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ObjectivesThis study aimed to define the antimicrobial peptide (AMP) expression pattern of the equine ocular surface and amniotic membrane using a targeted qPCR approach and 3'Tag-sequencing. It will serve as a reference for future studies of ocular surface innate immunity and amniotic membrane therapies.ProceduresA targeted qPCR approach was used to investigate the presence of orthologs for three of the most highly expressed beta-defensins (DEFB1, DEFB4B, and DEFB103A) of the human ocular surface and amniotic membrane in equine corneal epithelium, conjunctiva, and amniotic membrane. 3'Tag-sequencing was performed on RNA from one sample of corneal epithelium, conjunctiva, and amniotic membrane to further characterize their AMP expression.ResultsEquine corneal epithelium, conjunctiva, and amniotic membrane expressed DEFB1, DEFB4B, and DEFB103A. DEFB103A was expressed at the highest amounts in corneal epithelium, while DEFB4B was most highly expressed in conjunctiva and amniotic membrane. 3'Tag-sequencing from all three tissues confirmed these findings and identified expression of five additional beta-defensins, 11 alpha-defensins and two cathelicidins, with the alpha-defensins showing higher normalized read counts than the beta-defensins.ConclusionsThis study identified AMP expression in the equine cornea and conjunctiva, suggesting that they play a key role in the protection of the equine eye, similar to the human ocular surface. We also determined that equine amniotic membrane expresses a substantial number of AMPs suggesting it could potentiate an antimicrobial effect as a corneal graft material. Future studies will focus on defining the antimicrobial activity of these AMPs and determining their role in microbial keratitis.

Purpose To evaluate the effectiveness of cryopreserved human amniotic membrane transplantation (hAMT) in patients with infectious ulcerative keratitis. Methods A retrospective cohort study based on medical records of patients who underwent hAMT procedure over a 2-year period (2020-2021) was conducted at Ege University Hospital, Turkey. Cryopreserved hAM was used. Best corrected visual acuity (BCVA) was measured with the Snellen chart. Detailed ophthalmological examination was performed at all visits. Treatment success was defined as complete healing including disappearance of corneal infiltrates, epithelial defect closure, reduction in anterior segment reaction. Results A total of 13 eyes of 12 patients were diagnosed with ulcerative keratitis between 2020 and 2021. hAMT was performed in severe keratitis patients in which corneal infiltrates extending to middle to deep stromal and patients with neurotrophic component, in other words, having trouble for wound healing. The mean age was 60.9 +/- 18.7 years, and 66.6% of the patients were male. Average ulcer horizontal diameter was 9.7 +/- 2.8 mm. A single-layer human amniotic membrane (hAM) was transplanted 92.3% of eyes. Overlay technique was used in all patients for hAMT. The mean residence time of the amniotic membrane on the ocular surface was 14.1 +/- 10.2 days. The mean time from the first presentation to the hAM transplantation was 9.7 +/- 6.8 days. The average hospitalization time of all patients was 15 +/- 12.1 days, pre-hAMT was 10.2 +/- 7.3 days and post-hAMT was 4.8 +/- 9.1 days. The mean follow-up time was 3 months. Recovery response was positive in 76.9%. Average healing time was 32.1 +/- 14.04 days. Conclusions hAMT was effective in the treatment of infectious ulcerative keratitis by accelerating and supporting wound healing. Performing hAM transplantation at the early stages of the disease and prolonged residence of membrane on the ocular surface increase the success of the treatment and moreover shortens hospitalization time.

Perinatal derivatives (PnD) are birth-associated tissues, such as placenta, umbilical cord, amniotic and chorionic membrane, and thereof-derived cells as well as secretomes. PnD play an increasing therapeutic role with beneficial effects on the treatment of various diseases. The aim of this review is to elucidate the modes of action of non-hematopoietic PnD on inflammation, angiogenesis and wound healing. We describe the source and type of PnD with a special focus on their effects on inflammation and immune response, on vascular function as well as on cutaneous and oral wound healing, which is a complex process that comprises hemostasis, inflammation, proliferation (including epithelialization, angiogenesis), and remodeling. We further evaluate the different in vitro assays currently used for assessing selected functional and therapeutic PnD properties. This review is a joint effort from the COST SPRINT Action (CA17116) with the intention to promote PnD into the clinics. It is part of a quadrinomial series on functional assays for validation of PnD, spanning biological functions, such as immunomodulation, anti-microbial/anti-cancer activities, anti-inflammation, wound healing, angiogenesis, and regeneration.

Graphical Abstract Type 2 diabetes mellitus (T2DM), one of the most common carbohydrate metabolism disorders, is characterized by chronic hyperglycemia and insulin resistance (IR), and has become an urgent global health challenge. Mesenchymal stem cells (MSCs) originating from perinatal tissues such as umbilical cord (UC) and amniotic membrane (AM) serve as ideal candidates for the treatment of T2DM due to their great advantages in terms of abundant source, proliferation capacity, immunomodulation and plasticity for insulin-producing cell differentiation. However, the optimally perinatal MSC source to treat T2DM remains elusive. This study aims to compare the therapeutic efficacy of MSCs derived from AM and UC (AMMSCs and UCMSCs) of the same donor in the alleviation of T2DM symptoms and explore the underlying mechanisms. Our results showed that AMMSCs and UCMSCs displayed indistinguishable immunophenotype and multi-lineage differentiation potential, but UCMSCs had a much higher expansion capacity than AMMSCs. Moreover, we uncovered that single-dose intravenous injection of either AMMSCs or UCMSCs could comparably reduce hyperglycemia and improve IR in T2DM db/db mice. Mechanistic investigations revealed that either AMMSC or UCMSC infusion could greatly improve glycolipid metabolism in the liver of db/db mice, which was evidenced by decreased liver to body weight ratio, reduced lipid accumulation, upregulated glycogen synthesis, and increased Akt phosphorylation. Taken together, these data indicate that the same donor-derived AMMSCs and UCMSCs possessed comparable effects and shared a similar hepatoprotective mechanism on the alleviation of T2DM symptoms. Supplementary Information The online version contains supplementary material available at 10.1007/s12015-021-10320-w.

Unsuccessful wound closure in chronic wounds can be linked to altered keratinocyte activation and their inability to re-epithelize. Suggested mechanisms driving this impairment involve unbalanced cytokine signaling. However, the molecular events leading to these aberrant responses are poorly understood. Among cytokines affecting keratinocyte responses, Transforming Growth Factor-β (TFG-β) is thought to have a great impact. In this study, we have used a previously characterized skin epidermal in vitro model, HaCaT cells continuously exposed to TGF-β1, to study the wound recovery capabilities of chronified/senescent keratinocytes. In this setting, chronified keratinocytes show decreased migration and reduced activation in response to injury. Amniotic membrane (AM) has been used successfully to manage unresponsive complicated wounds. In our in vitro setting, AM treatment of chronified keratinocytes re-enabled migration in the early stages of wound healing, also promoting proliferation at later stages. Interestingly, when checking the gene expression of markers known to be altered in TGF-β chronified cells and involved in cell cycle regulation, early migratory responses, senescence, and chronic inflammation, we discovered that AM treatment seemed to reset back to keratinocyte status. The analysis of the evolution of both the levels of keratinocyte activation marker cytokeratin 17 and the spatial-temporal expression pattern of the proliferation marker Ki-67 in human in vivo biopsy samples suggests that responses to AM recorded in TGF-β chronified HaCaT cells would be homologous to those of resident keratinocytes in chronic wounds. All these results provide further evidence that sustained TGF-β might play a key role in wound chronification and postulate the validity of our TGF-β chronified HaCaT in vitro model for the study of chronic wound physiology.

Introduction: Therapeutic contact lenses (TCLs) are important tools for the treatment of ocular surface diseases and other corneal conditions. The main difference between a normal contact lens and a TCL is that the latter is not controlled by the patient.Aim: The aim of this article is to evaluate the effect of TCLs on amniotic membrane (AM) transplantation and outline the benefits.Materials and Methods: A prospective study was conducted on 30 clinical cases of recurrent erosion syndrome (RES) or superficial corneal trauma collected over one year treated with AM. The study was randomized and 15 of the patients were treated with AM and TCL and 15 with AM alone.Results: Patients treated with AM/TCL rated postoperative comfort with an av. 5.5 (from a scale of 2–6). In comparison, AM patients without TCL rated it 3.5. Twelve of the TCL cases had complete or better epithelialization from day 1, while 5 non-TCL patients had worse results. The study showed AM survived for about 1 week when TCL was included. In the absence of TCL, the membrane lasted less than 48 hours. The most common complications in a period of 7 days were suture rupture (AM&TCL: 1/AM: 8), ruptured membrane (AM&TCL: 2/AM: 12), hemorrhage (AM&TCL: 1/AM: 5), inflammation (AM&TCL: 0/AM: 2), infection (AM&TCL: 0/AM: 1).Conclusion: Therapeutic contact lens placement is a promising method for improving ocular surface health recovery, increases comfort, protects AM, and has long-term outcomes.

The fetal membranes provide a supportive environment for the growing embryo and later fetus. Due to their versatile properties, the use of fetal membranes in tissue engineering and regenerative medicine is increasing in recent years. Moreover, as microbial infections present a crucial complication in various treatments, their antimicrobial properties are gaining more attention. The antimicrobial peptides (AMPs) are secreted by cells from various perinatal derivatives, including human amnio-chorionic membrane (hACM), human amniotic membrane (hAM), and human chorionic membrane (hCM). By exhibiting antibacterial, antifungal, antiviral, and antiprotozoal activities and immunomodulatory activities, they contribute to ensuring a healthy pregnancy and preventing complications. Several research groups investigated the antimicrobial properties of hACM, hAM, and hCM and their derivatives. These studies advanced basic knowledge of antimicrobial properties of perinatal derivatives and also provided an important insight into the potential of utilizing their antimicrobial properties in a clinical setting. After surveying the studies presenting assays on antimicrobial activity of hACM, hAM, and hCM, we identified several considerations to be taken into account when planning future studies and eventual translation of fetal membranes and their derivatives as antimicrobial agents from bench to bedside. Namely, (1) the standardization of hACM, hAM, and hCM preparation to guarantee rigorous antimicrobial activity, (2) standardization of the antimicrobial susceptibility testing methods to enable comparison of results between various studies, (3) investigation of the antimicrobial properties of fetal membranes and their derivatives in the in vivo setting, and (4) designation of donor criteria that enable the optimal donor selection. By taking these considerations into account, future studies will provide crucial information that will enable reaching the optimal treatment outcomes using the fetal membranes and their derivatives as antimicrobial agents.

Objective: To report a case of bilateral Mooren's ulcer with impending corneal perforation in the right eye and perforated peripheral corneal ulcer in the left eye which successfully underwent Tenon's patch graft (TPG) with multilayered amniotic membrane grafting (AMG) as globe salvaging procedure during COVID-19 pandemic. Methods: Conjunctival resection was done on both the eyes followed by autologous Tenon's grafting with overlay amniotic membrane grafting in the left eye with perforation and multilayered AMG with AMG overlay in the right eye with impending perforation. Post-operatively topical antibiotics and steroids were prescribed. Results: At one month follow-up, the Tenon's graft and multilayered AMG were well-integrated with the surrounding healthy cornea with a good anatomical and visual outcome. Conclusion: Autologous Tenon's patch graft is a simple and cost-effective alternative to preserve globe integrity and prevent complications in emergency cases when immediate access to cornea transplantation is practically challenging.

Type 1 diabetes mellitus (T1DM) is a complex metabolic disease characterized by a massive loss of insulin-producing cells due to an autoimmune reaction. Currently, daily subcutaneous administration of exogenous insulin is the only effective treatment. Therefore, in recent years considerable interest has been given to stem cell therapy and in particular to the use of three-dimensional (3D) cell cultures to better reproduce in vivo conditions. The goal of this study is to provide a reliable cellular model that could be investigated for regenerative medicine applications for the replacement of insulin-producing cells in T1DM. To pursue this aim we create a co-culture spheroid of amniotic epithelial cells (AECs) and Wharton’s jelly mesenchymal stromal cells (WJ-MSCs) in a one-to-one ratio. The resulting co-culture spheroids were analyzed for viability, extracellular matrix production, and hypoxic state in both early- and long-term cultures. Our results suggest that co-culture spheroids are stable in long-term culture and are still viable with a consistent extracellular matrix production evaluated with immunofluorescence staining. These findings suggest that this co-culture may potentially be differentiated into endo-pancreatic cells for regenerative medicine applications in T1DM. Type 1 diabetes mellitus (T1DM) is a complex metabolic disease characterized by a massive loss of insulin-producing cells due to an autoimmune reaction. Currently, daily subcutaneous administration of exogenous insulin is the only effective treatment. Therefore, in recent years considerable interest has been given to stem cell therapy and in particular to the use of three-dimensional (3D) cell cultures to better reproduce in vivo conditions. The goal of this study is to provide a reliable cellular model that could be investigated for regenerative medicine applications for the replacement of insulin-producing cells in T1DM. To pursue this aim we create a co-culture spheroid of amniotic epithelial cells (AECs) and Wharton’s jelly mesenchymal stromal cells (WJ-MSCs) in a one-to-one ratio. The resulting co-culture spheroids were analyzed for viability, extracellular matrix production, and hypoxic state in both early- and long-term cultures. Our results suggest that co-culture spheroids are stable in long-term culture and are still viable with a consistent extracellular matrix production evaluated with immunofluorescence staining. These findings suggest that this co-culture may potentially be differentiated into endo-pancreatic cells for regenerative medicine applications in T1DM.

Human amniotic membranes (hAMs) are extraembryonic tissues currently employed in the treatment of many ocular and systemic diseases. Several reports indicate that hAMs can suppress the signaling pathway of tissue growth factor beta (TGF-β), a cytokine that plays a major role in the pathogenesis of proliferative vitreoretinopathy (PVR) through the induction of epithelial-mesenchymal transition (EMT) in exposed retinal pigmented epithelium (RPE) cells. The present study was conducted to evaluate the efficacy of a modified vitrectomy procedure (hAMP-V) involving the extensive coverage of exposed RPE with hAM patches to prevent postoperative PVR in a series of 15 cases of retinal detachment complicated by severe preoperatory PVR. The primary outcome was to assess the rate of successful retinal reattachment of a single hAMP-V procedure at 6 months from silicone oil removal. Secondary outcomes included the collection of intraoperative data concerning the quantity, size, and scope of hAM patches, and the assessment of postoperative improvements in mean LogMar BCVA at 3 and 6 months. Successful retinal reattachment was obtained in 14 out of 15 eyes (93.3%). Surgical failure due to major recurrence of PVR occurred in 1 out of 15 eyes (6.7%). Postoperative improvements in mean LogMar BCVA were statistically significant (p < 0.05, paired t-test). No intraoperative and postoperative adverse effects were reported. The study helped to refine the surgical technique while also offering cues for future improvements.

The potential clinical applications of human amniotic membrane (hAM) and human amniotic epithelial cells (hAECs) in the field of regenerative medicine have been known in literature since long. However, it has yet to be elucidated whether hAM contains different anatomical regions with different plasticity and differentiation potential. Recently, for the first time, we highlighted many differences in terms of morphology, marker expression, and differentiation capabilities among four distinct anatomical regions of hAM, demonstrating peculiar functional features in hAEC populations. The aim of this study was to investigate in situ the ultrastructure of the four different regions of hAM by means of transmission electron microscopy (TEM) to deeply understand their peculiar characteristics and to investigate the presence and localization of secretory products because to our knowledge, there are no similar studies in the literature. The results of this study confirm our previous observations of hAM heterogeneity and highlight for the first time that hAM can produce extracellular vesicles (EVs) in a heterogeneous manner. These findings should be considered to increase efficiency of hAM applications within a therapeutic context.

Background: Peripheral neuropathy usually leads to a major cause of motor disability, but the functional restoration after treatment continues to show modest results. Acupoint thread-embedding therapy is a subtype of acupuncture treatment in which biodegradable threads are inserted into skin, subcutaneous tissue or muscles at specific points for long stimulation. Different biodegradable materials have been developed and widely used. Human amniotic membrane is rich in collagen, extracellular matrix proteins and growth factors. The avascular, low immunogenic, anti-inflammatory, antibacterial, anti-fibrotic and non-tumorigenic properties of amniotic membrane make it valuable in medical applications and its use has no ethical problems. Elasticity, stiffness and other biomechanical properties also make it possible to use the amniotic membrane for various medical purposes. AM is almost always considered as discarded substance, it satisfies most of the criteria of an ideal biological tissue and shows almost zero rejection phenomenon. Conclusions: The human amniotic membrane, the cellular compounds and extracellular matrix have a lot of benefic proprieties that are or could be used in treatment of many human diseases. Its biological and biomechanical properties are promising in the manufacture and use of filaments in acupoint thread embedding therapy.

Mesenchymal stem cell therapy is considered as a proper tool for biological activities and treatment of diseases and cancer. Here, the suppressing effect of mesenchymal stem cells derived from the amniotic membrane (AM-MSCs) on colorectal cancer (HT29 cell line) was studied. MSCs were isolated from the human amniotic membrane and identity tests were performed. AM-MSC and HT29 cells were co-cultured and MTT assay was used to determine proper inhibitory concentration. The apoptotic effect of AM-MSC on HT29 cells was studied by Acridine orange staining. Expression of CDK2 protein and the antioxidant effect of AM-MSCs on HT29 was performed by immunocyto chemistry test. The effect of MSC on the HT29 cell line migration was determined by Scratch test. The result showed that AM-MSC had proliferation-inhibitory effects and caused apoptosis of the HT29 cell line. Real Time-PCR assay showed the increase of gene expression of p53, Caspase3 in apoptosis, and p21 in tumor cell line treated with AM-MSC. AM-MSC arrested the cell cycle of tumor cells by reduction of CDK2 protein. The migration of HT29 and ROS expression levels was decreased. Altogether, AM-MSC may play a role in suppressing colorectal cancer HT29 cell line by inhibiting angiogenesis, cell cycle, and induction of apoptosis and has antioxidant properties.

Objective: This study was designed to fabricate a suitable permanent scaffold for the normal aligned myotube formation and improve the process of myogenic differentiation of selected stem cells. Materials and Methods: In this experimental study, an engineered scaffold composed of decellularized human amniotic membrane (DHAM) and electrospun fibers of poly(ε-caprolactone) (PCL) was fabricated and characterized. PCL nanofibers were superimposed on DHAM (PCL-DHAM) in two different patterns, including randomized fibers (Random) and aligned fibers (Aligned). Adipose derived stem cells (ADSCs) were isolated from adult Wistar rats and cultured on designed scaffold and induced to myotube differentiation. Using an MTT assay, the vitality of cells was determined. Then, myogenic cell differentiation was assessed using scan electron microscopy (SEM), immunofluorescence assay, and reverse transcription-polymerase chain reaction (RT-PCR). Results: The mechanical properties of engineered PCL-DHAM composite improved significantly compared to DHAM as a control. The engineered PCL-DHAM promoted cell growth and high expression of myosin, Mhc2 and myogenin and thus enhanced the myotube formation. Conclusion: These findings revealed that bio-composite membrane prepared from PCL nanofibers and DHAM, may represent a promising biomaterial as a desirable scaffold for applying in the bioengineered muscle repair.

Joana Roque,1 Fernando Trancoso Vaz,1 Rita Basto,1 Susana Henriques,1 Ana Sofia Lopes,1 Diana Silva,1 Jorge Santos,2 Graça Pires,1 Maria Lisboa,1 Isabel Prieto1 1Ophthalmology Department, Prof. Doutor Fernando Fonseca Hospital, Amadora, Lisbon, Portugal; 2Department of Mathematics ECT, CIMA IIFA Évora University, Évora, PortugalCorrespondence: Joana RoqueOphthalmology Department, Prof. Doutor Fernando Fonseca Hospital, Amadora, Lisbon, IC19 2720-276, PortugalTel +351932828099Email joana.roque@hff.min-saude.ptPurpose: Amniotic membrane transplantation (AMT) has shown promising results as an antifibrotic agent in trabeculectomy. We aimed to evaluate the additional effect of AMT in MMC-augmented trabeculectomy.Patients and Methods: This retrospective study analyzed the results of the first 12 postoperative months of glaucomatous eyes submitted to Moorfields Safer Surgery Trabeculectomy with MMC alone (non-AMT group) compared to MMC and AMT (AMT group). Both groups were compared in terms of intraocular pressure (IOP), number of antihypertensive medications and need for surgical reinterventions. Absolute and relative success rates 12 months after surgery were defined as IOP < 18 mmHg, without and with the use of antihypertensive medications, respectively.Results: The analysis included 51 eyes of 45 glaucoma patients (29 eyes in the non-AMT group and 22 in the AMT group). Mean IOP decreased from 24.72± 5.11 mmHg and 26.86± 10.62 mmHg preoperatively in non-AMT and AMT groups to 12.86± 4.22 mmHg and 12.60± 4.43 mmHg, respectively, at 12 months (p = 0.84). Postoperative number of medications decreased significantly in both groups. Absolute success was seen in 71% of non-AMT eyes and 55% of AMT eyes (p = 0.46), whereas relative success was obtained in 14% and 30%, respectively (p = 0.55). Reinterventions were needed in 28% of the eyes (11 bleb injection/needling and 4 Ahmed tube implantation) in the non-AMT group and in 27% of the AMT group (10 bleb injection/needling and 1 Ahmed tube implantation) (p = 0.89).Conclusion: Trabeculectomy combined with MMC and AMT did not show better results than trabeculectomy with MMC alone.Keywords: trabeculectomy, glaucoma, mitomycin C, amniotic membrane, antifibrotics, scarring

Joana Roque,1 Fernando Trancoso Vaz,1 Rita Basto,1 Susana Henriques,1 Ana Sofia Lopes,1 Diana Silva,1 Jorge Santos,2 Graça Pires,1 Maria Lisboa,1 Isabel Prieto1 1Ophthalmology Department, Prof. Doutor Fernando Fonseca Hospital, Amadora, Lisbon, Portugal; 2Department of Mathematics ECT, CIMA IIFA Évora University, Évora, PortugalCorrespondence: Joana RoqueOphthalmology Department, Prof. Doutor Fernando Fonseca Hospital, Amadora, Lisbon, IC19 2720-276, PortugalTel +351932828099Email joana.roque@hff.min-saude.ptPurpose: Amniotic membrane transplantation (AMT) has shown promising results as an antifibrotic agent in trabeculectomy. We aimed to evaluate the additional effect of AMT in MMC-augmented trabeculectomy.Patients and Methods: This retrospective study analyzed the results of the first 12 postoperative months of glaucomatous eyes submitted to Moorfields Safer Surgery Trabeculectomy with MMC alone (non-AMT group) compared to MMC and AMT (AMT group). Both groups were compared in terms of intraocular pressure (IOP), number of antihypertensive medications and need for surgical reinterventions. Absolute and relative success rates 12 months after surgery were defined as IOP < 18 mmHg, without and with the use of antihypertensive medications, respectively.Results: The analysis included 51 eyes of 45 glaucoma patients (29 eyes in the non-AMT group and 22 in the AMT group). Mean IOP decreased from 24.72± 5.11 mmHg and 26.86± 10.62 mmHg preoperatively in non-AMT and AMT groups to 12.86± 4.22 mmHg and 12.60± 4.43 mmHg, respectively, at 12 months (p = 0.84). Postoperative number of medications decreased significantly in both groups. Absolute success was seen in 71% of non-AMT eyes and 55% of AMT eyes (p = 0.46), whereas relative success was obtained in 14% and 30%, respectively (p = 0.55). Reinterventions were needed in 28% of the eyes (11 bleb injection/needling and 4 Ahmed tube implantation) in the non-AMT group and in 27% of the AMT group (10 bleb injection/needling and 1 Ahmed tube implantation) (p = 0.89).Conclusion: Trabeculectomy combined with MMC and AMT did not show better results than trabeculectomy with MMC alone.Keywords: trabeculectomy, glaucoma, mitomycin C, amniotic membrane, antifibrotics, scarring

Perinatal derivatives are drawing growing interest among the scientific community as an unrestricted source of multipotent stromal cells, stem cells, cellular soluble mediators, and biological matrices. They are useful for the treatment of diseases that currently have limited or no effective therapeutic options by means of developing regenerative approaches. In this paper, to generate a complete view of the state of the art, a comprehensive 10-years compilation of clinical-trial data with the common denominator of PnD usage has been discussed, including commercialized products. A set of criteria was delineated to challenge the 10-years compilation of clinical trials data. We focused our attention on several aspects including, but not limited to, treated disorders, minimal or substantial manipulation, route of administration, dosage, and frequency of application. Interestingly, a clear correlation of PnD products was observed within conditions, way of administration or dosage, suggesting there is a consolidated clinical practice approach for the use of PnD in medicine. No regulatory aspects could be read from the database since this information is not mandatory for registration. The database will be publicly available for consultation. In summary, the main aims of this position paper are to show possibilities for clinical application of PnD and propose an approach for clinical trial preparation and registration in a uniform and standardized way. For this purpose, a questionnaire was created compiling different sections that are relevant when starting a new clinical trial using PnD. More importantly, we want to bring the attention of the medical community to the perinatal products as a consolidated and efficient alternative for their use as a new standard of care in the clinical practice.

Background: Heart failure is one of the most common cardiovascular disorders and is considered a chronic, progressive and debilitating disorder. The medical treatment of this disease is accompanied by many problems. Today, stem cells are being used increasingly to reduce the problems of heart failure treatments. Since pro-inflammatory cytokines play an important role in the prognosis and progression of cardiovascular disease, the present study aimed to investigate the effect of intravenous injection of human amniotic membrane mesenchymal stem cells on the levels of interleukins 4 and 12 in the serum of male rats in the heart failure model. Methods: This is an experimental study that was conducted from October 2018 to May 2019 in the Physiology Research Center of Iran University of Medical Sciences. In this study, 28 male wistar rats (180-200 gr) were randomly divided into four groups: control group, heart failure group, heart failure group that received culture medium and heart failure group that received mesenchymal stem cells by intravenous injection. After 30 days, echocardiography was done and then serum levels of interleukin 4 and 12 were measured in these groups by Elisa test. Results: The results of this study showed that intravenous injection of human amniotic membrane mesenchymal stem cells into male rats with heart failure, improved echocardiographic parameters such as ejection fraction (EF) and fractional shortening (FS) in the cell injection group compared to the heart failure group (P

Background Locoregional stem cell delivery is very important for increasing the efficiency of cell therapy. Amnisite BA (Amnisite) is a freeze-dried amniotic membrane harvested from bovine placenta. The objective of this study was to investigate the retention of cells of the stromal vascular fraction (SVF) on Amnisite and to determine the effects of cell-loaded Amnisite in a porcine radiation-induced chronic wound model.Methods Initially, experiments were conducted to find the most suitable hydration and incubation conditions for the attachment of SVF cells extracted from pig fat to Amnisite. Before seeding, SVFs were labeled with PKH67. The SVF cell-loaded Amnisite (group S), Amnisite only (group A), and polyurethane foam (group C) were applied to treat radiation-induced chronic wounds in a porcine model. Biopsy was performed at 10, 14, and 21 days post-operation for histological analysis.Results Retaining the SVF on Amnisite required 30 minutes for hydration and 1 hour for incubation. A PKH67 fluorescence study showed that Amnisite successfully delivered the SVF to the wounds. In histological analysis, group S showed increased re-epithelialization and revascularization with decreased inflammation at 10 days post-operation.Conclusions SVFs had acceptable adherence on hydrated Amnisite, with successful cell delivery to a radiation-induced chronic wound model.

The use of perinatal mesenchymal stem cells (MSCs) in bone tissue regeneration and engineering to substitute bone marrow MSCs has drawn great interest due to their high yield, ease of procurement, multilineage differentiation potential and lack of ethical concerns. Although amniotic membrane (AM) and Wharton’s jelly (WJ)-derived MSCs have been widely shown to possess osteogenic differentiation potential, the intrinsic properties determining their osteogenic capacity remain unclear. Here, we compared gene expression profiles of AM- and WJ-MSCs at basal and osteogenic conditions by using the NanoString Stem Cell Panel containing regulatory genes associated with stemness, self-renewal, Wnt, Notch and Hedgehog signalling pathways. At basal condition, WJ-MSCs displayed higher expression in most genes regardless of their functional roles in self-renewal, adhesion, or differentiation signalling pathways. After osteo-induction, elevated expression of self-renewal genes ADAR and PAFAH1B1 was observed in AM-MSCs, while stemness genes MME and ALDH1A1 were upregulated in WJ-MSC. Both MSCs showed differences in genes associated with ligands, receptors and ubiquitin ligases of the Notch pathway. In addition, further evidence was demonstrated in some signalling molecules including CTBPs, protein kinases, phosphatases, RHOA, RAC1. Downstream targets HES1 and JUN especially showed higher expression in non-induced WJ-MSCs. Hedgehog genes initially expressed in both MSCs were downregulated in WJ-MSCs during osteogenesis. This study has provided insights into the intrinsic biological differences that may lead to their discrimination in therapeutic intervention.

Perinatal derivatives (PnD) are birth-associated tissues, such as placenta, umbilical cord, amniotic and chorionic membrane, and thereof-derived cells as well as secretomes. PnD play an increasing therapeutic role with beneficial effects on the treatment of various diseases. The aim of this review is to elucidate the modes of action of non-hematopoietic PnD on inflammation, angiogenesis and wound healing. We describe the source and type of PnD with a special focus on their effects on inflammation and immune response, on vascular function as well as on cutaneous and oral wound healing, which is a complex process that comprises hemostasis, inflammation, proliferation (including epithelialization, angiogenesis), and remodeling. We further evaluate the different in vitro assays currently used for assessing selected functional and therapeutic PnD properties. This review is a joint effort from the COST SPRINT Action (CA17116) with the intention to promote PnD into the clinics. It is part of a quadrinomial series on functional assays for validation of PnD, spanning biological functions, such as immunomodulation, anti-microbial/anti-cancer activities, anti-inflammation, wound healing, angiogenesis, and regeneration.

Objective: The amniotic membrane, a thin membrane, may be used as a temporary cover on deep burns. The objective of this study is to identify the differences regarding bacterial contamination between membranes of vaginal and cesarean deliveries, as well as to assess the possibility of the clinical use of stored membranes.Methods: Twelve membranes were obtained from women submitted to vaginal and cesarean deliveries at the Obstetric Center of Hospital de Clínicas de Porto Alegre. Each amniotic membrane was stored in five different flasks containing a physiological solution. Samples were obtained from these flasks for analysis on days 1, 7, 14, 21 and 28. These samples were tested for bacterial contamination, analyzing its relation to time of storage and type of delivery. This is a pilot study with a transversal design.Results: The comparison between types of delivery yielded a relative risk of contamination in vaginal delivery (RR) of 2.67 (95% CI: 1.09-6.52) and no significance (P = 0.08). No contamination was found on day 1 flasks.Conclusion: All membranes derived from vaginal deliveries ended up showing bacterial contamination during the storage period, which lead to a theoretical unavailability for its use on Amniotic Membrane Banks., Objetivo: A membrana amniótica, uma membrana fina, pode ser utilizada como cobertura temporária em queimaduras profundas. O objetivo deste estudo é o de verificar as possíveis diferenças quanto à contaminação bacteriana entre as membranas de partos cesáreo e vaginal, assim como avaliar a viabilidade ou não do uso clínico-cirúrgico da membrana armazenada.Métodos: Foram coletadas 12 membranas amnióticas de mulheres submetidas a parto cesáreo e vaginal no Centro Obstétrico do HCPA. Cada membrana amniótica foi armazenada em cinco frascos diferentes contendo soro fisiológico, dos quais foram obtidas amostras para análise no momento da coleta e nos dias 7, 14, 21 e 28. Essas amostras foram testadas quanto à contaminação bacteriana, analisando sua relação com o tempo de armazenamento e com o tipo de parto realizado. O estudo é um piloto e tem um delineamento transversal.Resultados: A comparação entre os tipos de parto mostrou um risco relativo (RR) de 2,67 de contaminação no parto vaginal em relação à cesariana (IC de 95%: 1,09 a 6,52), P = 0,08. Não foi verificada contaminação em nenhum dos frascos no momento da coleta.Conclusão: Todas as membranas coletadas de parto vaginal apresentaram crescimento bacteriano no processo de estocagem, levando à sua inviabilidade teórica para uso em Bancos de Membrana Amniótica.

Background The concept of periodontal regeneration has been revolutionised since the introduction of growth factors and bioactive bone substitutes which ensures optimal regeneration of the diseased periodontium. The aim of the present study was to evaluate the efficacy of Amniotic membrane + Biphasic Calcium phosphate as compared to Collagen membrane + Biphasic Calcium phosphate for the management of periodontal intrabony defects. Methods 50 systemically healthy patients with localised moderate to severe periodontitis, sites which had a Probing Pocket Depth (PPD) ≥ 6 mm and an intrabony component of ≥ 3 mm as detected on Intra oral periapical radiographs (IOPAR) and bone sounding were recruited based on specific inclusion and exclusion criteria. They were randomly allocated by computer generated tables to Collagen membrane + Biphasic Calcium phosphate and Amniotic membrane + Biphasic Calcium phosphate groups. The amount of bone fill and changes in Probing Pocket Depth, Clinical Attachment Level were measured at baseline and six months. Results The results of the present study showed a mean reduction in the PPD of 2.89 ± 0.69 mm in the Collagen membrane + Biphasic Calcium phosphate group and 2.95 ± 0.57 mm in the Amniotic membrane + Biphasic Calcium phosphate group and CAL gain of 2.60 ± 1.43 mm in Collagen membrane + Biphasic Calcium phosphate group 3.18 ± 1.13 mm in the Amniotic membrane + Biphasic Calcium phosphate group at 6 months follow-up with no statistical significance between the groups. In terms of Defect resolution, 98.62 ± 6.51 % was achieved in Collagen membrane + Biphasic Calcium phosphate group and 98.25 ± 7.21 % in Amniotic membrane + Biphasic Calcium phosphate group. Conclusions Within the limitations of the present study, it can be concluded that AM can be used as a barrier membrane, in conjunction with Biphasic calcium phosphate, and provides comparable results to Collagen membrane with Biphasic calcium phosphate when used in the management of periodontal intrabony defects.

This case report illustrates the successful use of the double-layer amniotic membrane technique in a child with glaucoma and aniridia. A 3.5-year-old girl with bilateral congenital glaucoma as well as aniridia, lens coloboma, nystagmus, and strabismus had been followed up since birth. Medical treatment did not result in the desired intraocular pressure improvement. An Ahmed glaucoma valve was implanted bilaterally. In a follow-up visit at 2 years of age, there were complaints of redness with watery discharge in the right eye. A biomicroscopic evaluation revealed tube erosion of the conjunctiva. The eroded area was dissected from the surrounding tissue and the area was closed using the new double-layer amnion membrane technique. After the surgery, the intraocular pressure was normal. Subsequent follow-up indicated that the erosion defect was closed and stabilized. The double-layer amniotic membrane technique can be used successfully in cases of conjunctival tube erosion. To the authors' knowledge, this is the first published pediatric case report of using the novel technique of amniotic membrane transplantation for tube erosion.

Recent reviews suggest that amniotic membrane products may accelerate healing of diabetic foot ulcers. A new dried human amniotic membrane (dHAM) has been used for ocular ulcers but not for diabetic foot ulcers. This was a multi‐centre, prospective, patient and observer blind, randomised controlled pilot trial, to investigate whether 2 weekly addition of the dHAM to standard care versus standard care alone increased the proportion of healed participants' index foot ulcers within 12 weeks. Thirty‐one people (mean age 59.8 years, 81% male, 87% type 2 diabetes) were randomised (15 dHAM, 16 usual care). Within 12 weeks, healing occurred in 4 (27%) ulcers in the dHAM group versus 1 (6.3%) usual care group (P = .1). Percentage wound area reduction was higher in the dHAM versus control group. (P = .0057). There was no difference in AEs between the two groups. Six participants allocated to dHAM correctly identified their treatment group, although 5 in usual care incorrectly thought they were in the intervention arm. This pilot trial result is encouraging showing that this dHAM preparation is safe and promising treatment. These results will be used to design a statistically powered, definitive double blind randomised controlled trial.

Amnijska membrana (AM) predstavlja alternativno možnost za zdravljenje bakterijskih okužb, med njimi tudi okužb urinarnega trakta. Z difuzijskim testom na agarju smo testirali učinek homogenata humane amnijske membrane (hAM), pripravljenega na različne načine, na rast sevov bakterije Escherichia coli DH5α, DL88, DL90, DL94, DL101, DL102 ter na rast bakterij Staphylococcus aureus (S. aureus), Enterobacter species (Enterobacter sp.) in Serratia mercescens (S. mercescens). Ugotovili smo, da izbira homogenizatorja ter sam postopek priprave hAM (npr. filtriranje skozi 0,22 µm filter), vpliva na protimikrobni učinek homogenata hAM. S poliakrilamidno gelsko elektroforezo v prisotnosti natrijevega dodecilsulfata (SDS-PAGE) smo ugotovili, da obstajajo razlike v proteinski sestavi homogenatov hAM, pripravljenih na različne načine. Pridobljeni rezultati o aktivnosti pripravkov iz hAM so nam tako razkrili, katerim korakom pri pripravi homogenata hAM je treba nameniti posebno pozornost v nadaljnjih raziskavah. Amniotic membrane (AM) is an alternative solution for the treatment of bacterial infections, including urinary tract infections. Antimicrobial effect of various preparations of human amniotic membrane (hAM) on different bacterial strains was tested using the diffusion method. Included were Escherichia coli strains DH5α, DL88, DL90, DL94, DL101, DL102 and bacteria Staphylococcus aureus (S. aureus), Enterobacter species (Enterobacter sp.) and Serratia mercescens (S. mercescens). We found that the choice of homogenizer and the process of hAM preparation (e.g. filtration through a 0.22 μm filter) influenced the antimicrobial effect of hAM homogenate. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) we found differences in the composition of the different hAM preparations. The obtained results on the activity of hAM preparations thus revealed to which steps in the preparation of the hAM homogenate special attention in further research should be given.

The usage of canine amniotic membrane (cAM) is mainly of interest in veterinary ophthalmology. Topical formulations of cAM could deliver the beneficial properties of cAM without the need for surgical intervention. The present study aimed to investigate biological compositions of cAM and its extracts, including their corneal wound healing efficacy. In this study, canine amniotic membrane extract (cAME) and lyophilized canine amniotic membrane extract (cAMX) were developed. Bioactive molecules related to corneal wound healing, including hepatocyte growth factor, tissue inhibitor of metalloproteinase-1 and -2, Thrombospondin-1 and Interleukin-1 receptor antagonist were studied at both gene and protein expression levels. Cell viability and wound healing assays were investigated for the possibility of cAME and cAMX as topical applications. The results demonstrated that all of the relevant genes and proteins were detected in cAM, cAME and cAMX. Both cAME and cAMX showed wound healing properties in vitro and cAME at 1.0 mg/mL concentration appeared to have the best healing efficacy. In conclusion, cAME and cAMX generated for topical use provided promising results in the healing of corneal defects.

Superparamagnetic iron oxide nanoparticles (SPIONs) have made extensive advances in nanotechnology. The unique properties of these particles have expanded their application in various fields, including medicine. One of these applications is non-invasive analysis for cell tracking. However, the possibility of toxicity in cells is reported by these nanoparticles. Due to the fact that cellular damage caused by iron oxide nanoparticles is concentration-dependent, the determination of the appropriate concentration of iron oxide nanoparticles is very important to prevent cell damage or cell death due to apoptosis. The aim of this study was to find a concentration of SPIONs which does not result in apoptosis. Therefore, the effects of different concentrations of iron oxide nanoparticles on cell survival were investigated, and the their effects on increased gene expression involved in apoptosis (p53) in human amniotic membrane derived mesenchymal stem cells (hAMSCs) were evaluated. First, stem cells were extracted from human amniotic membrane tissue and cultured. To demonstrate the multipotent characteristic of hAMSCs, these cells were differentiated into adipose, bone, and chondrocyte cell lines. Then, the viability of the cells treated with different concentrations of iron oxide nanoparticles (200, 150, 100, 50, 0 μg / ml) over a period of 24 and 48 hours was evaluated by MTT method. The effect of the concentrations of 0, 100,150 and 200 μg / ml of nanoparticles after 24 hours in hAMSCs was investigated for the expression of p53 gene by Real-Time PCR. hAMSCs were spindle-shaped in a two-dimensional culture. Flow cytometry examination of surface markers revealed that these cells were able to express CD 29, CD90 and CD105 but they were unable to express CD34 and CD45. The results of the multi-potency assay of hAMSCs showed that these cells were capable of being differentiated into adipocyte, bone and chondrocyte cell lines. Iron oxide nanoparticles had no significant effect on cell survival at the concentrations of 50 and 100 μg / ml in 24 hours. However, cell viability decreased significantly after the concentration of 150 μg / ml (42 ± 1.4%, p

Objectives. Human amniotic membrane extract (AME) is known to contain numerous bioactive factors and anti-inflammatory substances. However, the anti-inflammatory effects of AME on the middle ear (ME) mucosa are unclear. This study assessed the effects of AME on the growth of the ME mucosa in response to bacterially-induced otitis media (OM).Methods. OM was induced by inoculating nontypeable Haemophilus influenzae (NTHi) into the ME cavity of rats. ME mucosal explants were cultured in AME concentrations of 0, 5, 10, or 50 μg/mL. The area of explant outgrowth was measured in culture and analyzed at 1, 3, 5, and 7 days after explantation. The expression of Ki-67, mucin 5AC (MUC5AC), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) in the explants was also evaluated using quantitative polymerase chain reaction (PCR) and immunocytochemistry (ICC).Results. The NTHi-induced ME mucosa growth increased gradually over the 7-day culture period in all explants at different AME concentrations. There was a trend for mucosal growth inhibition at higher concentrations of AME, although the growth was not significantly different among the groups until day 5. The ME mucosal explants treated with the 50 μg/mL concentration of AME showed significantly suppressed growth on postexplantation day 7 compared with other explants on the same day. PCR and ICC staining revealed that the expression of Ki-67, MUC5AC, TNF-α, and IL-10 further decreased in the explants with higher concentrations of AME than in those with lower concentrations of AME.Conclusion. Our results showed that higher concentrations of AME reduced the mucosal proliferative response in bacterial OM in rats. These findings provide evidence that AME has an influence on the inflammatory and proliferative responses to NTHi infection in ME mucosa.

Olivia G Mead, Leon P Mead Leon P Mead MD PA, Naples, FL, USACorrespondence: Leon P MeadLeon Mead MD PA, 730 Goodlette Road North, Suite 201, Naples, FL 34102, USATel +1 239-262-1119Fax +1 239-204-3104Email orthomead@aol.comObjective: To evaluate the long-term benefit of a single injection of amniotic membrane/umbilical cord (AM/UC) particulate in patients with moderate to severe knee osteoarthritis (OA).Methods: A single-center, investigator-initiated, retrospective study of patients who received intra-articular injection of 100 mg lyophilized and micronized AM/UC for radiographically confirmed, Kellgren–Lawrence (KL) grade 3 or 4 symptomatic knee OA. Data regarding demographics, OA severity, comorbidities, treatment regimens, complications, and patient-reported outcomes were collected and assessed. Patient Global Impression of Change (PGIC) was assessed on a 7-point scale, and Global Perceived Improvement (GPI), expressed as percent improvement relative to baseline, was used to further quantify the degree of symptomatic change. Clinically important response to treatment was assessed at 12 months using simplified OMERACT–OARSI responder criteria.Results: A total of 42 patients with KL grade 3 (36%) and 4 (64%) knee OA were included for analysis. Prior to injection, patient-rated pain was 6.6 ± 1.5 out of 10 (median: 7, range: 3– 10) despite prior treatment with oral/topical NSAIDs (62%) and intra-articular injection(s) of corticosteroids (57%) and/or hyaluronic acid (48%). Twelve months following AM/UC injection, 31 patients (74%) reported significant clinical improvement via PGIC, and the OMERACT–OARSI treatment response rate was 81% (34/42). GPI of pain and function was 62 ± 24%, 69 ± 27%, 69 ± 27%, and 64 ± 31% at 1, 3, 6, and 12 months, respectively. Symptomatic improvement was maintained for an average duration of 12.1 ± 4.5 months (median: 12, range: 3– 22). One patient developed swelling in the knee within 36 hours of injection. No other adverse events or complications were reported.Conclusion: Intra-articular injection of AM/UC particulate may be effective in alleviating pain and improving function in patients with moderate to severe knee OA, with the potential to delay total knee replacement for up to 12 months.Keywords: amniotic membrane, injection, knee, osteoarthritis, umbilical cord

Olivia G Mead, Leon P Mead Leon P Mead MD PA, Naples, FL, USACorrespondence: Leon P MeadLeon Mead MD PA, 730 Goodlette Road North, Suite 201, Naples, FL 34102, USATel +1 239-262-1119Fax +1 239-204-3104Email orthomead@aol.comObjective: To evaluate the long-term benefit of a single injection of amniotic membrane/umbilical cord (AM/UC) particulate in patients with moderate to severe knee osteoarthritis (OA).Methods: A single-center, investigator-initiated, retrospective study of patients who received intra-articular injection of 100 mg lyophilized and micronized AM/UC for radiographically confirmed, Kellgren–Lawrence (KL) grade 3 or 4 symptomatic knee OA. Data regarding demographics, OA severity, comorbidities, treatment regimens, complications, and patient-reported outcomes were collected and assessed. Patient Global Impression of Change (PGIC) was assessed on a 7-point scale, and Global Perceived Improvement (GPI), expressed as percent improvement relative to baseline, was used to further quantify the degree of symptomatic change. Clinically important response to treatment was assessed at 12 months using simplified OMERACT–OARSI responder criteria.Results: A total of 42 patients with KL grade 3 (36%) and 4 (64%) knee OA were included for analysis. Prior to injection, patient-rated pain was 6.6 ± 1.5 out of 10 (median: 7, range: 3– 10) despite prior treatment with oral/topical NSAIDs (62%) and intra-articular injection(s) of corticosteroids (57%) and/or hyaluronic acid (48%). Twelve months following AM/UC injection, 31 patients (74%) reported significant clinical improvement via PGIC, and the OMERACT–OARSI treatment response rate was 81% (34/42). GPI of pain and function was 62 ± 24%, 69 ± 27%, 69 ± 27%, and 64 ± 31% at 1, 3, 6, and 12 months, respectively. Symptomatic improvement was maintained for an average duration of 12.1 ± 4.5 months (median: 12, range: 3– 22). One patient developed swelling in the knee within 36 hours of injection. No other adverse events or complications were reported.Conclusion: Intra-articular injection of AM/UC particulate may be effective in alleviating pain and improving function in patients with moderate to severe knee OA, with the potential to delay total knee replacement for up to 12 months.Keywords: amniotic membrane, injection, knee, osteoarthritis, umbilical cord

AIM:To evaluate the clinical effect of human fibrin glue in pterygium excision combined with fresh amniotic membrane transplantation and its clinical value. METHODS: This study was a prospective analysis of 153 pterygium excision patients(153 cases, 153 eyes)from March 2018-October 2018. The patients were randomly and evenly divided into two groups: observation group(78 eyes)and control group(75 eyes). In fibrin glue group, patients(78 eyes)underwent pterygium excision, and the bare selera was covered with fresh amniotic membrane using fibrin glue for graft adherence, while in suture group, patients(75 eyes), the an amaiotic membrane grafts was attached to the selera using 10-0 nylon sutures after pterygium excision. Record the surgical time and the degree of pain after surgery were evaluated at 2h, 1, 3, 5 and 10d(before sutures out)after surgery. RESULTS: Average surgical time(14.5±3.6)min in fibrin glue group was significantly less than suture group(18.3±4.2)min(PF=39.939, 419.026, 28.410, all PPPP>0.05).CONCLUSION: It not only significantly reduces the surgical time, but also minimizes postoperative discomfort when we use fibrin glue in pterygium excisions. But it cannot decrease the recurrence rate.

Amniotic membrane stem cells are considered as a good alternative to embryonic stem cells, but their use in clinical studies is still not common. Here, exosomes from canine amniotic membrane mesenchymal stem cells (cAmMSCexo) were used for dog sperm cryopreservation. Upon cryopreserved straws using cryoprotectant containing 0, 0.5, 1, or 2 μg/mL of cAmMSC-exo were thawed, motility and membrane integrity were analyzed. However, results showed no significant differences between the groups. We concluded that cAmMSC-exo with lower than 2 µg/mL have no effects on sperm cryopreservation, and further studies to get higher concentrations of cAmMSC-exo should be conducted for clinical application.

Peripheral ulcerative keratitis (PUK) is a group of corneal disorders that cause peripheral corneal thinning, threatening globe integrity in advance stages. It is usually associated with systemic autoimmune diseases and management is based on local and systemic approaches. We present the case of a 47-year-old man with a previous history of bacterial keratitis in his left eye presenting with 1 month of bilateral ocular pain and redness. At examination, diffuse bilateral globe inflammation with paracentral corneal thinning in his left eye was observed. He was diagnosed with bilateral scleritis and PUK in his left eye. Workup for associated systemic autoimmune disease yielded negative results. The patient was started on pulses of intravenous methylprednisolone followed by oral prednisone failing to achieve sufficient control of the inflammatory syndrome. Subsequently, periodic intravenous cyclophosphamide was administered with a favorable response. A multilayer amniotic membrane graft was applied, but there was rapid melting with reabsorption of the tissue, resulting in extreme corneal thinning at the inferior paracentral cornea. A decentered 8.5-mm superficial anterior lamellar keratoplasty (SALK) was then performed obtaining the donor graft with a femtosecond laser but performing manual trepanation in the recipient. At 12 months, visual and biomicroscopic measures do not show deterioration and inflammation remains under control with oral azathioprine as maintenance regime. The management of PUK includes both systemic immunosuppression and tectonic procedures to preserve the globe integrity. Diverse surgical techniques have been attempted, but no definitive guidelines are available. Decentered large SALK is a simple technique that can yield acceptable visual results.

Background: Corneal neovascularization can result from many pathological processes affecting the ocular surface leading to disturbances and opacifications that reduce corneal clarity and may impact vision. In veterinary medicine, the use of topical corticosteroid is contraindicated in the presence of ulcerative keratitis, and there is sparse research regarding safe medical alternatives to inhibit corneal neovascularization in dogs to improve visual outcome.Aim: To investigate the pigment epithelium-derived factor (PEDF) concentration in equine amniotic membrane homogenate (EAMH) and its in-vitro vascular endothelial growth factor (VEGF) inhibition in tears of dogs with vascularized ulcerative keratitis.Methods: Homogenates from 10 equine amniotic membranes (AM) were analyzed by sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of equine PEDF and VEGF. Forty tear samples were collected from both eyes of dogs diagnosed with vascularized ulcerative keratitis, and 50 samples from healthy dogs. Samples from affected eyes were allocated to G1 – affected undiluted tears; G2 – affected tears diluted with phosphate-buffer solution; G3 – affected tears treated with low-concentrated EAMH; and G4 – affected tears treated with high-concentrated EAMH. Tears from the unaffected contralateral eyes were composed in G5, while G6 was composed by tears from healthy dogs (control). The presence and levels of VEGF were evaluated in all groups by Western blot and ELISA.Results: The PEDF:VEGF ratio in EAMH was 110:1. An increase in VEGF levels was observed in tears from eyes with vascularized corneal ulcers (G1) as well as in contralateral tears (G5), compared to normal dogs (G6). Highconcentrated EAMH provided a greater decrease in VEGF levels in-vitro compared to low-concentrated EAMH.Conclusion: EAMHs exhibited high concentrations of PEDF in comparison to VEGF and were able to partially decrease VEGF levels in tears of dogs with vascularized ulcers, in-vitro. Our results suggest that VEGF concentration is elevated in tears of dogs with active vascularized ulcerative keratitis in both affected and contralateral eyes compared to that of healthy dogs. Keywords: Amniotic membrane, Corneal neovascularization, Dog, PEDF, VEGF.

We compared cellular viability between cryopreserved and lyopreserved amniotic membranes and clinical outcomes of the lyopreserved construct in a prospective cohort study of 40 patients with neuropathic foot ulcers. Patients received weekly application of lyopreserved membrane for 12 weeks with standard weekly debridement and offloading. We evaluated the proportion of foot ulcers that closed, time to closure, closure trajectories, and infection during therapy. We used chi‐square tests for dichotomous variables and independent t‐tests for continuous variables with an alpha of α = .10. Cellular viability was equivalent between cryo‐ and lyopreserved amniotic tissues. Clinically, 48% of subjects' wounds closed in an average of 40.0 days. Those that did not close were older (63 vs 59 years, P = .011) and larger ulcers at baseline (7.8 vs 1.6 cm2, P = .012). Significantly more patients who achieved closure reached a 50% wound area reduction in 4 weeks compared with non‐closed wounds (73.7% vs 47.6%, P = .093). There was no difference in the slope of the wound closure trajectories between closed and non‐closed wounds (0.124 and 0.159, P = .85), indicating the rate of closure was similar. The rate of closure was 0.60 mm/day (SD = 0.47) for wounds that closed and 0.50 mm/day (SD = 0.58) for wounds that did not close (P = .89).

Primary Sjogren's syndrome is a complex systemic autoimmune disorder that primarily affects exocrine glands such as the lacrimal glands. Dry eye disease is one of the most prevalent complications of Sjogren's syndrome, affecting most patients. It significantly impairs quality of life and management is often difficult and unsatisfactory, in part due to weak correlation between symptoms and signs and poor recognition of the three main subtypes aqueous-deficient, evaporative and neuropathic dry eye. This review provides an overview of key aspects of dry eye disease, such as its multifactorial aetiology and recent insights into pathophysiology. The uses and pitfalls of commonly-used diagnostic tests for dry eye are reviewed, as well as the increasing number of new imaging technologies and biomarkers to refine diagnosis. There are many current and emerging treatment options for dry eye in Sjogren's syndrome, but high-level evidence of efficacy is mostly lacking, as are evidence-based treatment algorithms. All these aspects make the management of dry eye in Sjogren's syndrome challenging.

Perinatal tissues are an abundant source of human extracellular matrix proteins, growth factors and stem cells with proved potential use in a wide range of therapeutic applications. Due to their placental origin, these tissues possess unique biological properties, including being angiogenic, anti-inflammatory, anti-fibrotic, anti-microbial and immune privileged. Additionally, as a temporary organ, placenta is usually discarded as a medical waste, thus providing an easily available, cost effective, 'unlimited' and ethical source of raw materials. Although some of these tissues, such as the amniotic membrane and umbilical cord, have been used in clinical practices, most of them continue to be highly under explored. This review aims to outline the most relevant applications of perinatal tissues as a source of biomaterials and stem cells in the exciting fields of tissue engineering and regenerative medicine (TERM), as well as highlight how these solutions can be used to overcome the shortage of adequate scaffolds and cell sources that currently hampers the translation of TERM strategies towards clinical settings. STATEMENT OF SIGNIFICANCE: Stem cells and extracellular matrix derived from perinatal tissues such as placenta and umbilical cord, have drawn great attention for use in a wide variety of applications in the biomedical field. Due to their origin, these tissues possess unique biological properties, including being angiogenic, anti-inflammatory, anti-fibrotic, anti-microbial and immune privileged. Also they are typically considered medical waste, thus providing an easily available, cost effective, 'unlimited' and ethical source of raw materials. This work aims to present and discuss the most relevant applications of perinatal tissues as a source of biomaterials and stem cells in the exciting fields of tissue engineering and regenerative medicine (TERM). published

Andrew Walkden1,2 1Manchester Royal Eye Hospital, Manchester University Foundation Trust, Manchester, UK; 2University of Manchester Faculty of Medical and Human Sciences, Manchester, Greater Manchester, UKCorrespondence: Andrew Walkden Email walkdenandrew@gmail.comAim: The aim of this paper is to provide a succinct literature review of the different clinical applications for AMT usage in an ophthalmic setting, ranging from commonly used applications to less mainstream approaches. The hope is that this review enables the reader to have a better understanding of the biological properties of amnion as well as the indications and scenarios in which AMT can be used, whilst presenting relevant evidence from within the literature which may be of interest. We also provide an update on the methods of preservation of amniotic membrane and the application methodologies.Methods: Literature search. A PubMed search was performed using the search terms “amniotic membrane transplant”, “amnion AND cornea”, amnion AND ophthalmology”, “amnion AND ocular surface” and “Amnion AND eye”. A full review of the literature using the PubMed database was conducted up until 01/05/20. The articles used were written in English, with all articles accessed in full. Both review articles and original articles were used for this review. All full publications related to ophthalmology were considered.Keywords: amnion, amniotic membrane transplant, amniotic membrane graft, amniotic membrane

Primary Sjogren's syndrome is a complex systemic autoimmune disorder that primarily affects exocrine glands such as the lacrimal glands. Dry eye disease is one of the most prevalent complications of Sjogren's syndrome, affecting most patients. It significantly impairs quality of life and management is often difficult and unsatisfactory, in part due to weak correlation between symptoms and signs and poor recognition of the three main subtypes aqueous-deficient, evaporative and neuropathic dry eye. This review provides an overview of key aspects of dry eye disease, such as its multifactorial aetiology and recent insights into pathophysiology. The uses and pitfalls of commonly-used diagnostic tests for dry eye are reviewed, as well as the increasing number of new imaging technologies and biomarkers to refine diagnosis. There are many current and emerging treatment options for dry eye in Sjogren's syndrome, but high-level evidence of efficacy is mostly lacking, as are evidence-based treatment algorithms. All these aspects make the management of dry eye in Sjogren's syndrome challenging.

Vyshnevska H. O., Kakabadze Z. Sh., Schnayder S. A., Zayats L. M., Tiron O. I., Vastyanov R. S. A comparative study on the effectiveness of application of different types of biological matrixes on mandibular alveolar mucosa lesion healing. Journal of Education, Health and Sport. 2020;10(6):382-393. eISSN 2391-8306. DOI http://dx.doi.org/10.12775/JEHS.2020.10.06.040 https://apcz.umk.pl/czasopisma/index.php/JEHS/article/view/JEHS.2020.10.06.040 https://zenodo.org/record/4056927 The journal has had 5 points in Ministry of Science and Higher Education parametric evaluation. § 8. 2) and § 12. 1. 2) 22.02.2019. © The Authors 2020; This article is published with open access at Licensee Open Journal Systems of Nicolaus Copernicus University in Torun, Poland Open Access. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author (s) and source are credited. This is an open access article licensed under the terms of the Creative Commons Attribution Non commercial license Share alike. (http://creativecommons.org/licenses/by-nc-sa/4.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited. The authors declare that there is no conflict of interests regarding the publication of this paper. Received: 29.05.2020. Revised: 02.06.2020. Accepted: 30.06.2020. UDC 616.314-092.9:577.152.3 A COMPARATIVE STUDY ON THE EFFECTIVENESS OF APPLICATION OF DIFFERENT TYPES OF BIOLOGICAL MATRIXES ON MANDIBULAR ALVEOLAR MUCOSA LESION HEALING H. O. Vyshnevska1, Z. Sh. Kakabadze2, S. A. Schnayder1, L. M. Zayats3, O. I. Tiron4, R. S. Vastyanov4 1SI “Institute of Dentistry and Maxillofacial Surgery of the National Academy of Medical Sciences of Ukraine”, Odessa, Ukraine; 2Tbilisi State Medical University, Tbilisi, Georgia; 3Ivano-Frankivsk National Medical University, Ivano-Frankivsk, Ukraine 4Odessa National Medical University, Odessa, Ukraine Abstract Periodontal diseases is one of the most relevant and important problem in modern dentistry due to their increasing prevalence in population, association with the occurrence of focus of chronic infection, progressive development and irreversibility which, in case of severe damage of periodontal tissues, eventually leads to a loss of the number of teeth. All of the named factors eventually lead to the development of irreversible inflammatory-dystrophic and destructive changes in periodontal tissues. During the recent years the method of guided tissue regeneration that uses membranes is being successfully used for the treatment of periodontal diseases. A comparative study on the mandibular alveolar mucosa lesion healing during application of different types of biological matrixes containing decellularized amniotic membrane, decellularized amniotic membrane in combination with platelet-rich plasma (PRP) growth factors, and decellularized amniotic membrane in combination with PRP growth factors and hyaluronic acid was performed. The study proved the acceleration of the processes of wound area reduction. Under experimental conditions, the wound healing lasted from day 3 to day 5 of the study; however, the rate of reduction of the wound area was slower, which indicates an alterative stage of the acute inflammatory process. During days 5 – 7 (stage of regeneration, the 2nd phase of wound process) the wound area reduced rapidly and by day 14 a complete epithelization of the surgical wound was observed in all groups using decellularized amniotic membrane. In the group using the combination of PRP growth factors and hyaluronic acid the epithelization of wound was already observed on the day 5. In the control group healing occurres with scar formation. Key words: amniotic membrane; PRP growth factors; hyaluronic acid; epithelization; wound healing.

Arun C Gulani, Aaishwariya A Gulani Gulani Vision Institute, Jacksonville, FL, USACorrespondence: Arun C GulaniGulani Vision Institute, 8075 Gate Parkway West Suite 102, Jacksonville, FL 32216, USATel +1 904 296 7393Fax +1888 650-0744Email gulanivision@gulani.comBackground: To demonstrate the long-term results of enhanced cosmetic pterygium surgery with extensive Tenonectomy, adjunctive fibrin-glued amniotic membrane transplantation (AMT), and mitomycin C (MMC).Methods: Retrospective chart review of patients who had pterygium surgery with AMT and MMC between January 2001 to July 2017 and had completed at least 6 months of follow-up. Early and long-term postoperative cosmetic outcomes, recurrence rate, and complications were analyzed. Cosmetic outcomes were evaluated based on patient and surgeon reported outcome measures.Results: The study was conducted on a total of 603 eyes of 578 patients (316 males, 262 females) with an average age of 52.9 ± 15.1 years. At post-op day 1, patients reported no discomfort and could not tell which eye had surgery based on patient reported subjective grading scales. Over an average follow-up period of 23.1 ± 35 months (range: 6– 216 months), there was one pterygium recurrence (0.2%), eighteen granulomas (2.9%), one self-resolving scleral melt (0.2%), one correctable restricted ocular motility (0.2%), one pupil abnormality (0.2%), one dellen (0.2%) and one correctable upper lid abnormality (0.2%). Planned laser vision correction was used for residual corneal scar in eleven eyes (1.8%) as a staged refractive approach.Conclusion: This study highlights an improved technique of an old concept of pterygium surgery that not only reduces the recurrence but also enhances cosmetic excellence and improves the quality of vision.Keywords: amniotic membrane, mitomycin C, pterygium

Background: The amniotic membrane plays an important role in maintaining a healthy pregnancy. The main population cells from amniotic membrane include human amnion epithelial cells (hAECs) which have been shown to possess immunomodulatory properties. Objective: The proximity of hAECs with monocyte leads to the generation of tollerogenic dendritic cells. Materials and Methods: hAECs were obtained from normal pregnancy. Peripheral blood monocytes were isolated by anti-CD14 MACS method. Co-cultures of monocytes and hAECs were established in Transwell chambers supplemented with granulocytemacrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) in the absence and presence of lipopolysaccharide (LPS) to produce immature and mature DCs, respectively. Immunophenotyping of the obtained DCs was done through flow cytometry and the production of cytokines was measured by ELISA. Mixed leukocyte Reaction (MLR) was also performed for the functional assessment of DCs. Results: Immunophenotyping of [hAECs - Immature DC (iDC)] and [hAECs - iDC] + LPS cells revealed that the expression of CD1a, CD80, CD86, CD40, HLA-DR, and CD83 markers showed no significant difference as compared with the control group (iDCs and mDCs alone). In the [hAECs-iDCs] + LPS cells, the percentage of CD14 cells at the ratio of 1:2.5 showed significant differences compared to the control group. The production of IL-10 and IL-12 showed no significant difference in any of the cultures as compared to the control groups. Also, co-cultured DCs did not inhibit proliferation of lymphocyte. Conclusion: Our findings show that factors secreted from cultured hAECs are unable to generate of tollerogenic dendritic cells. To achieve a better understanding of other mechanisms more investigations are needed. Key words: Amniotic membrane, Dendritic cells, Human placenta, Immunomodulation, Monocyte.

Summary Introduction. The amniotic membrane is used in transplant surgery, ophthalmology and dermatology. Various methods have been developed to preserve amniotic membrane: hypothermic storage, cryopreservation, lyophilization. Transplantation of fresh amniotic membrane showed low inflammatory response. The efficient antibiotic solutions are carefully chosen for the hypothermic storage of amniotic membranes. The aim of this study was to compare the efficacy of two antibiotic solutions for the hypothermic amniotic membrane preservation and the structure of the amniotic membrane after the preservation process. Methods. Fifteen amniotic membranes were prepared and hypothermically stored in penicillin solution in BSS (2000 IU/ml) while the remaining fifteen in an antibiotic solution containing: benzylpenicillin (50 µg/ml), gentamicin (100 µg/ml), ciprofloxacin (200 µg/ml) and fluconazole (100 µg/ml). All amniotic membranes were microbiologically tested after preparation and after hypothermic storage for two weeks. Histological analysis of thirty amniotic membranes was performed after the process of preservation. Results. Fifteen amniotic membranes were sterile after hypothermic preservation in the penicillin solution. Also, fifteen amniotic membranes were sterile after hypothermic preservation in the solution of antibacterial agents (penicillin, gentamicin, ciprofloxacin) and antifungal agent fluconazole. The amniotic membrane had a normal structure and thickness of 35.33±11.03 μm. Conclusion. Both antibiotic solutions, one that contains only penicillin and one that contains multiple antibacterial agents and fluconazole, provide sterility of fresh hypothermically stored amniotic membranes for two weeks. In the preparation of fresh hypothermically stored amniotic membrane, a solution with multiple antibiotics is preferred. The normal tissue structure of the amniotic membrane was histologically confirmed after the process of preservation.

Daniel Brocks,1 Olivia G Mead,2 Sean Tighe,3,4 Scheffer CG Tseng2 1BostonSight, Needham, MA, USA; 2Ocular Surface Center and TissueTech Inc, Miami, FL, USA; 3Department of Ophthalmology, Florida International University, Miami, FL, USA; 4Department of Biochemistry and Molecular Biology, University of Miami, Miami, FL, USACorrespondence: Daniel BrocksBostonSight, 464 Hillside Ave Suite 205, Needham, MA 02494, USATel +1 781 726 7337Email danielbrocks@yahoo.comPurpose: To evaluate the clinical outcomes of self-retained cryopreserved amniotic membrane (cAM) for the treatment of corneal ulcers.Methods: This was a single-center, retrospective review of consecutive patients with non-healing corneal ulcers that underwent treatment with self-retained cAM (PROKERA® Slim). The primary outcome measure was time to complete corneal epithelialization. Ocular discomfort, corneal staining, corneal signs, and visual acuity were assessed at 1 week, 1 month, 3 months, and 6 months. Complications, adverse events, and ulcer recurrence were also recorded.Results: A total of 13 eyes (13 patients) with recalcitrant corneal ulcers were included for analysis, 9 (69%) of which progressed from neurotrophic keratitis (NK). Prior to cAM application, patients used conventional treatments such as artificial tears (n = 11), antibiotics (n = 11), ointment (n = 11), steroids (n = 6), and antivirals (n = 3). Self-retained cAMs (n = 1.5 ± 0.8) were placed for 6.8 ± 3.4 days, during which time antibiotics were continued. Four cases (31%) were subsequently treated with bandage contact lens (n = 3) and tarsorrhaphy (n = 1). All corneal ulcers healed in a median of 14 days (range: 4– 43). This was accompanied by a significant improvement in ocular discomfort, corneal staining, and corneal signs at 1 week, 1 month, 3 months, and 6 months (P

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.

Human Amniotic Epithelial Cells (hAEC) isolated from term placenta are a promising source for regenerative medicine. However, it has long been debated whether the hAEC population consists of heterogeneous or homogeneous cells. In a previous study, we investigated the characteristics of hAEC isolated from four different regions of the amniotic membrane finding significant heterogeneity. The aim of this study was to evaluate the hepatic differentiation capability of hAEC isolated from these four regions. Human term placentae were collected after caesarean section and hAEC were isolated from four regions of the amniotic membrane (R1‐R4, according to their relative distance from the umbilical cord) and treated in hepatic differentiation conditions for 14 days. hAEC‐derived hepatocyte‐like cells showed marked differences in the expression of hepatic markers: R4 showed higher levels of Albumin and Hepatocyte Nuclear Factor (HNF) 4α whereas R1 expressed higher Cytochrome P450 enzymes, both at the gene and protein level. These preliminary results suggest that hAEC isolated from R1 and R4 of the amniotic membrane are more prone to hepatic differentiation. Therefore, the use of hAEC from a specific region of the amniotic membrane should be taken into consideration as it could have an impact on the outcome of therapeutic applications.

Chunjie Liu, 1,* Siyu Tian, 2,* Jiangbo Bai, 2 Kunlun Yu, 2 Lei Liu, 3 Guoli Liu, 4 Ruiyi Dong, 5 Dehu Tian 2 1Department of Orthopedics, Tangshan Workers Hospital, Tangshan, Hebei 063000, People’s Republic of China; 2Department of Hand Surgery, The Third Affiliated Hospital of Hebei Medical University, Shijiazhuang, Hebei 050051, People’s Republic of China; 3Department of Orthopedics, Changping District Hospital, Beijing 102200, People’s Republic of China; 4Department of Orthopedics, The Second Hospital of Tangshan, Tangshan, Hebei 063000,People’s Republic of China; 5Department of Orthopedics, Cangzhou Integrated Traditional Chinese and Western Medicine Hospital, Cangzhou, Hebei 061001, People’s Republic of China*These authors contributed equally to this workCorrespondence: Dehu TianDepartment of Hand Surgery, The Third Affiliated Hospital of Hebei Medical University, 139 Ziqiang Road, Shijiazhuang, Hebei 050017, People’s Republic of ChinaTel +86-151-3056-6366Fax +86-311-8860-3818Email tiandehu_1961@126.comBackground: Adhesion after tendon injury is a common complication in clinical practice. The lack of effective prevention mechanisms seriously affects the functional rehabilitation of patients. This research aimed to optimise the amniotic membrane and explain the mechanism of tendon–amniotic membrane by imitating the tendon sheath to construct a multilayer electrospun polycaprolactone (PCL) nanofibre membrane.Materials and Methods: Fresh amnions were subjected to freezing and vacuum drying. The two surfaces of freeze-dried amnions were coated with PCL nanofibres by electrospinning, thereby forming a multilayer composite membrane and constructing a growth factor-sustained release system conforming to the tendon-healing cycle. The new materials were characterised, and the biological effects on tenocytes and fibroblasts were evaluated. The tendon injury model of New Zealand rabbits was constructed to observe the effects on tendon adhesion and healing.Results: After freezing and vacuum drying, fresh amnions were found to effectively remove most of the cell components but retained the active components TGF-β 1, bFGF, VEGF, and PDGF, as well as the fibrous reticular structure of the basement membrane. After coating with PCL nanofibres, a composite membrane mimicking the structure of the tendon sheath was constructed, thereby strengthening the tensile strength of the amnion. By up-regulating the phosphorylation of ERK1/2 and SMAD2/3, the adhesion and proliferation of tenocytes and fibroblasts were promoted, and collagen synthesis was enhanced. In the rabbit tendon repair model, the composite membrane effectively isolated the exogenous adhesion tissue and promoted endogenous tendon healing.Conclusion: The composite membrane mimicking the structure of tendon sheath effectively isolated the exogenous adhesion tissue and achieved good tendon slip. By slowly releasing the growth factors TGF-β 1, bFGF, VEGF and PDGF, the ERK1/2 and SMAD2/3 pathways were regulated. Consequently, endogenous tendon healing was promoted. This strategy can alternatively address the clinical problem of tendon adhesion.Keywords: amniotic membrane, electrospinning, nanofibers, composite membrane, tendon adhesion

Background Adhesion after tendon injury is a common complication in clinical practice. The lack of effective prevention mechanisms seriously affects the functional rehabilitation of patients. This research aimed to optimise the amniotic membrane and explain the mechanism of tendon-amniotic membrane by imitating the tendon sheath to construct a multilayer electrospun polycaprolactone (PCL) nanofibre membrane. Materials and methods Fresh amnions were subjected to freezing and vacuum drying. The two surfaces of freeze-dried amnions were coated with PCL nanofibres by electrospinning, thereby forming a multilayer composite membrane and constructing a growth factor-sustained release system conforming to the tendon-healing cycle. The new materials were characterised, and the biological effects on tenocytes and fibroblasts were evaluated. The tendon injury model of New Zealand rabbits was constructed to observe the effects on tendon adhesion and healing. Results After freezing and vacuum drying, fresh amnions were found to effectively remove most of the cell components but retained the active components TGF-β1, bFGF, VEGF, and PDGF, as well as the fibrous reticular structure of the basement membrane. After coating with PCL nanofibres, a composite membrane mimicking the structure of the tendon sheath was constructed, thereby strengthening the tensile strength of the amnion. By up-regulating the phosphorylation of ERK1/2 and SMAD2/3, the adhesion and proliferation of tenocytes and fibroblasts were promoted, and collagen synthesis was enhanced. In the rabbit tendon repair model, the composite membrane effectively isolated the exogenous adhesion tissue and promoted endogenous tendon healing. Conclusion The composite membrane mimicking the structure of tendon sheath effectively isolated the exogenous adhesion tissue and achieved good tendon slip. By slowly releasing the growth factors TGF-β1, bFGF, VEGF and PDGF, the ERK1/2 and SMAD2/3 pathways were regulated. Consequently, endogenous tendon healing was promoted. This strategy can alternatively address the clinical problem of tendon adhesion.

Background: The human amniotic membrane (HAM) is a suitable and effective scaffold for cell culture and delivery, and adipose-derived stem cells (ADSCs) are an important source of stem cells for transplantation and chondrogenic differentiation. Objective: To assess the practicability of a cryopreserved HAM as a scaffold in cell proliferation and differentiation in vitro. Materials and Methods: In this experimental study, adipose tissue samples were harvested from the inguinal region of male patients aged 15-30 years. Flow cytometry was used to identify CD31, CD45, CD90, and CD105 markers in adipose stem cells. HAM was harvested from donor placenta after cesarean section, washed, trypsin-based decellularized trypsinized decellularized, and used as a scaffold via three methods: 1) ADSCs were differentiated into chondrocytes on cell culture flasks (monolayer method), and after 14 days of culture, the cells were transferred and cultured on both sides of the HAM; 2) ADSCs were cultured and differentiated directly on both sides of the HAM for 14 days (scaffold-mediated differentiation); and 3) chondrocytes were differentiated with micromass culture for 14 days, transferred on HAM, and tissue slides were histologically analyzed qualitatively. Results: Flow cytometry confirmed the presence of mesenchymal stem cells. Histological findings revealed that the cells adhered and grew well on the stromal layer of HAM. Among the three methods, scaffold-mediated differentiation of ADSCs showed the best results. Conclusion: ADSCs have excellent attachment, viability, and differentiation capacity in the stromal side of HAM. Additionally, the direct culture and differentiation of ADSCs on HAM is more suitable than the culture of differentiated cells on HAM. Key words: Amniotic membrane, Scaffold, Chondrogenesis, Differentiation, Mesenchymal stem cell.