Amniotic Membrane Matrix Research Articles

The Combination of Decellularized Cartilage and Amniotic Membrane Matrix Enhances the Production of Extracellular Matrix Elements in Human Chondrocytes

The Combination of Decellularized Cartilage and Amniotic Membrane Matrix Enhances the Production of Extracellular Matrix Elements in Human Chondrocytes

Comparison of human acellular amniotic membranes with acellular amniotic membranes pretreated with MPLA for repair of fascia in rats.

A decellularized amniotic membrane matrix is an ideal scaffolding system. This study compared the effectiveness of amniotic membrane and amniotic membrane pretreated with monophosphoryl lipid A (MPLA) for repairing the fascia of the abdomen with tension sutures. A total of 24 rats were randomly divided into three groups of eight each. In the control group, the fascia was repaired only with non-absorbable monofilament yarn under tension. In the second and third groups, in addition to the control treatment, acellular amniotic membrane was used and MPLA pretreated amniotic membrane was used, respectively. At 3, 7 and 14 days after treatment, the fascia repair was examined macroscopically and microscopically in all groups. Macroscopic examination showed that the use of MPLA pretreated significantly different from the other groups only in the fibrin exudate. Changes in the fascia rupture pressure showed significant differences between groups. Group three, which was sutured with MPLA pretreated acellular membrane, showed greater amounts of collagen, monocytes and neovascularization, especially at days 7 and 14. The results show that MPLA pretreated acellular amniotic membrane helped to repair abdominal fascia to some extent.

Α Novel Sutureless Pterygium Excision Surgery Using Human-Derived Dehydrated Amniotic Membrane

There are many surgical techniques available for the management of pterygium. Excision with limbal conjunctival autograft is currently the most popular surgical procedure and has been shown to have low recurrence rate and fewer complications. The fixation of limbal conjunctival autograft is performed with either sutures or fibrin glue. The use of sutures results in longer operating time and intense postoperative irritation and epiphora. We describe a new technique for pterygium excision using a human amniotic membrane-derived dry matrix (Omnigen®; Nottingham, UK: Nu-Vision Biotherapies). The use of Omnigen instead of conjunctival autograft in pterygium surgery is a new promising technique and results in shorter operation time, easier graft fixation, reduction in complications, and less postoperative discomfort. This is a simple technique for pterygium surgery with dry amniotic membrane matrix and without the need for anchoring limbal sutures or an assistant holding the graft whilst it is glued in place.

Amniotic membrane matrix effects on calcineurin-NFAT-related gene expressions of SHED treated with VEGF for endothelial differentiation.

The nuclear factor of activated T-cell (NFAT) signaling pathway is involved in angiogenesis following initiation by vascular endothelial growth factor (VEGF). A number of angiogenic genes have been associated with calcineurin in the NFAT pathway, forming a calcineurin-NFAT pathway. This study aims to investigate the involvement of four angiogenic genes within the calcineurin-NFAT pathway in the endothelial-like differentiation of stem cells from human exfoliated deciduous teeth (SHED) cultured on a human amniotic membrane (HAM) induced by VEGF. SHED were induced with VEGF for 24 h, then cultured on the stromal side of HAM. The cells were then further induced with VEGF until days 1 and 14. To understand the role of calcineurin, its potent inhibitor, cyclosporin A (CsA), was added into the culture. Results from SEM and H&E analyses showed SHED grew on HAM surface. Gene expression study of Cox-2 showed a drastically reduced expression with CsA treatment indicating Cox-2 involvement in the calcineurin-NFAT pathway. Meanwhile, IL-8 was probably controlled by another pathway as it showed no CsA inhibition. In contrast, high expression of ICAM-1 and RCAN1.4 by VEGF and CsA implied that these genes were not controlled by the calcineurin-NFAT-dependent pathway. In conclusion, the results of this study suggest the involvement of Cox-2 in the calcineurin-NFAT-dependent pathway while RCAN1.4 was controlled by NFAT molecule in endothelial-like differentiation of SHED cultured on HAM with VEGF induction.

Comparison of Vestibular Depth Relapse and Wound Healing After Reconstructive Preprosthetic Surgery Using Cryopreserved Amniotic Membrane and Acellular Dermal Matrix - A Comparative Study.

Introduction:The significance of membranes as wound dressing in oral surgeries has been reported by previous studies. The aim of the present split-mouth randomized clinical study was to assess and compare the wound dressing properties of acellular dermal matrix (ADM) and cryopreserved human amniotic membrane (AM) after reconstructive preprosthetic oral surgery.Materials and Methods:Twenty-eight patients with complete mandibular edentulism and resorbed alveolar bone were included. After taking mandibular impression, a clear acrylic splint with increased labial flange height was created. In each participant, labial vestibular depth was elevated using the Clark's technique. Subsequently, half of the exposed periosteum was covered with ADM while the other half was covered with cryopreserved human AM. Vestibule depth and relapse in the two sides were measured immediately after vestibuloplasty and at the end of the 1st week, 2nd week, 1st month, and 3rd months with graduations of 0.1 mm. Furthermore, after 3 and 7 days, samples were collected from graft material, and the macrophage population was analyzed by flow cytometry.Results:There was no significant difference in the relapse of vestibule depth between the two grafts at different time intervals. However, the frequency of wound-infiltrating macrophages (CD68+ cells) was significantly higher in areas covered by ADM after 3 and 7 days.Discussion:ADM is as effective as cryopreserved AM in terms of maintaining the postoperative vestibular depth. On the other hand, our results suggested that the onset of healing phase in ADM-covered areas occurs faster compared to the periosteum covered with cryopreserved human AM. This clinical trial showed significantly faster postoperative healing onset when ADM was used than when cryopreserved human AM was applied on the periosteum.

The Cells and Extracellular Matrix of Human Amniotic Membrane Hinder the Growth and Invasive Potential of Bladder Urothelial Cancer Cells.

Bladder cancer is one of the most common cancers among men in industrialized countries and on the global level incidence and mortality rates are increasing. In spite of progress in surgical treatment and chemotherapy, the prognosis remains poor for patients with muscle-invasive bladder cancer. Therefore, there is a great need for the development of novel therapeutic approaches. The human amniotic membrane (hAM) is a multi-layered membrane that comprises the innermost part of the placenta. It has unique properties that make it suitable for clinical use, such as the ability to promote wound healing and decrease scarring, low immunogenicity, and immunomodulatory, antimicrobial and anticancer properties. This study aimed to investigate the effect of (i) hAM-derived cells and (ii) hAM scaffolds on the growth dynamics, proliferation rate, and invasive potential of muscle-invasive bladder cancer T24 cells. Our results show that 24 and 48 h of co-culturing T24 cells with hAM-derived cells (at 1:1 and 1:4 ratios) diminished the proliferation rate of T24 cells. Furthermore, when seeded on hAM scaffolds, namely (1) epithelium of hAM (e-hAM), (2) basal lamina of hAM (denuded; d-hAM), and (3) stroma of hAM (s-hAM), the growth dynamic of T24 cells was altered and proliferation was reduced, even more so by the e-hAM scaffolds. Importantly, despite their muscle-invasive potential, the T24 cells did not disrupt the basal lamina of hAM scaffolds. Furthermore, we observed a decrease in the expression of epithelial-mesenchymal transition (EMT) markers N-cadherin, Snail and Slug in T24 cells grown on hAM scaffolds and individual T24 cells even expressed epithelial markers E-cadherin and occludin. Our study brings new knowledge on basic mechanisms of hAM affecting bladder carcinogenesis and the results serve as a good foundation for further research into the potential of hAM-derived cells and the hAM extracellular matrix to serve as a novel bladder cancer treatment.

Mineralized collagen scaffolds fabricated with amniotic membrane matrix increase osteogenesis under inflammatory conditions.

Defects in craniofacial bones occur congenitally, after high-energy impacts, and during the course of treatment for stroke and cancer. These injuries are difficult to heal due to the overwhelming size of the injury area and the inflammatory environment surrounding the injury. Significant inflammatory response after injury may greatly inhibit regenerative healing. We have developed mineralized collagen scaffolds that can induce osteogenic differentiation and matrix biosynthesis in the absence of osteogenic media or supplemental proteins. The amniotic membrane is derived from placentas and has been recently investigated as an extracellular matrix to prevent chronic inflammation. Herein, we hypothesized that a mineralized collagen–amnion composite scaffold could increase osteogenic activity in the presence of inflammatory cytokines. We report mechanical properties of a mineralized collagen–amnion scaffold and investigated osteogenic differentiation and mineral deposition of porcine adipose-derived stem cells within these scaffolds as a function of inflammatory challenge. Incorporation of amniotic membrane matrix promotes osteogenesis similarly to un-modified mineralized collagen scaffolds, and increases in mineralized collagen–amnion scaffolds under inflammatory challenge. Together, these findings suggest that a mineralized collagen–amnion scaffold may provide a beneficial environment to aid craniomaxillofacial bone repair, especially in the course of defects presenting significant inflammatory complications.

Amniotic Membrane Enhance the Effect of Vascular Endothelial Growth Factor on the Angiogenic Marker Expression of Stem Cells from Human Exfoliated Deciduous Teeth

Previously, it was reported that human amniotic membrane (AM) induced stem cells from human deciduous exfoliated teeth (SHED) endothelial-like-cell differentiation. This interesting effect of AM matrix on SHED demands further elucidation. Objective of this in vitro work was to study the effect of 24-h VEGF induced on SHED endothelial differentiation when seeded on acellular stromal side (SS) of AM matrix. Stemness of SHED was identified by flow cytometry. Cell attachment and morphological changes towards the matrix was observed by scanning electron microscopy. Protein expression of endothelial marker was examined by Western blot. The expression of stem cells and endothelial-specific gene markers of VEGF-induced SHED cultured on human AM was inspected via reverse transcriptase-polymerase chain reaction. Results showed SHED at both passages retain stemness property. Ang-1 protein was expressed in SHED. Cells treated with VEGF and cultured on AM transformed attached well to AM. VEGF-induced SHED expressed both stem cell and endothelial-specific markers throughout the treatments and timeline. Interestingly, prolonged VEGF treatment increased the expression of Cox-2 and VE-Cadherin genes in all treated groups when compared to SHED. It was concluded that the VEGF-induced SHED showed better expression of endothelial-specific markers when cultured on SS of AM, with prolonged VEGF treatment.

Development of Injectable Amniotic Membrane Matrix for Postmyocardial Infarction Tissue Repair.

Ischemic heart disease represents the leading cause of death worldwide. Heart failure following myocardial infarction (MI) is associated with severe fibrosis formation and cardiac remodeling. Recently, injectable hydrogels have emerged as a promising approach to repair the infarcted heart and improve heart function through minimally invasive administration. Here, a novel injectable human amniotic membrane (hAM) matrix is developed to enhance cardiac regeneration following MI. Human amniotic membrane is isolated from human placenta and engineered to be a thermoresponsive, injectable gel around body temperature. Ultrasound-guided injection of hAM matrix into rat MI hearts significantly improves cardiac contractility, as measured by ejection fraction (EF), and decrease fibrosis. The results of this study demonstrate the feasibility of engineering as an injectable hAM matrix and its efficacy in attenuating degenerative changes in cardiac function following MI, which may have broad applications in tissue regeneration.

Assessment of Antibiotic Influence on Structural Modifications of Amniotic Membrane by FTIR Spectroscopy

The amniotic membrane is a readily available biomaterial with an important potential for tissue regeneration in dermatology and ophtalmology, with anti-inflammatory and anti-microbial properties. The extracellular matrix of the amniotic membrane is composed mainly of collagen, fibronectin and laminin. The purpose of our study was to investigate the structural modifications of collagen extracellular matrix of amniotic membrane upon interaction with two different antibiotics, frequently used in surgical and post- surgical procedure, respectively ciprofloxacin and gentamicin. SEM micrographs evidenced the ultrastructure features of dried amniotic membrane, with laminar structure, flexible, transparent, with no blood vesels or nerves. FTIR spectroscopy combined with deconvolution techniques was applied with the aim to determine the extent of denaturation upon treatment with different antibiotics. By spectral analysis, we concluded that gentamicin treatment is more favorable compared to ciprofloxacin, as the denaturation process is reflected by the lower sheet/turns ratio of the secondary structure composition.

Incorporation of the Amniotic Membrane as an Immunomodulatory Design Element in Collagen Scaffolds for Tendon Repair.

Tendon injuries often require surgical intervention and even then result in poor outcomes due to scar formation and repeated failure. Biomaterial implants offer the potential to address multiple underlying concerns preventing improved tendon repair. Here, we describe modifications to the composition of an anisotropic collagen-glycosaminoglycan (CG) scaffold biomaterial, incorporating amniotic membrane (AM)-derived matrix to alter the inflammatory response and establish conditions for improved regenerative repair. We explored two methods of AM matrix incorporation to address multiple concerns associated with tendon repair. Amniotic membrane-derived matrix was incorporated directly into the scaffold microstructure during fabrication to form a C/AM composite. Alternatively, decellularized amniotic matrix was wrapped around the traditional collagen-chondroitin sulfate (C/CS) scaffold to form a core-shell composite (C/CS plus AM wrap) in a manner similar to current collagen membrane wraps used in rotator cuff and Achilles tendon surgeries to improve the mechanical strength of the repair. Human mesenchymal stem cells (MSCs) cultured within these materials were evaluated for metabolic health and immunomodulatory gene expression in response to inflammatory media challenge of interleukin 1 β and tumor necrosis factor α. The scaffolds were able to maintain MSC metabolic activity in all media conditions over the course of a 7 day culture. Expression of genes encoding for pro-inflammatory cytokines were down-regulated in AM containing scaffolds, suggesting the potential to employ AM-modified CG scaffolds for tendon-repair applications.

Histoanalysis and sterility assay of fresh human amniotic membrane for use in urogynecology

Human amniotic membrane has long been used as a biomaterial for wound/burn and varicose/diabetic ulcer dressing to prevent endometrial adhesion, as a graft for the cornea, periosteum, and bladder, or as a material to generate a neovagina. Several clinics in Indonesia opt for fresh amniotic membrane for grafts because it is widely available and easy to obtain. This study investigated the reliability of using fresh amniotic membrane as a biomaterial. Placenta samples were obtained from patients who underwent cesarean without complications, and human amniotic membrane was separated from the chorion and cut into a 3 × 3 cm2 size. Specimens were categorized into two groups: before (0.9% NaCl) and after (2 g.L−1 kanamycin) antibiotic wash as the control and fresh amniotic membrane groups, respectively. Cells in the amniotic membrane matrix were visualized using hematoxylin and eosin and DAPI staining. DNA in the matrix was quantified using Quick-DNA™ Miniprep Plus Kit, measured using a BioDrop nanospectrophotometer. DNA length was determined using gel electrophoresis. Sterility assay was performed using nutrient broth, followed by plating on agarose to identify any strain. Sections of both groups showed an intact cuboidal epithelial monolayer at the fetal surface. DNA residue in the matrix after NaCl and kanamycin washes was 41.36 ± 8.48 ng.mg−1 and 42.53 ± 9.57 ng.mg−1 tissue weight, respectively. All samples were sterile. Fresh amniotic membrane for clinical use might not be ideal because nuclei were identified across the membrane and the dsDNA residue length was >200bp.

Effect of Acellular Amniotic Membrane Matrix Patch on Healing of Cut Surface After Sleeve Gastrectomy in Rats

ABSTRACTBackground: The aim of this study was to evaluate the effect of acellular amniotic membrane matrix patch on healing cut surface after sleeve gastrectomy in rats. Methods: Thirty male Wistar albino rats were divided into three groups: control (n = 10), Experiment-1 (n = 10), and Experiment-2 (n = 10) groups. Control group rats underwent only sleeve gastrectomy with primary gastrorrhaphy. Experiment-1 group rats underwent sleeve gastrectomy, primary gastrorrhaphy and covered with acellular amniotic membrane matrix patch. Experiment-2 group rats underwent sleeve gastrectomy, incomplete primary gastrorrhaphy and covered with acellular amniotic membrane matrix patch. All rats were sacrificed on the fifth postoperative day. Macroscopic findings and histopathologic alterations were evaluated and compared. Results: There was a statistically significant difference between levels of PMNs, granulation formation and vascularization, distributions of edema, type of mucosal epithelium, and fibroblastic proliferation according to groups (p < 0.05). The PMNs level in the Experiment-1 group was significantly lower than the control group (p < 0.05). In experiment groups, the level of granulation tissue, vascularization and fibroblastic proliferation was significantly higher than the control group (p < 0.05). The levels of edema and type of mucosal epithelium of the experiment groups were significantly lower than the control group (p < 0.05). Conclusion: Covering sleeve gastrectomy cut surface area with acellular amniotic membrane matrix results better healing. Moreover, acellular amniotic membrane matrix provides safe healing even in incomplete sutured area.

Interpositional Arthroplasty Technique for Hallux Rigidus Using Amniotic Membrane and Regenerative Tissue Matrix

Hallux rigidus is a painful osteoarthritic condition of the first metatarsophalangeal joint. It is the most prevalent arthritic condition of the foot, affecting up to 1 in 40 adults over 50 years of age. Mild cases of hallux rigidus are frequently treated with cheilectomy, whereas severe cases are often treated with arthrodesis. Interposition arthroplasty is a joint-sparing alternative to fusion that has been used effectively in moderate to severe cases of hallux rigidus. This paper discusses a novel modification of interpositional arthroplasty technique using amniotic membrane graft and regenerative tissue matrix to successfully treat patients who have failed cheilectomy. Levels of Evidence: Diagnostic Level 4. See Instructions for Authors for a complete description of levels of evidence.

Efficacy of Particulate Amniotic Membrane and Umbilical Cord Tissues in Attenuating Cartilage Destruction in an Osteoarthritis Model.

Osteoarthritis (OA) is a progressive degenerative joint disease, and to date, no disease-modifying OA drug exists. Amniotic membrane and umbilical cord products have been used clinically in several diseases due to their anti-inflammatory and antiscarring properties. In the present study, we sought to evaluate whether a particulate amniotic membrane and umbilical cord (AM/UC) matrix could aid in attenuating disease progression. Lewis rats underwent medial meniscus transection (MMT) to induce OA. Two weeks after surgery, animals received intra-articular injections (50 μL) of either 50 or 100 μg/μL particulate AM/UC or saline control and were subsequently euthanized 1 or 4 weeks later. Cartilage degeneration was assessed using both histological scoring methods and equilibrium partitioning of an ionic contrast agent-microcomputed tomography (EPIC-μCT). EPIC-μCT analysis demonstrated that overall cartilage destruction was attenuated, with a significant increase in both cartilage thickness and volume as well as a significant decrease in total lesion area in animals injected with either dose of particulate AM/UC at 1 week, but only a high dose at 4 weeks postinjection. Osteoarthritis Research Society International (OARSI) histology scores of tibial sections corroborated EPIC-μCT results. Overall joint destruction was attenuated in animals injected with either dose of AM/UC tissue compared with saline-injected control animals at 1 week postinjection. Only high-dose AM/UC-injected animals continued to show less overall joint destruction by 4 weeks postinjection. Intra-articular injection of particulate AM/UC tissue attenuates cartilage degradation in a rat MMT model of OA, suggesting that it may be able to slow joint destruction in patients with OA.

Angiogenic potential of extracellular matrix of human amniotic membrane.

Combination between tissue engineering and other fields has brought an innovation in the area of regenerative medicine which ultimate aims are to repair, improve, and produce a good tissue construct. The availability of many types of scaffold, both synthetically and naturally have developed into many outstanding end products that have achieved the general objective in tissue engineering. Interestingly, most of this scaffold emulates extracellular matrix (ECM) characteristics. Therefore, ECM component sparks an interest to be explored and manipulated. The ECM featured in human amniotic membrane (HAM) provides a suitable niche for the cells to adhere, grow, proliferate, migrate and differentiate, and could possibly contribute to the production of angiogenic micro-environment indirectly. Previously, HAM scaffold has been widely used to accelerate wound healing, treat bone related and ocular diseases, and involved in cardiovascular repair. Also, it has been used in the angiogenicity study, but with a different technical approach. In addition, both side of HAM could be used in cellularised and decellularised conditions depending on the objectives of a particular research. Therefore, it is of paramount importance to investigate the behavior of ECM components especially on the stromal side of HAM and further explore the angiogenic potential exhibited by this scaffold.

Effects of Acellular Amniotic Membrane Matrix and Bone Marrow-Derived Mesenchymal Stem Cells in Improving Random Skin Flap Survival in Rats.

BackgroundThe necrotic skin flap represents a great challenge in plastic and reconstructive surgery. In this study, we evaluated the effect of bioscaffolds, acellular amniotic membranes (AAMs), and bone marrow-derived mesenchymal stem cells (BM-MSCs) on random skin flap (RSF) survival in rats by applying a cell-free extracellular matrix scaffold as a supportive component for the growth and proliferation of BM-MSCs on RSFs. AAM matrix scaffolds were created by incubating AMs in ethylenediaminetetraacetic acid 0.05% at 37°C, and cell scrapers were used.ObjectivesThe aim of the present study was to assess the effect of AAM as a scaffold in TE, and combined with transplanted BM-MSCs, on the survival of RSFs and on the biomechanical parameters of the incision-wound flap margins 7 days after flap elevation.Materials and MethodsBM-MSCs and AAMs were transplanted into subcutaneous tissue in the flap area. On the 7th postoperative day, the surviving flap areas were measured using digital imaging software, and the flap tissue was collected for evaluation. Forty rats were randomly divided into four groups of 10 each: group 1 received an AAM injection; group 2 underwent BM-MSC transplantation; group 3 received both AAM injection + BM-MSC transplantation; and group 4 was the control group, receiving only saline.ResultsThe survival area in the AAM/BM-MSC group was significantly higher than in the control group (18.49 ± 1.58 versus 7.51 ± 2.42, P < 0.05). The biomechanical assessment showed no significant differences between the experimental groups and the control group (P > 0.05), and there was no correlation with flap survival.ConclusionsOur findings showed that the treatment of flaps with BM-MSC and AAM transplantations significantly promoted flap survival compared to a control group. The viability of the flap was improved by combining BM-MSCs with AAM matrix scaffolds.

Immunomodulatory effects of amniotic membrane matrix incorporated into collagen scaffolds.

Adult tendon wound repair is characterized by the formation of disorganized collagen matrix which leads to decreases in mechanical properties and scar formation. Studies have linked this scar formation to the inflammatory phase of wound healing. Instructive biomaterials designed for tendon regeneration are often designed to provide both structural and cellular support. In order to facilitate regeneration, success may be found by tempering the body's inflammatory response. This work combines collagen-glycosaminoglycan scaffolds, previously developed for tissue regeneration, with matrix materials (hyaluronic acid and amniotic membrane) that have been shown to promote healing and decreased scar formation in skin studies. The results presented show that scaffolds containing amniotic membrane matrix have significantly increased mechanical properties and that tendon cells within these scaffolds have increased metabolic activity even when the media is supplemented with the pro-inflammatory cytokine interleukin-1 beta. Collagen scaffolds containing hyaluronic acid or amniotic membrane also temper the expression of genes associated with the inflammatory response in normal tendon healing (TNF-α, COLI, MMP-3). These results suggest that alterations to scaffold composition, to include matrix known to decrease scar formation in vivo, can modify the inflammatory response in tenocytes. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1332-1342, 2016.

Immunomodulatory effects of amniotic membrane matrix incorporated into collagen scaffolds

Immunomodulatory effects of amniotic membrane matrix incorporated into collagen scaffolds

Extract of fetal membrane would inhibit thrombosis and hemolysis

The innermost layer of fetal membranes is amnion which has anti-adhesive, anti-inflammation and viscoelastic properties, as well as low immunogenicity. Amniotic membrane has been employed in variety of clinical fields as a natural biomaterial. Amniotic epithelial cells possess stem cell characteristics and capability to differentiate into endothelial cells. The basement membrane of amnion is an extracellular matrix enriched scaffold to support adhesion of endothelial cells. The matrix of amniotic membrane contains two kinds of glycosaminoglycans including perlecan (a heparan sulfate proteoglycan) and hyaluronic acid which both inhibit blood coagulation. Moreover, the other ingredients of amniotic membrane such as pigment-epithelium derived factor (PEDF), IL-10, MMP-9 inhibit platelet aggregation. Based on some biochemical and biomechanical evidences, we hypothesized in this paper that amniotic membrane could prevent thrombosis and hemolysis; therefore, has the capability to be applied in blood contacting devices and implants.