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For more than 100 years, the human amniotic membrane (hAM) has been used in multiple tissue regeneration applications. The hAM consists of cells with stem cell characteristics and a rich layer of extracellular matrix. Undoubtedly, the hAM with viable cells has remarkable properties such as the differentiation potential into all three germ layers, immuno-modulatory, and anti-fibrotic properties. At first sight, the hAM seems to be one structural entity. However, by integrating its anatomical location, the hAM can be divided into placental, reflected, and umbilical amniotic membrane. Recent studies show that cells of these amniotic sub-regions differ considerably in their properties such as morphology, structure, and content/release of certain bioactive factors. The aim of this review is to summarize these findings and discuss the relevance of these different properties for tissue regeneration. In summary, reflected amnion seems to be more immuno-modulatory and could have a higher reprogramming efficiency, whereas placental amnion seems to be pro-inflammatory, pro-angiogenic, with higher proliferation and differentiation capacity (e.g., chondrogenic and osteogenic), and could be more suitable for certain graft constructions. Therefore, we suggest that the respective hAM sub-region should be selected in consideration of its desired outcome. This will help to optimize and fine-tune the clinical application of the hAM.

Suture and autologous nerve transplantation are the primary therapeutic measures for completely severed nerves. However, imbalances in the microenvironment and adhesion of surrounding tissues can affect the quality of nerve regeneration and repair. Previous studies have shown that human amniotic membrane can promote the healing of a variety of tissues. In this study, the right common peroneal nerve underwent a 5-mm transection in rats. Epineural nerve repair was performed using 10/0 non-absorbable surgical suture. The repair site was wrapped with a two-layer amniotic membrane with α-cyanoacrylate rapid medical adhesive after suture. Hindlimb motor function was assessed using footprint analysis. Conduction velocity of the common peroneal nerve was calculated by neural electrical stimulation. The retrograde axoplasmic transport of the common peroneal nerve was observed using fast blue BB salt retrograde fluorescent staining. Hematoxylin-eosin staining was used to detect the pathological changes of the common peroneal nerve sputum. The mRNA expression of axon regeneration-related neurotrophic factors and inhibitors was measured using real-time polymerase chain reaction. The results showed that the amniotic membrane significantly improved the function of the injured nerve; the toe spread function rapidly recovered, the nerve conduction velocity was restored, and the number of fast blue BB salt particles were increased in the spinal cord. The amniotic membrane also increased the recovery rate of the tibialis anterior muscle and improved the tissue structure of the muscle. Meanwhile, mRNA expression of nerve growth factor, growth associated protein-43, collapsin response mediator protein-2, and brain-derived neurotrophic factor recovered to near-normal levels, while Lingo-1 mRNA expression decreased significantly in spinal cord tissues. mRNA expression of glial-derived neurotrophic factor did not change significantly. Changes in mRNA levels were more significant in amniotic-membrane-wrapping-treated rats compared with model and nerve sutured rats. These results demonstrate that fresh amniotic membrane wrapping can promote the functional recovery of sutured common peroneal nerve via regulation of expression levels of neurotrophic factors and inhibitors associated with axonal regeneration. The study was approved by the Committee on Animal Research and Ethics at the Affiliate Hospital of Zunyi Medical University, China (approval No. 112) on December 1, 2017.

To establish the efficacy and safety of ex vivo cultured autologous human conjunctival epithelial cell (hCjEC) transplantation for treatment of pterygia. Twenty-five patients with pterygia were recruited at different centers across the country. Autologous hCjEC grafts were prepared from conjunctival biopsy specimens excised from the healthy eye and cultured ex vivo on human amniotic membrane mounted on inserts using a unique mounting device. The hCjEC grafts were then transported in an in-house designed transport container for transplantation. Post-surgery, the patients were followed up on days 1, 7, 14, 30, 90, and 180 as per the approved study protocol. Clinical outcomes were assessed by slit lamp examination, visual acuity, imprint cytology, fluorescein/rose bengal staining, Schirmer's test, and photographic evaluation three and 6 months post-transplantation. Two patients were lost to follow-up and final analysis included 23 cases. No recurrence of pterygium was observed in 18 (78.3%) patients; all of these eyes showed a smooth conjunctival surface without epithelial defects. Recurrence was observed in 5 (21.7%) patients at 3 months post-treatment. No conjunctival inflammation, secondary infections or other complications were reported. Adequate goblet cells were present in 19 (82.6%) patients at the site of transplantation. We have, for the 1(st) time, standardized a protocol for preparing autologous hCjEC grafts that can be safely transported to multiple centers across the country for transplantation. The clinical outcome was satisfactory for treating pterygia.

BackgroundThe classification of macular hole closure patterns (MHCPs) currently relies on time domain OCT allowing only “open” and “closed” statuses or is based on inner foveal contour shape. Both classification types give no information on retinal layer reconstitution. Novel sophisticated surgical techniques lead to previously unknown MHCPs, outdating existing classifications and urging new ones. The purpose of the present study is to introduce a new classification allowing proper description of all MHCPs resulting from newer surgeries and based on the restoration of retinal layers.MethodsRetrospective analysis of patients undergoing MH surgery with five different surgical techniques was performed. MHCPs were classified according to spectral domain optical coherence tomography (SD-OCT). Type 0: open MH (0A: flat margin, 0B: elevated, 0C: oedematous); type 1: closed MHs (1A: reconstitution all retinal layers; 1B interruption of the external layers; 1C interruption of internal layers); type 2: MH closed with autologous or heterologous filling tissue interrupting the normal foveal layered anatomy (2A: filling tissue through all layers; 2B reconstitution of normal inner retinal layers; 2C reconstitution of normal outer retinal layers; 2D H-shaped bridging of filling tissue).ResultsClosure rate was 95.2% (241/253). Surgical technique and vision correlated to closure pattern (p < 0.001). Type 1 MHCPs had the best post-operative visual acuity (VA) compared with type 2 and type 0 (p < 0.001). MHCPs 1A and 1C performed better than all others. MHCP at months 1 and 3 changed in 42/254 (16.5%) and remained stable in 212/254 (83.5%). Improvement in vision was higher in eyes with shifting closure pattern (0.57 ± 0.33 vs 0.51 ± 0.48 logMAR; p 0.021).ConclusionMHCP classification based on retinal layer restoration properly comprises post-operative anatomic morphologies. MHCPs correlate the surgical technique and post-operative visual outcomes.

Intra-amniotic infection and inflammation (IAI) affect fetal development and are highly associated with preterm labor and premature rupture of membranes, which often lead to adverse neonatal outcomes. Human amniotic membrane (hAM), the inner part of the amnio-chorionic membrane, protects the embryo/fetus from environmental dangers, including microbial infection. However, weakened amnio-chorionic membrane may be breached or pathogens may enter through a different route, leading to IAI. The hAM and human amniotic fluid (hAF) respond by activation of all components of the innate immune system. This includes changes in 1) hAM structure, 2) presence of immune cells, 3) pattern recognition receptors, 4) cytokines, 5) antimicrobial peptides, 6) lipid derivatives, and 7) complement system. Herein we provide a comprehensive and integrative review of the current understanding of the innate immune response in the hAM and hAF, which will aid in design of novel studies that may lead to breakthroughs in how we perceive the IAI.

Background Increasing evidence has shown that mesenchymal stem cells (MSCs) yield a favorable therapeutic benefit for thermal burn skin wounds. Human amniotic MSCs (hAMSCs) derived from amniotic membrane have multilineage differentiation, immunosuppressive, and anti-inflammatory potential which makes them suitable for treating skin wounds. However, the exact effects of hAMSCs on the healing of thermal burn skin wounds and their potential mechanisms are not explored. Methods hAMSCs were isolated from amniotic membrane and characterized by RT-PCR, flow cytometry, immunofluorescence, and tumorigenicity test. We assessed the effects of hAMSCs and hAMSC conditional medium (CM) on wound healing in a deep second-degree burn injury model of mice. We then investigated the biological effects of hAMSCs and hAMSC-CM on the apoptosis and proliferation of heat stress-injured human keratinocytes HaCAT and dermal fibroblasts (DFL) both in vivo and in vitro. Next, we explored the underlying mechanisms by assessing PI3K/AKT and GSK3β/β-catenin signaling pathways in heat injured HaCAT and DFL cells after hAMSCs and hAMSC-CM treatments using PI3K inhibitor LY294002 and β-catenin inhibitor ICG001. Antibody array assay was used to identify the cytokines secreted by hAMSCs that may activate PI3K/AKT signaling pathway. Results Our results showed that hAMSCs expressed various markers of embryonic stem cells and mesenchymal stem cells and have low immunogenicity and no tumorigenicity. hAMSC and hAMSC-CM transplantation significantly promoted thermal burn wound healing by accelerating re-epithelialization with increased expression of CK19 and PCNA in vivo. hAMSCs and hAMSC-CM markedly inhibited heat stress-induced apoptosis in HaCAT and DFL cells in vitro through activation of PI3K/AKT signaling and promoted their proliferation by activating GSK3β/β-catenin signaling. Furthermore, we demonstrated that hAMSC-mediated activation of GSK3β/β-catenin signaling was dependent on PI3K/AKT signaling pathway. Antibody array assay showed that a panel of cytokines including PAI-1, C-GSF, periostin, and TIMP-1 delivered from hAMSCs may contribute to the improvement of the wound healing through activating PI3K/AKT signaling pathway. Conclusion Our results demonstrated that hAMSCs and hAMSC-CM efficiently cure heat stress-induced skin injury by inhibiting apoptosis of skin cells and promoting their proliferation through activating PI3K/AKT signaling pathway, suggesting that hAMSCs and hAMSC-CM may provide an alternative therapeutic approach for the treatment of skin injury.

Amniotic membrane transplantation (AMT) is an effective treatment for refractory macular hole closure, yet the underlying mechanism supporting that effectiveness remains unclear. Here, we investigated the role of retinal regeneration in this process using an in vivo rabbit retinal-hole (RH) model in which AMT was performed on one of the holes. Histological and immunofluorescence examinations with anti-glial fibrillary acidic protein and anti-glutemine synthetase antibodies were conducted to evaluate cell migration and hole closure. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to analyze the tissues after implantation. Optical coherence tomography scans revealed that AMT promoted RH closure. Histological analyses demonstrated that retinal pigment epithelium (RPE) cells migrated onto the AM, and immunofluorescence analyses showed that retinal glial cells, including Müller cells, contributed to RH closure. SEM and TEM findings revealed cellular coverage and extracellular matrix formation at the AMT site, suggesting successful integration with the surrounding retinal tissue. Furthermore, the loss of mitochondrial cristae within the RPE caused by the creation of RH was suppressed by AMT. In summary, AMT promoted RH closure in a rabbit model by serving as a scaffold for retinal cell migration and by preserving RPE integrity, although regeneration of the retinal-layers was not observed.

Biological tissues from various anatomical sources have been utilized for tissue transplantation and have developed into an important source of extracellular scaffolding material for regenerative medicine applications. Tissue scaffolds ideally integrate with host tissue and provide a homeostatic environment for cellular infiltration, growth, differentiation, and tissue resolution. The human amniotic membrane is considered an important source of scaffolding material due to its 3D structural architecture and function and as a source of growth factors and cytokines. This tissue source has been widely studied and used in various areas of tissue repair including intraoral reconstruction, corneal repair, tendon repair, microvascular reconstruction, nerve procedures, burns, and chronic wound treatment. The production of amniotic membrane allografts has not been standardized, resulting in a wide array of amniotic membrane products, including single, dual, and tri-layered products, such as amnion, chorion, amnion–chorion, amnion–amnion, and amnion–chorion–amnion allografts. Since these allografts are not processed using the same methods, they do not necessarily produce the same clinical responses. The aim of this review is to highlight the properties of different human allograft membranes, present the different processing and preservation methods, and discuss their use in tissue engineering and regenerative applications.

Background Human amniotic membrane (AM) transplantation has been applied to treat ocular surface diseases, including corneal trauma. The focus of much deliberation is to balance the mechanical strength of the amniotic membrane, its resistance to biodegradation, and its therapeutic efficacy. It is commonly observed that the crosslinked human decellularized amniotic membranes lose the functional human amniotic epithelial cells (hAECs), which play a key role in curing the injured tissues. Methods and results In this study, we crosslinked human decellularized amniotic membranes (dAM) with genipin and re-planted the hAECs onto the genipin crosslinked AM. The properties of the AM were evaluated based on optical clarity, biodegradation, cytotoxicity, and ultrastructure. The crosslinked AM maintained its transparency. The color of crosslinked AM deepened with increasing concentrations of genipin. And the extracts from low concentrations of genipin crosslinked AM had no toxic effect on human corneal epithelial cells (HCECs), while high concentrations of genipin exhibited cytotoxicity. The microscopic observation and H&E staining revealed that 2 mg/mL genipin-crosslinked dAM (2 mg/mL cl-dAM) was more favorable for the attachment, migration, and proliferation of hAECs. Moreover, the results of the CCK-8 assay and the transwell assay further indicated that the living hAECs’ tissue-engineered amniotic membranes could facilitate the proliferation and migration of human corneal stromal cells (HCSCs) in vitro. Conclusions In conclusion, the cl-dAM with living hAECs demonstrates superior biostability and holds significant promise as a material for ocular surface tissue repair in clinical applications.

Human amniotic membrane (hAM) is the innermost layer of fetal membranes, which surrounds the developing fetus and forms the amniotic cavity. hAM and hAM-derived cells possess many properties that make them suitable for use in regenerative medicine, such as low immunogenicity, promotion of epithelization, anti-inflammatory properties, angiogenic and antiangiogenic properties, antifibrotic properties, antimicrobial properties, and anticancer properties. Many pathological conditions of the urinary tract lead to organ damage or complete loss of function. Consequently, the reconstruction or replacement of damaged organs is needed, which makes searching for new approaches in regenerative and reconstructive urology a necessity. The use of hAM for treating defects in kidneys, ureters, urinary bladder, and urethra was tested in vitro in cell cultures and in vivo in mice, rats, rabbits, cats, dogs, and also in humans. These studies confirmed the advantages and the potential of hAM for use in regenerative and reconstructive urology as stated above. However, they also pointed out a few concerns we have to take into consideration. These are (1) the lack of a standardized protocol in hAM preparation and storage, (2) the heterogeneity of hAM, and especially (3) low mechanical strength of hAM. Before any wider use of hAM for treating urological defects, the protocols for preparation and storage will need to be standardized, followed by more studies on larger animals and clinical trials, which will altogether extensively assess the potential of hAM use in urological patients.