 2001;12:269–81. [125] Marsit N, Dwejen S, Saad I, Abdalla S, Shaab A, Salem S, et al. Substantiation of 25 kGy radiation sterilization dose for banked air dried amniotic membrane and evaluation of personnel skill in influencing finished product bioburden. Cell Tissue Bank 2014;15:603–11. [126] Singh R, Gupta P, Kumar P, Kumar A, Chacharkar M. Properties of air dried radiation processed amniotic membranes under different storage conditions. Cell Tissue Bank 2003;4:95–100. [127] von Versen-Hoeynck F, Steinfeld AP, Becker J, Hermel M, Rath W, Hesselbarth U. Sterilization and preservation influence the biophysical properties of human amnion grafts. Biologicals 2008;36:248–55. [128] Nakamura T, Yoshitani M, Rigby H, Fullwood NJ, Ito W, Inatomi T, et al. Sterilized, freeze-dried amniotic membrane: a useful substrate for ocular surface reconstruction. Invest Ophthalmol Vis Sci 2004;45:93–9. [129] Rodríguez-Ares MT, López-Valladares MJ, Tourino , Vieites B, Gude F, Silva MT, et al. Effects of lyophilization on human amniotic membrane. Acta Ophthalmol 2009;87:396–403. [130] Lim LS, Poh RW, Riau AK, Beuerman RW, Tan D, Mehta JS. Biological and ultrastructural properties of acelagraft, a freeze-dried g-irradiated human amniotic membrane. Arch Ophthalmol 2010;128:1303–10. [131] Mehta J, Riau A, Tan D, Beuerman R. Analysis of matrix proteins, growth factors and membrane surface in commercial available freeze-dried amniotic membrane. Invest Ophthalmol Vis Sci 2008;49:5745. [132] Ahn J-I, Jang I-K, Lee D-H, Seo Y-K, Yoon H-H, Shin Y-H, et al. A comparison of lyophilized amniotic membrane with cryopreserved amniotic membrane for the reconstruction of rabbit corneal epithelium. Biotechnol Bioprocess Eng 2005;10:262–9. [133] Libera RD, GBd Melo, AdS Lima, Haapalainen EF, Cristovam P, Gomes JÁP. Assessment of the use of cryopreserved x freezedried amniotic membrane (AM) for reconstruction of ocular surface in rabbit model. Arq Bras Oftalmol 2008;71:669–73. [134] Jang IK, Ahn JI, Shin JS, Kwon YS, Ryu YH, Lee JK, et al. Transplantation of reconstructed corneal layer composed of corneal epithelium and fibroblasts on a lyophilized amniotic membrane to severely alkali-burned cornea. Artif Organs 2006;30:424–31. [135] Guo N, Puhlev I, Brown DR, Mansbridge J, Levine F. Trehalose expression confers desiccation tolerance on human cells. Nat Biotechnol 2000;18:168. [136] Nakamura T, Sekiyama E, Takaoka M, Bentley AJ, Yokoi N, Fullwood NJ, et al. The use of trehalose-treated freeze-dried

Please cite this article in press as: M. Gholipourmalekabadi, et al., How preparation and preservation procedures affect the properties of amniotic membrane? How safe are the procedures?, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.005

JBUR 5867 No. of Pages 18

burns xxx (2019) xxx –xxx

[137]

[138] [139]

[140]

[141]

[142]

[143]

[144]

[145]

[146]

[147]

[148]

[149]

[150]

[151]

[152]

[153]

[154]

[155]

[156]

amniotic membrane for ocular surface reconstruction. Biomaterials 2008;29:3729–37. Tanaka Y, Kubota A, Yokokura S, Uematsu M, Shi D, Yamato M, et al. Optical mechanical refinement of human amniotic membrane by dehydration and cross-linking. J Tissue Eng Regen Med 2012;6:731–7. Slansky H, Dohlman C. Collagenase and the cornea. Surv Ophthalmol 1970;14:402. Spoerl E, Wollensak G, Reber F, Pillunat L. Cross-linking of human amniotic membrane by glutaraldehyde. Ophthalmic Res 2004;36:71–7. Sekar S, Sasirekha K, Krishnakumar S, Sastry T. A novel crosslinked human amniotic membrane for corneal implantations. Proc Inst Mech Eng H 2013;227:221–8. Lai J-Y, Ma DH-K. Glutaraldehyde cross-linking of amniotic membranes affects their nanofibrous structures and limbal epithelial cell culture characteristics. Int J Nanomed 2013;8:4157. Lai J-Y. Evaluation of cross-linking time for porous gelatin hydrogels on cell sheet delivery performance. J Mech Med Biol 2011;11:967–81. Lai J-Y, Li Y-T. Influence of cross-linker concentration on the functionality of carbodiimide cross-linked gelatin membranes for retinal sheet carriers. J Biomater Sci Polym Ed 2011;22:277–95. Lai J-Y, Li Y-T. Functional assessment of cross-linked porous gelatin hydrogels for bioengineered cell sheet carriers. Biomacromolecules 2010;11:1387–97. Ma L, Gao C, Mao Z, Zhou J, Shen J. Enhanced biological stability of collagen porous scaffolds by using amino acids as novel cross-linking bridges. Biomaterials 2004;25:2997–3004. Fujisato T, Tomihata K, Tabata Y, Iwamoto Y, Burczak K, Ikada Y. Cross-linking of amniotic membranes. J Biomater Sci Polym Ed 1999;10:1171–81. Lai J-Y, Wang P-R, Luo L-J, Chen S-T. Stabilization of collagen nanofibers with l-lysine improves the ability of carbodiimide cross-linked amniotic membranes to preserve limbal epithelial progenitor cells. Int J Nanomed 2014;9:5117. Jayakrishnan A, Jameela S. Glutaraldehyde as a fixative in bioprostheses and drug delivery matrices. Biomaterials 1996;17:471–84. Lai J-Y. Interrelationship between cross-linking structure, molecular stability, and cytocompatibility of amniotic membranes cross-linked with glutaraldehyde of varying concentrations. RSC Adv 2014;4:18871–80. Lai J-Y, DH-K Ma, Cheng H-Y, Sun C-C, Huang S-J, Li Y-T, et al. Ocular biocompatibility of carbodiimide cross-linked hyaluronic acid hydrogels for cell sheet delivery carriers. J Biomater Sci Polym Ed 2010;21:359–76. Bigi A, Cojazzi G, Panzavolta S, Rubini K, Roveri N. Mechanical and thermal properties of gelatin films at different degrees of glutaraldehyde crosslinking. Biomaterials 2001;22:763–8. Matsuda S, Iwata H, Se N, Ikada Y. Bioadhesion of gelatin films crosslinked with glutaraldehyde. J Biomed Mater Res 1999;45:20–7. Kitagawa K, Okabe M, Hayashi A, Nikaido T. Combined use of a novel dried cross-linked amniotic membrane and tissue adhesive to conjunctival defect following multiple trabeculectomy. Toyama Med J 2009;20:1–3. Farrington M, Wreghitt T, Matthews I, Scarr D, Sutehall G, Hunt C, et al. Processing of cardiac valve allografts: 2. Effects of antimicrobial treatment on sterility, structure and mechanical properties. Cell Tissue Bank 2002;3:91–103. Hodde J, Hiles M. Virus safety of a porcine-derived medical device: evaluation of a viral inactivation method. Biotechnol Bioeng 2002;79:211–6. Morejón-Alonso L, Carrodeguas RG, García-Menocal JÁD, Pérez JAA, Manent SM. Effect of sterilization on the properties of CDHA-OCP-beta-TCP biomaterial. Mater Res 2007;10:15–20.

17

[157] Sikka RS, Fischer DA, Swiontkowski MF. Reprocessing singleuse devices: an orthopaedic perspective. JBJS 2005;87:450–7. [158] Munting E, Wilmart J-F, Wijne A, Hennebert P, Delloye C. Effect of sterilization on osteoinduction comparison of five methods in demineralized rat bone. Acta Orthop Scand 1988;59:34–8. [159] Bhatia M, Pereira M, Rana H, Stout B, Lewis C, Abramson S, et al. The mechanism of cell interaction and response on decellularized human amniotic membrane: implications in wound healing. Wounds A Compend Clin Res Pract 2007;19:207–17. [160] Singh R, Purohit S, Chacharkar M. Effect of high doses of gamma radiation on the functional characteristics of amniotic membrane. Radiat Phys Chem 2007;76:1026–30. [161] Paolin A, Trojan D, Leonardi A, Mellone S, Volpe A, Orlandi A, et al. Cytokine expression and ultrastructural alterations in fresh-frozen, freeze-dried and g-irradiated human amniotic membranes. Cell Tissue Bank 2016;17:399–406. [162] Djefal A, Tahtat D, Khodja AN, Bouzid SS, Remane N. Validation and substantiation of 25 kGy as sterilization dose for lyophilized human amnion membrane. Cell Tissue Bank 2007;8:9–12. [163] von Versen-Höynck F, Syring C, Bachmann S, Möller D. The influence of different preservation and sterilisation steps on the histological properties of amnion allografts—light and scanning electron microscopic studies. Cell Tissue Bank 2004;5:45–56. [164] Yatim RM, Kannan TP, Ab Hamid SS. Effect of gamma radiation on the expression of mRNA growth factors in glycerol cryopreserved human amniotic membrane. Cell Tissue Bank 2016;17:643–51. [165] Ab Hamid SS, Zahari NK, Yusof N, Hassan A. Scanning electron microscopic assessment on surface morphology of preserved human amniotic membrane after gamma sterilisation. Cell Tissue Bank 2014;15:15–24. [166] Mrázová H, Koller J, Kubišová K, Fujeríková G, Klincová E, Babal P. Comparison of structural changes in skin and amnion tissue grafts for transplantation induced by gamma and electron beam irradiation for sterilization. Cell Tissue Bank 2016;17:255–60. [167] Singh R, Purohit S, Chacharkar M, Bhandari P, Bath A. Microbiological safety and clinical efficacy of radiation sterilized amniotic membranes for treatment of seconddegree burns. Burns 2007;33:505–10. [168] Baldry M. The bactericidal, fungicidal and sporicidal properties of hydrogen peroxide and peracetic acid. J Appl Microbiol 1983;54:417–23. [169] Lomas R, Cruse-Sawyer J, Simpson C, Ingham E, Bojar R, Kearney J. Assessment of the biological properties of human split skin allografts disinfected with peracetic acid and preserved in glycerol. Burns 2003;29:515–25. [170] Wehmeyer JL, Natesan S, Christy RJ. Development of a sterile amniotic membrane tissue graft using supercritical carbon dioxide. Tissue Eng C Methods 2015;21:649–59. [171] von Versen-Höynck F, Hesselbarth U, Möller D. Application of sterilised human amnion for reconstruction of the ocular surface. Cell Tissue Bank 2004;5:57–65. [172] Burger S. Current regulatory issues in cell and tissue therapy. Cytotherapy 2003;5:289–98. [173] Yano K, Speidel AT, Yamato M. Four Food and Drug Administration draft guidance documents and the REGROW Act: a litmus test for future changes in human cell- and tissue-based products regulatory policy in the United States? J Tissue Eng Regen Med 2018;12:1579–93. [174] Shapiro JK, Wesoloski BJ. FDA’s regulatory scheme for human tissue. Human Tissue Regul 2007;11:9–12. [175] Azuara-Blanco A, Pillai C, Dua HS. Amniotic membrane transplantation for ocular surface reconstruction. Br J Ophthalmol 1999;83:399–402.

Please cite this article in press as: M. Gholipourmalekabadi, et al., How preparation and preservation procedures affect the properties of amniotic membrane? How safe are the procedures?, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.005

JBUR 5867 No. of Pages 18

18

burns xxx (2019) xxx –xxx

[176] Chen H-J, Pires RT, Tseng SC. Amniotic membrane transplantation for severe neurotrophic corneal ulcers. Br J Ophthalmol 2000;84:826–33. [177] Ishino Y, Sano Y, Nakamura T, Connon CJ, Rigby H, Fullwood NJ, et al. Amniotic membrane as a carrier for cultivated human corneal endothelial cell transplantation. Invest Ophthalmol Vis Sci 2004;45:800–6. [178] Jin CZ, Park SR, Choi BH, Lee K-Y, Kang CK, Min B-H. Human amniotic membrane as a delivery matrix for articular cartilage repair. Tissue Eng 2007;13:693–702. [179] Kosuga M, Takahashi S, Sasaki K, Enosawa S, Li X-K, Okuyama S, et al. Phenotype correction in murine mucopolysaccharidosis type VII by transplantation of human amniotic epithelial cells after adenovirus-mediated gene transfer. Cell Transplant 2000;9:687–92. [180] Mligiliche N, Endo K, Okamoto K, Fujimoto E, Ide C. Extracellular matrix of human amnion manufactured into tubes as conduits for peripheral nerve regeneration. J Biomed Mater Res 2002;63:591–600. [181] Mohammad J, Shenaq J, Rabinovsky E, Shenaq S. Modulation of peripheral nerve regeneration: a tissue-engineering approach. The role of amnion tube nerve conduit across a 1centimeter nerve gap. Plast Reconstr Surg 2000;105:660–6.

[182] Takashima S, Ise H, Zhao P, Akaike T, Nikaido T. Human amniotic epithelial cells possess hepatocyte-like characteristics and functions. Cell Struct Funct 2004;29:73–84. [183] Tsai S-H, Liu Y-W, Tang W-C, Zhou Z-W, Hwang C-Y, Hwang G-Y, et al. Characterization of porcine arterial endothelial cells cultured on amniotic membrane, a potential matrix for vascular tissue engineering. Biochem Biophys Res Commun 2007;357:984–90. [184] Tseng SC, Prabhasawat P, Barton K, Gray T, Meller D. Amniotic membrane transplantation with or without limbal allografts for corneal surface reconstruction in patients with limbal stem cell deficiency. Arch Ophthalmol 1998;116:431–41. [185] Gholipourmalekabadi M, Seifalian AM, Urbanska AM, Omrani MD, Hardy JG, Madjd Z, et al. 3D protein-based bilayer artificial skin for the guided scarless healing of third-degree burn wounds in vivo. Biomacromolecules 2018;19:2409–22. [186] Lafzi A, Abolfazli N, Faramarzi M, Eyvazi M, Eskandari A, Salehsaber F. Clinical comparison of coronally-advanced flap plus amniotic membrane or subepithelial connective tissue in the treatment of Miller’s class I and II gingival recessions: a split-mouth study. J Dent Res Dent Clin Dent Prospects 2016;10:162.

Please cite this article in press as: M. Gholipourmalekabadi, et al., How preparation and preservation procedures affect the properties of amniotic membrane? How safe are the procedures?, Burns (2019), https://doi.org/10.1016/j.burns.2019.07.005