Preparation Of Amniotic Membrane Research Articles

A Novel Technique of Amniotic Membrane Preparation Mimicking Limbal Epithelial Crypts Enhances the Number of Progenitor Cells upon Expansion.

We aimed to investigate whether a novel technique of human amniotic membrane (HAM) preparation that mimics the crypts in the limbus enhances the number of progenitor cells cultured ex vivo. The HAMs were sutured on polyester membrane (1) standardly, to obtain a flat HAM surface, or (2) loosely, achieving the radial folding to mimic crypts in the limbus. Immunohistochemistry was used to demonstrate a higher number of cells positive for progenitor markers p63α (37.56 ± 3.34% vs. 62.53 ± 3.32%, p = 0.01) and SOX9 (35.53 ± 0.96% vs. 43.23 ± 2.32%, p = 0.04), proliferation marker Ki-67 (8.43 ± 0.38 % vs. 22.38 ± 1.95 %, p = 0.002) in the crypt-like HAMs vs. flat HAMs, while no difference was found for the quiescence marker CEBPD (22.99 ± 2.96% vs. 30.49 ± 3.33 %, p = 0.17). Most of the cells stained negative for the corneal epithelial differentiation marker KRT3/12, and some were positive for N-cadherin in the crypt-like structures, but there was no difference in staining for E-cadherin and CX43 in crypt-like HAMs vs. flat HAMs. This novel HAM preparation method enhanced the number of progenitor cells expanded in the crypt-like HAM compared to cultures on the conventional flat HAM.

The Effect of Amniotic Tissue on Spinal Interventions: A Systematic Review.

Amniotic membrane tissue has been thought to potentiate healing in many soft tissue conditions. Specifically, recent studies have shown its therapeutic potential for treatment in the setting of spinal pathologies. The purpose of this study is to thoroughly review the existing scientific literature and evidence concerning the clinical use of amniotic membrane-derived biologic agents on postoperative outcomes following spinal surgery. A systematic review was conducted following preferred reporting items for systematic reviews and meta-analyses guidelines using PubMed, Embase, and Cochrane databases up to December 2020 to identify animal and clinical studies examining the therapeutic potential for amniotic membrane tissue in the setting of spinal pathologies (including disc herniation, prevention of epidural fibrosis, and spinal fusion). Studies were broken down into 2 categories: experimental model type and the type of amnion product being analyzed. A total of 12 studies (4 clinical studies and 8 studies utilizing animal models) met inclusion criteria. Additionally, the major types of amnion product were divided into cryopreserved/freeze-dried amniotic membrane, human amniotic fluid, human amniotic membrane, cross-linked amniotic membrane, and amnion-derived epithelial cells. While heterogeneity of study design precludes definitive specific results reporting, most studies showed positive benefits on healing/outcomes with amniotic augmentation. Specifically, amnion products have shown promising effects in reducing epidural adhesions and scar tissue after spine surgery, improving spinal fusion rate and postoperative pain scores, and promoting better functional outcomes after spine surgery. A review of the limited number of reported studies revealed a wide variety of amniotic membrane preparations, treatment regimens, and indications, which limit definitive conclusions. To date, while there is no definitive clinical proof that amniotic tissues enhance tissue repair or regeneration, the aggregate results demonstrate promising basic science and outcomes potential in spinal surgery. Further study is warranted to determine whether this application is appropriate in the clinical setting. This systematic review provides a summary of the existing literature regarding the use of amniotic membrane preparations, treatment regimens, and indications within spinal surgery. With the growing popularity and utilization of biologic agents such as amniotic membrane-derived products in orthopedic and neurologic surgery, this systematic review gives physicians a concise summary on the outcomes and indications associated with amniotic membrane products.

A systematic review on clinical outcomes of human amniotic membrane preparations in the management of venous leg ulcers

Introduction: Venous leg ulcers are the commonest type of chronic lower leg ulcers worldwide. It impacts the patient’s quality of life significantly. Our objective was to assess current evidence onusing human amniotic membranes (HAM) in venous leg ulcer management. Methods: Google Scholar, PubMed, and the Cochrane library were utilized to search the following search terms (MeSH terms in PubMed) in the abstract field or in the title, “Amnion” OR “Placenta” AND “Varicose ulcer “OR “Stasis ulcer” OR “Chronic venous ulceration” in studies published until the 1st of March 2022. We used standard methods to assess the quality of the published articles. The articles thus included were cohort studies (both retrospective and prospective) and randomized control trials. Results: When the above criteria were used in the search, 12, 8, 15,6, and 4 citations were found in MEDLINE, Cochrane Library, Google Scholar, Embase, and Web of Science respectively. The 15 nonduplicate studies were screened with the inclusion and exclusion criteria, we selected 7 studies for this review. However, the amniotic membrane preparations used in these studies were not uniform. All randomized controlled trials (n=3) have concluded that there is an improvement in healed ulcer percentage at the end of the study in the interventional group when compared to the control group, which was statistically significant (p<0.05%). The percentage of ulcers that had healed at the end study was 60% in interventional groups of the above trials. One prospective study showed that the recurrence rate was less than 30% at a 3-year follow-up examination. We couldn't perform a meta-analysis due to study heterogeneity. Conclusion: Current scientific evidence indicates that amniotic membrane preparations can be utilized in promoting chronic forms of venous leg ulcers adjunctive to traditional treatment or as second-line therapy.

Injection of air to facilitate amniotic membrane preparation

Injection of air to facilitate amniotic membrane preparation

Amniotic Membrane Preparation Crucially Affects Its Broad-Spectrum Activity Against Uropathogenic Bacteria.

Urinary tract infections are among the most common bacterial infections in humans. Moreover, they are highly recurrent and increasingly often resistant to antibiotics. The antimicrobial properties of the amniotic membrane (AM), the innermost layer of fetal membranes, have been briefly reported in the literature, however, the results of published studies are often inconsistent and unclear; moreover, its effect on uropathogenic bacteria has not yet been investigated. Further, there is no data in the literature about the effect of AM preparation and storage on its antimicrobial properties. To examine the impact of several preparation procedures on the antimicrobial properties of AM, we prepared patches and homogenates of fresh (fAM) and cryopreserved (cAM) human AM and tested them on 14 selected Gram-positive and Gram-negative uropathogenic bacteria. By employing novel antimicrobial efficiency assays we showed that fAM and cAM homogenates have broad-spectrum antimicrobial activity against all here tested uropathogenic bacteria, except for Serratia marcescens. Moreover, they had a potent effect also on the multiple-resistant clinical strains of uropathogenic Escherichia coli. Interestingly, the patches of fAM and cAM had no antimicrobial effect on any of the tested strains. We therefore prepared and stored AM patches according to the standard procedure for clinical use in ophthalmology, which includes the cryopreservation of antibiotic-treated AM, and performed antimicrobial efficiency assays. Our findings suggest that the ultrastructure of AM patches could enable the retention of added antibiotics. In addition, we also prepared gentamicin-resistant uropathogenic E. coli strains, which confirmed that the antimicrobial effect of antibiotic-treated AM patches can be attributed to the antibiotic alone. To summarize, here we describe novel protocols for preparation and storage of AM to ensure the preservation of its antimicrobial factors. Moreover, we describe the mechanism of AM retention of antibiotics, based on which the AM could potentially be used as a drug delivery vehicle in future clinically applicable approaches.

Amniotic membrane in ophthalmology: properties, preparation, storage and indications for grafting-a review.

The use of amniotic membrane in ophthalmic surgery and other surgical procedures in the fields of dermatology, plastic surgery, genitourinary medicine and otolaryngology is on the increase. Furthermore, amniotic membrane and its epithelial and mesenchymal cells have broad use in regenerative medicine and hold great promise in anticancer treatment. Amniotic membrane is a rich source of biologically active factors and as such, promotes healing and acts as an effective material for wound dressing. Amniotic membrane supports epithelialization and exhibits anti-fibrotic, anti-inflammatory, anti-angiogenic and anti-microbial features. Placentas utilised in the preparation of amniotic membrane are retrieved from donors undergoing elective caesarean section. Maternal blood must undergo serological screening at the time of donation and, in the absence of advanced diagnostic testing techniques, 6months postpartum in order to cover the time window for the potential transmission of communicable diseases. Amniotic membrane is prepared by blunt dissection under strict aseptic conditions, then is typically transferred onto a nitrocellulose paper carrier, usually with the epithelial side up, and cut into multiple pieces of different dimensions. Amniotic membrane can be stored under various conditions, most often cryopreserved in glycerol or dimethyl sulfoxide or their mixture with culture medium or buffers. Other preservation methods include lyophilisation and air-drying. In ophthalmology, amniotic membrane is increasingly used for ocular surface reconstruction, including the treatment of persistent epithelial defects and non-healing corneal ulcers, corneal perforations and descemetoceles, bullous keratopathy, as well as corneal disorders with associated limbal stem cell deficiency, pterygium, conjunctival reconstruction, corneoscleral melts and perforations, and glaucoma surgeries.

Preparation of Amniotic Membrane and It’s Application in the Treatment of Skin Loss and Lyell's Syndrome (Toxic Epidermal Necrolysis): Current State and New Opportunities

Amniotic membrane is a good protection against bacterial infections, reduces losses of proteins, fluids and electrolytes, decreases pain from burns and accelerates healing. Due to its richness in nutrients and low immunogenicity, it is often used as a skin substitute. Human amnion may be prepared in tissue banks, as biostatic or biovital grafts. There are various methods of amniotic graft preparations developed to make them efficient in surgical treatment and prevent the risk of transmission of infectious diseases. This paper presents the current clinical application of human amnion in the treatment of skin losses, such as burns to the face and Lyell's syndrome (toxic epidermal necrolysis). It also describes new applications of fetal membranes for other diseases. Placental grafts are often an alternative therapy in the situations when standard treatment does not yield desirable effects. Such grafts are especially beneficial in comparison to many other bioactive therapies, including low cost, easy manipulation, capability of promoting cell migration and proliferation and stimulation of stem cell activity.

Substantiation of 25 kGy radiation sterilization dose for banked air dried amniotic membrane and evaluation of personnel skill in influencing finished product bioburden

Preparation of amniotic membrane (AM) by air drying method followed by radiation sterilization is simple and valuable approach; sterility and quality of the final AM product are depending on the quality management system at the tissue bank. Validation and substantiation of radiation sterilization dose (RSD) for tissue allografts is an essential step for the development and validation of the standard operating procedures (SOP). Application of SOP is perfectly relying on trained staff. Skills differences among personnel involved in AM preparation could have an effect on microbiological quality of the finished product and subsequently on the RSD required. AM were processed by four different couples of the tissue bank technicians. The AM grafts were randomly selected and subjected to bioburden test to validate and substantiate the 25 kGy RSD. Bioburden test for AM grafts were also useful to evaluate the skill of the tissue bank technicians and thus, to validate the current SOP for air dried AM. Moreover, the effect of placental source on bioburden counts on AM grafts was assessed. Substantiation of the 25 kGy RSD at a sterility assurance level of 10−1, and sample item portion = 1, was carried out using Method VDmax25 of the International Organization for Standardization, document no. 11137-2 (ISO in Sterilization of healthcare products—radiation—part 2: establishing the sterilization dose, Method VDmax—substantiation of 25 kGy or 15 kGy as the sterilization dose, International Standard Organization, 2006). The results showed that there were no significant differences in the bioburdens of the four batches (α = 1 %), this means no significant differences in the skill of the four couples of the tissue bank technicians in terms of their ability to process AM according to the air dried AM SOP. The 25 kGy RSD was validated and substantiated as a valid sterilization dose for the AM prepared with the current established SOP at the Biotechnology Research Center experimental tissue bank. The donor’s type of delivery, normal or caesarean, showed no significant effect on the levels of microbial counts on the tested AMs (α = 1 %).

A Simple Alkaline Method for Decellularizing Human Amniotic Membrane for Cell Culture

Human amniotic membrane is a standard substratum used to culture limbal epithelial stem cells for transplantation to patients with limbal stem cell deficiency. Various methods were developed to decellularize amniotic membrane, because denuded membrane is poorly immunogenic and better supports repopulation by dissociated limbal epithelial cells. Amniotic membrane denuding usually involves treatment with EDTA and/or proteolytic enzymes; in many cases additional mechanical scraping is required. Although ensuring limbal cell proliferation, these methods are not standardized, require relatively long treatment times and can result in membrane damage. We propose to use 0.5 M NaOH to reliably remove amniotic cells from the membrane. This method was used before to lyse cells for DNA isolation and radioactivity counting. Gently rubbing a cotton swab soaked in NaOH over the epithelial side of amniotic membrane leads to nearly complete and easy removal of adherent cells in less than a minute. The denuded membrane is subsequently washed in a neutral buffer. Cell removal was more thorough and uniform than with EDTA, or EDTA plus mechanical scraping with an electric toothbrush, or n-heptanol plus EDTA treatment. NaOH-denuded amniotic membrane did not show any perforations compared with mechanical or thermolysin denuding, and showed excellent preservation of immunoreactivity for major basement membrane components including laminin α2, γ1-γ3 chains, α1/α2 and α6 type IV collagen chains, fibronectin, nidogen-2, and perlecan. Sodium hydroxide treatment was efficient with fresh or cryopreserved (10% dimethyl sulfoxide or 50% glycerol) amniotic membrane. The latter method is a common way of membrane storage for subsequent grafting in the European Union. NaOH-denuded amniotic membrane supported growth of human limbal epithelial cells, immortalized corneal epithelial cells, and induced pluripotent stem cells. This simple, fast and reliable method can be used to standardize decellularized amniotic membrane preparations for expansion of limbal stem cells in vitro before transplantation to patients.

Amniotic membrane as a tissue engineering substrate

AbstractAmniotic membrane (AM) is commonly used for cell culture and transplantation onto the ocular surface. AM serves the dual purpose of culture substrate and scaffold thus reducing cell manipulation. Crucially AM is believed to promote the retention of stem cell phenotypes essential for the long term restoration of functional tissue. As potential cellular therapies for corneal regeneration are being developed many of them continue to use AM. Its widespread usage in ophthalmology and extensive list of wound healing attributes has made AM a popular choice. Amniotic membrane is being used as a carrier for innovative research into the transplantation of non‐corneal cell types onto ocular surface burn models including mesenchymal stem cells and oral mucosal epithelium. A lack of standardisation in the preparation of amniotic membrane can cause considerable variation and some methods of preparation may reduce or enhance certain attributes. Various methods of processing including cross‐linking, denuding and freeze drying have sought to enhance amniotic membrane for tissue engineering purposes with varied levels of success.

Impact of human amniotic membrane preparation on release of angiogenic factors

Preserved amniotic membrane (AM) has been used in the field of ophthalmology and wound care due to its bacteriostatic, antiphlogistic, protease-inhibiting, re-epithelialization, wound-protecting and scar formation-reducing properties. Typically, AM is applied after banking in a glycerol-preserved or freeze-dried state. Cell viabilities in different forms of preparation vary substantially, which in consequence may also be reflected in the amount and type of growth factors released from the preserved material. Therefore, we characterized the angiogenic factor (AF) profile released from different AM preparations. For this, medium was conditioned with non-preserved, glycerol- and cryo-preserved AM for 48 h, which was screened for AFs using a protein array system. In parallel, the preparations were tested for cell viability. Non-preserved as well as cryo-preserved AM maintained viabilities at 106.5 +/- 23.9% and 21.9 +/- 23.3%, respectively, whereas glycerol-preserved AM was found to be non-viable. Of the 20 investigated factors, high levels of angiogenin, GRO, IL-6/8, TIMP-1/2 and MCP-1 and low levels of EGF, IFNgamma, IGF-1, leptin, RANTES, TGFbeta1 and thrombopoietin were identified to be secreted from non-preserved AM. Cryo-preserved AM secreted high levels of IL-8, intermediate levels of GRO and TIMP-1/2 but only low levels of angiogenin, IFNgamma, IL-6 and MCP-1 and no detectable EGF, IGF-1, leptin, RANTES, TGFbeta1 and thrombopoietin. After banking in glycerol, AM releases only minute amounts of TIMP-1/2. Along with viability, the AF profile of amniotic membrane largely depends on the preparation method applied for banking. This should be considered for selection of an AM product for a specific clinical application.

The effect of amniotic membrane preparation method on its ability to serve as a substrate for the ex-vivo expansion of limbal epithelial cells

Human amniotic membrane (HAM) is employed as a substrate for the ex-vivo expansion of limbal epithelial cells (LECs) used to treat corneal epithelial stem cell deficiency in humans. The optimal method of HAM preparation for this purpose is unknown. This study evaluated the ability of different preparations of stored HAM to serve as substrates for LEC expansion ex-vivo. The effect of removing the amniotic epithelial cells (decellularisation) from HAM prior to seeding of LECs, the effect of glycerol cryopreservation and the effect of peracetic acid (PAA) sterilization and antibiotic disinfection were evaluated using different HAM test groups. Human LECs were cultured on each preparation and the following outcomes were assessed: confluence of growth, cell density, cell morphology and expression of the putative LESC markers deltaN-p63alpha and ABCG2. Removing amniotic epithelial cells prior to seeding of LECs resulted in a higher percentage of confluence but a lower cell density than intact HAM suggesting that decellularisation does not increase proliferation, but rather that it facilitates migration of LECs resulting in larger cells. Decellularisation did not affect the percentage of cells expressing the putative LESC markers deltaN-p63alpha (≤4% in both intact and acellular groups) and ABCG2 (≤3% in both intact and acellular groups). Glycerol cryopreservation of HAM resulted in poor morphology and a low proportion of cells expressing deltaN-p63alpha (≤6%) and ABCG2 (≤8%). HAM frozen at −80 °C in Hank's Balanced Salt Solution (HBSS) was superior, demonstrating excellent morphology of cultured LECs and high levels of deltaN-p63alpha (≤68%) and ABCG2 (≤62%) expression ( p < 0.001). The use of PAA or antibiotics to decontaminate HAM does not appear to affect this function. The variables affecting the ability of HAM to serve as a substrate for LEC expansion ex-vivo are poorly understood. The use of glycerol as a cryoprotectant impairs this ability whereas simple frozen HAM appears to work extremely well for this purpose.

Biomechanical Characterization of Human Amniotic Membrane Preparations for Ocular Surface Reconstruction

Purpose: To investigate the tensile and elastic properties of both commercially available and experimental human amniotic membrane preparations. Method: Nine preparations of human amniotic membrane were studied. The four dry preparations were untreated (nonirradiated, n = 20), and gamma (n = 25), low-dose (AmbioDry^®, Okto Ophtho Inc., Costa Mesa, Calif., USA, n = 20) and high-dose (n = 20) electron beam sterilized. The same dry membranes were moistened with balanced salt solution (n = 20, 34, 20 and 20, respectively). The ninth group consisted of thawed medium-frozen amniotic membrane (AmnioGraft^®, Bio-Tissue Inc., Miami, Fla., USA, n = 20). The membranes were cut into thin strips, loaded on a gram range load sensor, and stretched incrementally to the point of rupture. The modulus of elasticity, displacement until rupture and maximum tolerated stress were recorded and compared. Results: The dry preparations exhibited higher moduli of elasticity when compared with the moist samples, with the low-dose electron beam-irradiated samples having the greatest mean modulus of elasticity overall and maintaining a high modulus of elasticity as a moist sample (p < 0.05). Moist nonirradiated preparations and thawed medium-frozen preparations stretched the farthest before rupture and experienced the greatest mean stresses at the point of rupture. While 3 of 4 membranes had greater stretch when moistened as compared to their dry counterparts, there was no difference in the membrane stiffness between dry and moistened low-dose electron beam-irradiated samples (p > 0.8). Conclusions: Low-dose electron beam-irradiated amnion appeared to maintain desirable elastic characteristics in transition from a dry to rehydrated state and may thus provide an easy-to-manipulate transplant tissue for ocular surface reconstruction. Moist nonirradiated and thawed medium-frozen tissues, however, may provide surgical advantages as they required greater forces to rupture.

Further evaluation of amniotic membrane banking for transplantation in ocular surface diseases.

Objective: To define the best conditions for amniotic membrane preparation, storage and banking in its use for corneal reconstruction. Methods: Amniotic membrane pieces were prepared under sterile conditions from placentas selected on the basis of donor medical and social history, serology, microbiological tests and histology. The pieces were kept at -140 degrees C but before grafting they were thawed and stored at 4 degrees C in RPMI medium, to have a preparation usable within 72 h. This procedure was validated by testing its therapeutic effectiveness in 25 patients 13 of which had corneal ulcers of various origin, 3 had sequelae of herpes simplex keratitis, 3 band keratopathy and 6 corneal stem cell deficiency due to chemical or thermal burns. Results: The preparation showed appreciable anti-inflammatory and analgesic effects. In the absence of corneal stem cell deficiency a stable re-epithelialisation was achieved in 15 out of 19 patients. When the limbus was lesioned, the amniotic membrane decreased vascularization and increased the number of corneal epithelial cells only in 1 of the 6 patients. No adverse reactions attributable to the tissue were recorded. Conclusions: A ready-to-use amniotic membrane preparation stored at 4 degrees C after cryopreservation has been tested in corneal reconstruction. Like the amniotic membrane thawed immediately before grafting, this preparation displayed full therapeutic effect in epithelial defects with stromal ulceration but without severe limbal stem cell deficiency. In two years banking activity 463 pieces of the preparation were successfully distributed to 90 Italian hospitals.