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Diabetic wounds, as one of the most important complications of diabetes, face many challenges in treatment. Herein we investigated whether decellularized human amniotic membrane (dAM) loaded with epigallocatechin-3-gallate (EGCG) could promote healing in diabetic rats. Sixty diabetic rats were randomly planned into the untreated group, dAM group, EGCG group, and dAM + EGCG group. On days 7, 14, and 21, five rats from each group were sampled for stereological, molecular, and tensiometrical assessments. Our finding revealed that the wound closure rate, the total volumes of new epidermis and dermis, the numerical densities of fibroblasts, blood vessels, collagen density as well as tensiometrical parameters of the healed wounds were considerably increased in the treated groups than in the untreated group, and these changes were more obvious in the dAM + EGCG ones. Furthermore, the expression of TGF-β, bFGF, and VEGF genes were significantly upregulated in all treated groups compared to the untreated group and were greater in the dAM + EGCG group. This is while expression of TNF-α and IL-1β, as well as cell numerical densities of neutrophils and macrophages decreased more considerably in the dAM + EGCG group in comparison to the other groups. In conclusion, it was found that using both dAM transplantation and EGCG has more effect on diabetic wound healing. • The dAM and EGCG promote wound healing. • The dAM and EGCG increase the amount of collagen. • The dAM and EGCG regulate the inflammatory process. • The dAM and EGCG improve the stereological characteristics. • The dAM and EGCG upregulated regenerative genes. • The dAM and EGCG increase biomechanical characteristics. [ABSTRACT FROM AUTHOR]

To determine the efficacy of latelet-rich fibrin (PRF) and Amniotic membrane (AM) along with the coronally advanced flap (CAF) technique in treating Miller's class I gingival recession (GR) defects. A total of 32 sites with Miller's class I GR defects were distributed into Group A (CAF + PRF, n = 16) and Group B (CAF + AM, n = 16). Clinical parameters like gingival index (GI), plaque index (PI), gingival bleeding index (GBI), gingival sulcus depth (GSD), relative attachment level (RAL), and gingival marginal level (GML) were measured at baseline and at 3, 6 and 9 months after surgical intervention. PRF and AM with CAF were effective treatment modalities for treating Miller's class I GR defects, with an average root coverage value of 2.00 ± 0.75 mm in Group A and 1.5 ± 0.3 mm in Group B. Complete coverage (100 %) was obtained in 57 % sites of group A and 49 % sites of group B. At the 9-month follow-up, there was a significant increase in relative attachment levels in both groups when compared to baseline. In the present study it was observed that there was a clinically and statistically significant improvement in root coverage with both groups. PRF-treated sites showed > 50 % complete coverage and hence were superior. AM showed comparable results to PRF and could be used as an alternative. [ABSTRACT FROM AUTHOR]

Amniotic membrane (AM) is an attractive source for bone tissue engineering because of its low immunogenicity, contains biomolecules and proteins, and osteogenic differentiation properties. Hydroxyapatite is widely used as bone scaffolds due to its biocompatibility and bioactivity properties. The aim of this study is to design and fabricate scaffold based on hydroxyapatite-coated decellularized amniotic membrane (DAM-HA) for bone tissue engineering purpose. So human amniotic membranes were collected from healthy donors and decellularized (DAM). Then a hydroxyapatite-coating was created by immersion in 10X SBF, under variable parameters of pH and incubation time. Hydroxyapatite-coating was characterized and the optimal sample was selected. Human adipose-derived mesenchymal stem cell behaviors were assessed on control, amniotic membrane, and coated amniotic membrane. The results of the SEM, MTT assay, and Live-Dead staining showed that DAM and DAM-HA support cell adhesion, viability and proliferation. Osteogenic differentiation was evaluated by assessment of alkaline phosphatase activity and expression of osteogenic markers. Maximum gene expression values compared to control occurred in 14 days for alkalin phosphatase, while the highest values for osteocalcin and osteopontin in 21 days. These gene expression values in DAM and DAM-HA for alkalin phosphatase is 6.41 and 8.47, for osteocalcin is 3.95 and 5.94 and for osteopontin is 5.59 and 9.9 respectively. The results of this study indicated DAM supports the survival and growth of stem cells. Also, addition of hydroxyapatite component to DAM promotes osteogenic differentiation while maintaining viability. Therefore, hydroxyapatite-coated decellularized amniotic membrane can be a promising choice for bone tissue engineering applications. [Display omitted] • Amniotic membrane is a suitable source for bone tissue engineering application. • Prepared a homogeneous hydroxyapatite coating through immersion in 10X SBF. • Hydroxyapatite coated amniotic membrane promoted the viability of the cells. • Hydroxyapatite coated amniotic membrane promotes osteogenic differentiation. [ABSTRACT FROM AUTHOR]

Descemet's membrane endothelial keratoplasty (DMEK) may provide fast visual rehabilitation in the therapy of corneal endothelial disorders. However, due to shortage of donated corneas, how to construct a corneal endothelial substitute with powerful functions that can be used for DMEK is still unsolved. Herein, we introduced the method of corneal crosslinking (CXL) and conjugated the components of native Descemet's membrane (DM) to improve the mechanical properties and the biocompatibility of denuded amniotic membrane (dAM), further assessed their effects on cell adhesion, proliferation, YAP translocation, and metabolic activity in human corneal endothelial (HCE) cells. Using modified crosslinked dAM (mcdAM) and non-transfected HCE cells, we constructed a tissue-engineered HCE (TE-HCE) and evaluated its functions in cat and monkey models as well. Our results showed that the mechanical properties of mcdAM were improved effectively by CXL, and the adhesion, proliferation, and YAP translocation of HCE cells were dose-dependently improved after ECM modification. The combination of 0.01 mg/mL laminin with 0.1 mg/mL fibronectin showed the highest efficacy. Then, the TE-HCE was constructed in vitro , with a high density of 3612 ± 243 cells/mm2. Results of DMEK in animal models showed that corneal transparency was maintained, accompanied with normal morphology and histological structure of the regenerated corneal endothelium. Therefore, CXL combined with DM-mimic-coating methods could effectively improve the mechanical properties of dAM and enhance the biocompatibility with HCE cells. The constructed TE-HCE had normal histological structure and functioned well in animal models via DMEK, which could be used as a promising powerful equivalent of HCE. Using high-quality corneal endothelium and an appropriate endothelial keratoplasty is the most effective way for the treatment of corneal endotheliopathy. Descemet's membrane endothelial keratoplasty (DMEK) which can provide better visual acuity, lower immunological rejection rates, and improved graft survival is an ideal surgery at present. However, due to the shortage of donated corneas, it is urgent to find an equivalent substitute of corneal endothelial donor which is suitable for the DMEK surgery to solve the problem of corneal endothelial regeneration. Herein, we introduced the clinical cornea-crosslinking and Descemet's membrane-mimic-coating methods to build the modified crosslinked denuded amniotic membrane scaffold and further constructed a high-quality corneal endothelial functional substitute that can be used in DMEK surgery. [Display omitted] [ABSTRACT FROM AUTHOR]

In human body, biomaterials can be used to accelerate the wound healing process. Amniotic membrane-derived secretory leukocyte protease inhibitor (SLPI) is one of the biomaterials which able to accelerate the wound healing process by its several functions such as protease inhibition, leukocytes activity control, growth factor regulation such as transforming growth factor β (TGF-β), and its anti inflammatory, anti bacterial also anti retroviral properties. Objectives: To analyzed the SLPI effect on the TNF-α expression and the quantity of new blood vessel in the incisional wound healing. SLPI are produced from Essericea coli TOP10 as the cloning host, BL21 (DE3) strains as the expression host and pET30a plasmids were used for the expression system construction. The incision wound was made in dorsal skin area, then treated with SLPI dose 0.03 cc, 0.45 cc, 0.06 cc. After treatment, the wound covered with hypafix. The control group only covered with hypafix. In the next four days, the dorsal skin area is biopsied, then the quantity of TNF-α expression and blood vessels was analyzed with hematoxylin eosin and immunohistochemistry staining. The SLPI 0.045 cc showed the higher quantity of new blood vessel compared the SLPI 0.03 cc and control group (p < 0.05). While the TNF-α expression in the SLPI 0.045 cc is lower than SLPI 0.03 cc (p = 0.002) and control group (p = 0.000). The SLPI capable to accelerate the wound healing by decreasing the TNF-α expression and increased the new blood vessel quantity at 0.045 cc dose. [Display omitted] [ABSTRACT FROM AUTHOR]

• A microgel consisting of decellularized amniotic membrane is proposed to treat radiation-induced lung injury. • The microgels that preserve the structural integrity of the contained cargo can be inhaled into lung for topical treatment. • The microgels can switch the microenvironment from pro-inflammation status to pro-healing status. Radiation-induced lung injury (RILI) is one of the major complications in patients exposed to accidental radiation and radiotherapy for thoracic malignancies. However, there is no reliable approach to effectively treat RILI. With favorable biological and physical characteristics, decellularized extracellular matrix (dECM) from amniotic membrane can not only act as a reactive oxygen species scavenger to attenuate the damage of oxidative stress, but also release bio-factors to polarize the macrophages from M1-like phenotype to M2-like phenotype, thus switching the microenvironment from pro-inflammation status to pro-healing status. Meanwhile, as a promising drug delivery system, microgels with a small size and high surface/volume ratio can not only be inhaled into lung tissues with a desired concentration, but also preserve the structural integrity and the functionalities of the contained therapeutics. Therefore, it will be a potent strategy to combine microgels and amniotic dECM for the treatment of RILI, in which the inhaled microgels show a high surface area to rapidly remove reactive oxygen species and release cargos to reduce the inflammation-mediated damage by modulating macrophage phenotypes. We hypothesize that amniotic dECM microgels will be a promising and effective biomaterial for the attenuation of RILI. [ABSTRACT FROM AUTHOR]

Exfoliative cytology of human amniotic fluid (AF) has been extensively studied since 1940s, but no data exist in equine species. The AF compartment represents the environment in which the foetus grows and matures, and its composition changes, reflecting foetal well-being and development. The aim of this study was to describe for the first time the morphology of equine AF cells and amniotic membrane (AM) with light microscopy (LM) and transmission electron microscopy (TEM). AF was collected at parturition within 5 min after the appearance of the AM with a 60 mL syringe from 34 mares and samples of AM were collected from a subset of 7 mares with normal pregnancy hospitalized for attended parturition. For LM observation, a sample of cytocentrifuged fresh AF was stained with May-Grünwald Giemsa and AM sections were stained with H-E. For TEM observation, AF and AM were fixed, embedded in epoxy resins, then sectioned and stained with uranyl acetate and lead citrate solutions. Nucleated and anucleated squamous cells with basophilic cytoplasm, intensely basophilic cornified cells, polymorphonuclear cells, and clusters of eosinophilic amorphous substance were observed. Cells presumably derived from tracheal epithelium and small round nucleated cells with eosinophilic cytoplasm presumably derived from amniotic or urinary epithelium were occasionally found. Lamellar body-like structures (LBs) were present in some epithelial cells. In AM, epithelial, basal and mesenchymal layers were clearly visible with both techniques as previously described. Epithelial cells had several cytoplasmic vacuolization and microvilli were present on apical surface. The connective tissue presented fibroblasts, mesenchymal and rare polymorphonuclear cells, surrounded by abundant extracellular matrix, with distribution of collagen fibres. This is the first report about equine amniotic compartment description by LM and TEM. As recently reported in human medicine, the AM could be a second potential source of pulmonary surfactant, given the finding of LBs inside the cells which could have the same function as in humans. Further studies in samples collected at different gestational ages could increase the knowledge of AF cells and their modification during pregnancy, as well as a better comprehension of the role of AM as a secondary source of pulmonary surfactant in the horse. The diagnostic evaluation of AF cellular composition in high-risk pregnancies may also be investigated. • This is the first report about equine amniotic compartment description. • The amniotic compartment reflects fetal well-being and development. • Lamellar body-like structures were present in some epithelial cells. • Equine AM could be a second potential source of pulmonary surfactant. [ABSTRACT FROM AUTHOR]

• The properties of the amniotic membrane (AM) are ideal for its use as a dural substitute. • AM patches can be processed and stored at low costs and used without impacting operative time. • AM create a good separation plane between the dura mater, cerebral cortex, and myocutaneous layer. • AM pre and postoperative outcomes are the same as biological substitutes ones. A dura mater substitute in decompressive craniectomies must protect the brain while providing a dissection plane between the cortex and myocutaneous layer. The human amniotic membrane (AM) has anti-inflammatory, wound healing, and differentiation properties. We tested AM properties as a dural substitute by comparing the outcomes to biological ones. We prospectively collected data on 25 patients who randomly underwent decompressive craniectomy with lyophilized AM patches and 25 in which biological substitutes were utilized between 2015 and 2019. The AM was laid with the epithelial side facing the brain because of the anti-adhesive proprieties, while the chorion facing the myocutaneous flap. We collected data on demographics, neurological status, comorbidities, and surgical outcomes. Additionally, we created a score – dura mimicking score– and reviewed postoperative imaging and pathological specimens. The majority (96%) of AM grafts were integrated into native dura. Thirteen patients scored as excellent and 11 good on our "dura mimicking score", showing tissue integration ability but no cerebral cortex adhesion. The histopathological analysis showed that AM had thick plates of dense fibrous tissue with small reactive vessels, reactive fibroblasts, and lymphocytes infiltrate. The AM group's first outcomes were not different from the biological substitute patients but higher integration rate to the dura and less adhesion to the myocutaneous flap in AM patients. We documented the anti-adhesive, protective, and integrative properties of AM dural substitute patches in patients who underwent decompressive craniectomies, comparing the intraoperative differences and postoperative outcomes to biological dural substitutes. [ABSTRACT FROM AUTHOR]

The purpose of this study was to compare the clinical, aesthetic, and functional outcomes between amniotic membrane (test group) and split-thickness skin grafts (control group) used for radial forearm free flap defect closure. The primary outcome measurement for both groups was assessment of the defect closure healing process. In addition, aesthetic (Vancouver Scar Scale) and functional outcomes (skin sensitivity, hand/wrist functionality, grip strength) were evaluated. Fifty eligible patients with radial forearm free flap donor site defects were randomly assigned to two groups receiving either amniotic membrane (test group; n = 25) or split-thickness skin graft (control group; n = 25) for defect covering. Forty-seven of the 50 patients (n = 47) were able to be followed up for 6 months and showed a significantly longer healing process (p < 0.001) with amniotic membrane (64.5 ± 38.4 days; n = 24) than with split-thickness skin grafts (29.2 ± 8.9 days; n = 23); however, there were no differences in the prevalence of healing defects/dehiscence and/or wound infections. Forty-two of the 47 patients (21 in each group) were able to be continually followed up for 12 months, and showed no differences in terms of clinical outcome as well as the subjective and objective aesthetic and functional results evaluated. With regard to the clinical, aesthetic, and functional outcomes evaluated for radial forearm free flap defects, coverage with amniotic membrane offers an excellent alternative treatment approach, avoiding secondary induced donor site morbidity. [ABSTRACT FROM AUTHOR]

ACL injuries are common and the standard treatment remains to be ACL reconstruction. Controversy still remains around graft ligamentisation and various methods have been experimented to augment this process. In this series we discuss the outcome of using dehydrated umbilical cord derived membrane in ACL reconstruction. A prospective Level IV analysis of outcome in 30 consecutive patients of complete isolated ACL tear presenting to us between May 2022 to September 2022 was done. There was no age or sex restriction. Revision cases were excluded from this series. ACL reconstruction was done using standard all inside technique using the ipsilateral semitendinosus graft which was wrapped with dehydrated umbilical cord derived membrane (5*3 cm2) after soaking in normal saline (Cord Thick - Life Cell; LifeCell international private limited, Chennai, Tamil Nadu, India) and secured with Monocryl 3-0 sutures in a continuous manner. 18 patients completed the one year follow up and were analysed for final results. Eleven of the patients followed up were females and seven males, with an average age of 30.3 years.The average VAS score at 6 weeks was 2.2. The mean KOOS score at the follow up visits were 72.3, 82.3, 90.7, 95.8. The mean SANE score reported at 6 weeks, 3 months, 6 months and 1 year were 68.8, 80, 87.5, 95.1.The follow up MRI's confirmed good ligamentisation of the graft even as early as 6 weeks with minimal fluid in the tunnel. The use of dehydrated umbilical cord derived membrane augmentation to potentiate graft integration in ACL reconstruction seems promising and helps improve patient outcomes. [ABSTRACT FROM AUTHOR]

[Display omitted] • An ultrathin bilayer membrane is fabricated from amniotic membrane and silicon. • Bilayer membrane reveals a decreased degradation and improved mechanical property. • Silicon layer makes bilayer membrane suturable with increased transparency. • Silicon/amniotic bilayer membrane improves rabbit corneal epithelial defects. Amniotic membrane (AM) is an effective and widely used dressing in ocular injuries to reconstruct the cornea. Due to its low mechanical strength, high biodegradation rate, and difficult handling, its usage in medical interventions remains challenging. In this study, decellularized AM was covered with an ultrathin layer of Polydimethylsiloxane (PDMS) through a spinning method, which in turn resulted in an ultrathin (less than 80 µm in thickness) bilayer corneal wound dressing membrane with improved mechanical behavior and transparency. The biomechanical, biological, and antibacterial properties of the bilayer membranes were measured both in vitro and in vivo. The optimized microsized membrane was applied on a corneal defect wound created in a rabbit model to evaluate the corneal healing. The results demonstrated a significant decrease in degradation rate, improved mechanical properties, and AM/PDMS transparency compared with AM. The corneal transparency improved until 21 days post-surgery in AM/PDMS group. Histological evaluations revealed that AM/PDMS had better epithelial delaminated cell morphology. The results of the RT-PCR showed a significant increase in MMP9, a significant decrease in Col1A1, TGF-β1, TNF-α and IL-6 in both AM and AM/PDMS compared with control wounds. This study suggessts AM/PDMS membrane as an excellent corneal wound dressing. [ABSTRACT FROM AUTHOR]

Due to the increasing number of studies performed in the field of regenerative medicine during the last two decades, more analytic studies are still needed to clarify the future prospect of this area of science. The main aim of this research was to review the clinical applications of human Amniotic membrane in the field of regenerative medicine critically. Furthermore, in the light of increasing numbers of available products derived from amniotic membrane, we aimed look in depth to see whether regenerative medicine research strategies have a place in the clinical setting. More specifically, in the present study, we attempted to provide insight on developing the new indication for more research and in the next step, for market leaders companies to expand cost-effectiveness of new derived AM products. 20 companies or distributers have offered some commercial products in this field. Survey on more than 90 clinical trials in last five years showed dermatology (and more specific wound healing), orthopedic, and ophthalmology are heavily biased toward multibillion dollar industry. Moreover, urology and dentistry with fewer numbers of clinical data in comparison with the above-mentioned areas, currently are in the path of translation (especially dentistry). In addition, otolaryngology and oncology with the lowest number showed more potential of research thorough understanding the properties that will help guiding the use of AM-derived products in these two areas in future. More than 50% of clinical studies were done or are developing in USA, which have the biggest share in market products. Subsequently, China, Egypt, India, Iran, and Germany with the ongoing clinical trials in different phases may have more approved products in near future. [ABSTRACT FROM AUTHOR]

The diverse application of collagen has created a need to discover renewable and economical sources with prevailing/improved physico-chemical properties. To address this scenario, the present study has extracted collagen from Human Amniotic Membrane (AM) and Umbilical cord, which are treated as medical waste and compared its physico-chemical properties. Collagen was extracted by pepsin solubilization using various salt concentrations (1 M, 2 M and 4 M). Umbilical Cord Collagen (UC) yield was 10% higher than Amniotic Membrane Collagen (AC). UC reported 58% higher sulphated glycosaminoglycan content than AC. Electrophoretic pattern of AC and UC in both disulphide bond reducing and non-reducing conditions showed bands corresponding to collagen type I, III, IV, V and XV. Collagen morphology was examined using SEM and the amino acid content was quantified by HPLC and LC-MS/MS. Triple helicity was confirmed by CD and FTIR spectra. Thermal transition temperature of AC and UC was found equivalent to animal collagen. Self-assembly, fibril morphology and spatial alignment was studied using AFM and DLS. Biocompatibility was analyzed using 3T3 fibroblast cells. In conclusion, UC with higher yield, presented with better physico-chemical, structural and biological properties than AC could serve as an efficient alternative to the existing animal collagen for diverse applications. Unlabelled Image • The study utilized postpartum waste for collagen isolation. • Molecular weight of collagen types in amnion and umbilical cord was identified. • Collagens showed self-assembling ability and cytocompatibility. • Large size and overlap region of umbilical cord collagen fibril imparts stability. • Umbilical cord would be a better collagen source than amnion for clinical therapy. [ABSTRACT FROM AUTHOR]

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. 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). Image, graphical abstract [ABSTRACT FROM AUTHOR]

Objective: To evaluate the efficacy and safety of amniotic membrane (AM) for the healing of split-thickness skin graft donor sites (STSGDS).Method: Electronic search of PubMed, Cochrane library and EMBASE for randomized controlled trials (RCTs) or non-randomized clinical trials (NRCTs) of AM therapy in STSGDS. Review Manager5.3 was utilized to analyze and present the data.Results: Seven studies with 219 patients were included. Compared with other treatments, the mean difference (MD) in healing time was -3.87 days (95% CI -4.39, -3.35; P < 0.00001); Relative risk for the healing rate was 1.61 (95% CI 0.0.47-5.47; P = 0.44); There was no statistical difference in the sensation of pain (P > 0.05); The relative risk for infection rate was 0.66 (95% CI 0.29, 2.18; I2 = 0%; P = 0.65).Conclusion: This systematic review and meta-analysis indicate that it is effective and safe to use AM for treating STSGDS. [ABSTRACT FROM AUTHOR]

Human amniotic membrane (AM) offers unique advantages as a matrix to support the transplantation of limbal stem cells (LSCs) due to its inherent pro-regenerative and anti-inflammatory properties. However, the widespread use of AM in clinical treatments of ocular surface disorders is limited by its weak mechanical strength and fast degradation, and high cost associated with preserving freshly isolated AM. Here we constructed a composite membrane consisting of an electrospun bioabsorbable poly(ε-caprolactone) (PCL) nanofiber mesh to significantly improve the ultimate tensile strength, toughness, and suture retention strength by 4–10-fold in comparison with decellularized AM sheet. The composite membrane showed extended stability and conferred longer-lasting coverage on wounded cornea surface compared with dAM. The composite membrane maintained the pro-regenerative and immunomodulatory properties of dAM, promoted LSC survival, retention, and organization, improved re-epithelialization of the defect area, and reduced inflammation and neovascularization. This study demonstrates the translational potential of our composite membrane for stem cell-based treatment of ocular surface damage. Human decellularized amniotic membrane (dAM) has been widely shown as a biodegradable and bioactive matrix for regenerative tissue repair. However, the weak mechanical property has limited its widespread use in the clinic. Here we constructed a composite membrane using a layer of electrospun poly(ε-caprolactone) (PCL) nanofiber mesh to reinforce the dAM sheet through covalent interfacial bonding, while retaining the unique bioactivity of dAM. In a rabbit model of limbal stem cell (LSC) deficiency induced by alkaline burn, we demonstrated the superior property of this PCL-dAM composite membrane for repairing damaged cornea through promoting LSC transplantation, improving re-epithelialization, and reducing inflammation and neovascularization. This new composite membrane offers great translational potential in supporting stem cell-based treatment of ocular surface damage. [ABSTRACT FROM AUTHOR]

Human amniotic membranes (HAMs), as a biological membrane with healing, osteogenic, and cell therapy potential, has been in the spotlight to enhance the outcomes of treating bone defects. Present study aims to clinically assess the potential of HAM loaded with buccal fat pad-derived stem cells (BFSCs) as an osteogenic coverage for onlay bone grafts to maxillomandibular bone defects. Nine patients with jaw bone defects were enrolled in the present study. The patients were allocated to two study groups: Iliac crest bone graft with HAM coverage (n = 5), and Iliac bone grafts covered with HAM loaded with BFSCs (n = 4). Five months following the grafting and prior to implant placement, cone beam computed tomography was performed for radiomorphometric analysis. The mean increase in bone width was found to be significantly greater in the HAM + BFSCs group (4.42 ± 1.03 mm versus 3.07 ± 0.73 mm, p < 0.05). Further, the changes in vertical dimension were greater in the HAM + BFSCs group (4.66 ± 1.06 mm versus 4.14 ± 1.03 mm, p > 0.05). Combined use of HAM with mesenchymal stem cells may enhance bone regeneration specifically in the horizontal dimension. Moreover, this methodology reduces the amount of harvested autogenous bone and diminish secondary bone resorption. [ABSTRACT FROM AUTHOR]

Human amniotic membrane (AM) has intrinsic anti-inflammatory, anti-fibrotic and antimicrobial properties. Tissue preservation methods have helped to overcome the short shelf life of fresh AM allowing "on demand" use of AM grafts. Cryopreserved AM that retains all native tissue components, including viable cells, has clinical benefits in treating chronic wounds. However, cryopreservation requires ultra-low temperature storage, limiting the use of cryopreserved products. To overcome this limitation, a new lyopreservation method has been developed for ambient storage of living tissues. The goal of this study was to investigate the viability and functionality of AM cells following lyopreservation. Fresh AM and devitalized lyopreserved AM (DLAM) served as positive and negative controls, respectively. Using live/dead staining, we confirmed the presence of living cells in viable lyopreserved AM (VLAM) and showed that these cells persisted up to 21 days in culture medium. The functionality of cells in VLAM was assessed by their differentiation potential and anti-fibrotic activity in vitro. With osteogenic induction, cells in VLAM deposited calcium within the membrane, a marker of osteogenic cells, in a time-dependent manner. The migration of human lung fibrotic fibroblasts in a scratch wound assay was reduced significantly in the presence of VLAM-derived conditioned medium. Quantitative PCR analyses indicated that VLAM reduced the expression of pro-fibrotic factors such as type I collagen and increased the expression of anti-fibrotic factors such as hepatocyte growth factor and anti-fibrotic microRNA in fibrotic fibroblasts. Taken together, these results demonstrate that endogenous cells in VLAM remain viable and functional post-lyophilization. This study, for the first time, provides direct evidence showing that tissue viability and functional cells can be preserved by lyophilization. Similar to fresh amniotic membrane (AM), viable lyopreserved AM (VLAM) retains viable cells for extended periods of time. More importantly, these cells are functional and maintain their osteogenic differentiation potential and anti-fibrotic activity. Our results confirmed that the novel lyophilization method preserves tissue viability. [ABSTRACT FROM AUTHOR]

Objective: The search for safe and effective tissue fillers has been an ongoing effort for many years. Obtaining biological materials from patients can require additional incisions and brings surgical and cosmetic risks as a major disadvantage. The aim of this experimental study was, therefore, to evaluate the efficacy of folded amniotic membrane as a soft tissue filler and to assess its volume, and to investigate the possible usage of injectable amniotic membrane for augmentation.Methods: Multi-layer amniotic membranes are transplanted to the subcutaneous tunnels created in the rat's back, and the amniotic membrane transformed into the injection material is inoculated transcutaneously under the rat's skin. At the end of two months, the amount of volume loss and histopathological changes in the grafts were examined under light microscope.Results: The multi-layer amniotic membrane grafts maintain its volume around 92%. It has been shown that the injectable amniotic membrane maintains its presence in the tissue, can augment it, and can be used as a soft tissue filler. The availability of using amniotic membrane for injection material is shown. No foreign body reaction to the amnion grafts, fibrosis, and necrosis were observed in our study.Conclusion: According to our study results, multi-layer amnion graft and amnion injection seem to be used as camouflage grafts and soft tissue augmentation materials. Although further clinical trials are mandatory before considering the use of amniotic membrane as a tissue filler material in humans, we are hopeful about its long-term safety and efficacy. [ABSTRACT FROM AUTHOR]

Abstract Current gold standard treatment of Buschke-Löwenstein tumor (BLT) is surgical excision. Surgical excision in the perianal region leaves a large area to be covered and bears the risk of poor wound healing as well as infection. Dehydrated human amnion/chorion membrane (dHACM) has been proven to be beneficial in wound closure and has been used in many cases, such as deep ulcers, ophthalmological surgeries, tendon repairs, etc. We report a case of 14-month old boy presented with mass from perianal region since six months prior to visit in Dr Soetomo General Hospital Surabaya. From physical examination, pedunculated exophytic growth with cauliflower-like appearance was found and because of the mass size, the anal opening and the rectum was difficult to be evaluated. CT scan obtained showed heterogenous solid mass with multiple calcification with size of ±7,62 × 6,58 × 3,12 cm in perineal cutaneous and subcutaneous tissue that encases the anus and anorectal area. Surgery was performed to remove the mass and the wound was closed with dHACM. Patient healed well without signs of infection nor excessive scarring. Hence, dHACM could be an excellent option in closing wound post excision in BLT cases. [ABSTRACT FROM AUTHOR]

Abstract Background Several methods to cultivate limbal epithelial stem cells (LESCs) in vitro with the support of feeder layers and different growth medium formulations have been established for several years. The initial green medium consists of various ingredients that exhibit a non-optimal level of biosafety, therefore, different modifications have been made to suit it to safe clinical applications. However, the question of which formulation is the most appropriate remains to be answered. Aims This study evaluated the outgrowth kinetics and stemness of cells cultured from human limbal explants with the aim of preserving LESC characteristics in the human-derived platelet-rich fibrin (HPRF)–conditioned medium with no feeder cell layer or carrier for the first time. The final composition of the cell culture system included only human-derived products without any xenobiotic or chemical substances to minimize the potential risk for human health, which will be useful for clinical purposes. Methods To test our hypothesis, limbal explants were incubated with either Dulbecco's Modified Eagle's Medium (DMEM)/F12-10% human serum (HS), human-derived amniotic membrane (HAM)-conditioned DMEM/F12-10% HS or HPRF-conditioned DMEM/F12-10% HS to determine whether outgrowth kinetics and stemness of cells show any differences among groups. Results The results showed that the HPRF-conditioned medium showed higher concentration levels of growth factors, which may be involved in the promotion of LESC expansion while preserving the stem cell characteristics. HPRF-conditioned medium had significantly superior capacity to enhance the cell growth rate, the stem/progenitor cell phenotype and the expressions of putative stem cell markers. Conclusion This novel xeno-feeder-chemical-free, completely human-derived and biologically safe culture system including HPRF and HS would be of interest to replace conventional cell culture strategies to meet safety requirements mandatory for clinical use in humans. [ABSTRACT FROM AUTHOR]

In orthopedic sports medicine, amniotic-derived products have demonstrated promising preclinical and early clinical results for the treatment of tendon/ligament injuries, cartilage defects, and osteoarthritis. The amniotic membrane is a metabolically active tissue that has demonstrated anti-inflammatory, antimicrobial, antifibrotic, and epithelialization-promoting features that make it uniquely suited for several clinical applications. Although the existing clinical literature is limited, there are several ongoing clinical trials aiming to elucidate the specific applications and benefits of these products. This article reviews the current amniotic-derived treatment options and the existing literature on outcomes, complications, and safety profile of these products for use in sports medicine. [ABSTRACT FROM AUTHOR]

Abstract Clinical application of a large variety of biomaterials is limited by the imperfections in storage technology. Perspective approaches utilizing low-temperature storage are especially challenging for multicellular structures, such as tissues, organs, and bioengineered constructs. Placenta, as a temporary organ, is a widely available unique biological material, being among the most promising sources of various cells and tissues for clinical and experimental use in regenerative medicine and tissue engineering. The aim of this study was to analyse the mechanisms of cryoinjuries in different placental tissues and bioengineered constructs as well as to support the viability after low temperature storage, which would contribute to development of efficient biobanking technologies. This study shows that specificity of cryodamage depends on the structure of the studied object, intercellular bonds, as well as interaction of its components with cryoprotective agents. Remarkably, it was possible to efficiently isolate cells after thawing from all of the studied tissues. While the outcome was lower in comparison to the native non-frozen samples, the phenotype and expression levels of pluripotency genes remained unaffected. Further progress in eliminating of recrystallization processes during thawing would significantly improve biobanking technologies for multicellular constructs and tissues. [ABSTRACT FROM AUTHOR]

Abstract Simple limbal epithelial transplantation is a recently developed technique for treating limbal stem cell deficiency caused by ocular burns. A small limbal biopsy from the donor eye, usually from the patient's healthy eye, is excised and dissected into multiple pieces. An amniotic membrane is atttached using fibrin glue on the diseased eye after removing the conjunctivalized pannus from the corneal surface. The limbal biopsy pieces are placed onto the amniotic membrane, fixed with fibrin glue, followed by bandaging of the ocular surface with a contact lens. This technique (auto simple limbal epithelial transplantation) offers easier surgical manipulations and economic advantages over other techniques for the treatment of limbal stem cell deficiency. We therefore review simple limbal epithelial transplantation along with recent modifications in the technique and case studies, including challenges and failures. Graphical abstract [ABSTRACT FROM AUTHOR]

Highlights • Nerve injuries result from trauma or pathological lesions like tumors and cause functional and esthetic deficits. • Repairing treatments that promote nervous system plasticity mechanisms maintaining can improve functional recovery. • Amniotic membrane helps the regeneration treatment as with epithelization and decreases inflammation and neovascularization. • Allografts instead of xenografts results with less immunological reactions on the injured site supporting axonal regeneration. Abstract In this study we aimed to examine the effects on wound healing and nerve regeneration of human and rat amniotic membrane wraps around primary epineural anastomosis areas after a peripheral nerve transection injury in rats. We randomized 25 male adult rats with induced peripheral transection injuries into 5 groups (control, transection injury, primary epineural anastomosis [PEA] after injury, PEA with a human amniotic membrane [hAM] wrap, and PEA with a rat amniotic membrane [rAM] wrap groups and treated their injuries accordingly. We took tissue samples from the anastomosis regions, 12 weeks after the experiment, and analyzed them stereologically and ultrastructurally. We performed a statistical analysis with the recovered stereological counts and the measurement data. Our results showed that the use of amniotic membranes for allografts (between same species) instead of xenografts (between different species), along with microsurgery, provides a suitable microenvironment during the healing process with less immunological reaction on the injured site and supports axonal regeneration. [ABSTRACT FROM AUTHOR]

[Display omitted] • Polycaprolactone/amniotic membrane (PCL/AM) was fabricated as a biomaterial used in the regeneration of peripheral nerves. • PCL/AM promoted macrophage polarization and regulated inflammatory microenvironment. • PCL/AM induced myelin sheath and axon regeneration. • Application of PCL/AM in repairing peripheral nerve injuries is technically feasible. Appropriate levels of inflammation are an important part of functional repair of nerve damage. However, excessive inflammation can cause the continuous activation of immune inflammatory cells and degeneration of nerve cells. Regulating the temporal and spatial changes in M1/M2 macrophages can regulate the local inflammatory immune environment of the tissue to promote its transformation to a direction conducive to tissue repair.In the present study, a multi-layer multifunctional nanofiber composite membrane of polycaprolactone(PCL) and amniotic membrane (AM) was constructed using electrospinning. In vitro studies have shown that the PCL/AM composite promoted the axon growth of SH-SY5Y cells and induced their differentiation into neurons. The PCL/AM composite wrapped the nerve stump to form a microenvironment that was conducive to nerve regeneration, blocked the invasion of scar tissue, promoted the recruitment of macrophages and moderate polarization to M2, enhanced the expression of anti-inflammatory factors IL-10 and IL-13, inhibited the expression of pro-inflammatory factors IL-6 and TNF-α, and induced myelin sheath and axon regeneration. By releasing various bioactive substances to regulate the polarization of M2 macrophages and formation of anti-inflammatory factors, the PCL/AM composite can enhance axonal regeneration and improve nerve repair. [ABSTRACT FROM AUTHOR]

Injectable biomaterials that can completely fill the root canals and provide an appropriate environment will have potential application for pulp regeneration in endodontics. This study aimed to fabricate and characterize a novel injectable human amniotic membrane (HAM) hydrogel scaffold crosslinked with genipin, enabling the proliferation of Dental Pulp Stem Cells (DPSCs) and optimizing pulp regeneration. HAM extracellular matrix (ECM) hydrogels (15, 22.5, and 30 mg/ml) crosslinked with different genipin concentrations (0, 0.1, 0.5, 1, 5, and 10 mM) were evaluated for mechanical properties, tooth discoloration, cell viability, and proliferation of DPSCs. The hydrogels were subcutaneously injected in rats to assess their immunogenicity. The hydrogels were applied in a root canal model and subcutaneously implanted in rats to determine their regenerative potential for eight weeks, and histological and immunostaining analyses were performed. Hydrogels crosslinked with low genipin concentration demonstrated low tooth discoloration, but 0.1 mM genipin crosslinked hydrogels were excluded due to their unfavourable mechanical properties. The degradation ratio was lower in hydrogels crosslinked with 0.5 mM genipin. The 30 mg/ml-0.5 mM crosslinked hydrogel exhibited a microporous structure, and the modulus of elasticity was 1200 PA. In vitro, cell culture showed maximum viability and proliferation in 30 mg/ml-0.5 mM crosslinked hydrogel. All groups elicited minimum immunological responses, and highly vascularized pulp-like tissue was formed in human tooth roots in both groups with/without DPSCs. Genipin crosslinking improved the biodegradability of injectable HAM hydrogels and conferred higher biocompatibility. Hydrogels encapsulated with DPSCs can support stem cell viability and proliferation. In addition, highly vascularized pulp-like tissue formation by this biomaterial displayed potential for pulp regeneration. [ABSTRACT FROM AUTHOR]

Background The aim of this study was to evaluate the surgical handling and clinical applicability of a specific 3D-printed membrane design fabricated using a gelatin, elastin and sodium hyaluronate blend for conjunctival reconstruction and compare it with amniotic membrane (AM), which is normally used in such surgeries. Methods 3D printing technique was employed to fabricate the membrane based on gradient design. Prior to printing, rheometry was employed to optimize the ink composition. The printed membranes were then fully characterized in terms of physical and mechanical properties. In vitro viability, proliferation and adhesion of human limbal epithelial cells were assessed using MTT assay and scanning electron microscopy (SEM), respectively. Prior to in vivo experiment, surgical handling of each membrane was evaluated by three surgeons. In vivo evaluation was conducted through implanting the gelatin-based membranes and AM on induced conjunctival defects in rabbits (n = 8). Clinical observations, including epithelialization, inflammation severity, scar tissue formation and presence of granulation tissue, were recorded from day 1 through day 28. Histological examination was performed on all enucleated eyes on day 28. In addition to H&E staining, specific stains including Periodic Acid Schiff staining, Masson's Trichrome staining and immuno-histochemical staining for α-SMA were further used to assess goblet cell proliferation, healed sub-epithelial stroma and scar tissue formation and the presence of myofibroblasts, respectively. Results Among all the examined compositions, a blend of 8% w/v gelatin, 2% w/v elastin and 0.5% w/v sodium hyaluronate was found to be appropriate for printing. The printed membranes had favorable optical characteristics (colorless and transparent), and the surgical handling was significantly easier compared to AM. Epithelial cells cultivated on the membranes indicated suitable viability and proliferation, and SEM images presented appropriate cell adhesion on the surface of the membranes. Clinical observations suggested similar epithelialization time (approximately 3 weeks) for both the membrane and AM grafted eyes but significantly lower levels of clinical inflammation in the membrane group from day 1 through day 28 (p = 0.01), which is a key advantage of using the printed membranes over the AM. Histological examination showed similar qualities in the healed epithelium in terms of cell morphology and cell layers. However, twice the density of goblet cells per 100 cells was observed in the gelatin-based membrane grafted group. Remnant of the degraded implant was seen in only 3 of the membranes, but in 7 of the AM grafted eyes. Inflammation and granulomatous reaction was significantly higher in sections containing the AM compared to membrane (p < 0.01 and p = 0.01, respectively). α-SMA staining was more evident, but not significantly different from the gelatin-based membrane, for the AM group (p = 0.25). Conclusion The designed gelatin-based membrane offers the necessary physical and mechanical characteristics needed for successful ocular surface/conjunctival defect construction and may be considered a promising alternative to AM due to a more predictable degradation pattern, higher goblet cell density on the healed epithelium, less inflammation and reduced scar tissue formation. [ABSTRACT FROM AUTHOR]

In the field of neurosurgery, often the dura mater cannot be sutured, and consequently, it requires a duraplasty procedure using a dural fascial graft. Since 1890, various materials have been researched as dura mater substitutes. Amniotic membrane, for example, is suitable as a dural graft material and has been used in neurosurgery since 2012. However, there has been little research on human patient's dural healing after the use of amniotic membrane in their duraplasty procedure. To address this gap, a clinical experimental study was undertaken to evaluate the human dural healing of 16 patients who had undergone duraplasty in decompressive craniectomy surgery at Dr. Soetomo General Hospital, Surabaya. The amniotic membrane allograft, was sutured to cover the dural defect for eight randomly chosen patients (Group I). The fascial autograft from the temporal muscle had been applied for eight other patients (Group II). Between 10 and 20 weeks after surgery, the patients underwent cranioplasty and dural healing evaluation by cerebrospinal fluid (CSF) leakage testing through the edge of the dural defect. The fibrocyte infiltration around the edge of the dural defect was examined histologically. Statistical analysis, using an independent t -test, was performed with a confidence interval of 95%. The results of the clinical and histological analysis suggest that an amniotic membrane graft was able to provide watertight dural closure and adequate fibrocyte infiltration comparable with that provided by temporalis muscle fascia. This study shows that using an amniotic membrane in neurosurgery has a potential advantage over an alternative dural healing. [ABSTRACT FROM AUTHOR]

Conjunctival reconstruction is an integral component of ocular surface restoration. Decellularised tissues are frequently used clinically for tissue engineering. This study identifies porcine decellularised conjunctiva (PDC) and human decellularised conjunctiva (HDC) as promising substitutes for conjunctival reconstruction. PDC and HDC were nearly DNA-free, structurally intact and showed no cytotoxic effects in vitro, which was confirmed by DNA quantification, histology, transmission electron microscopy, collagen quantification and cytotoxicity assay. Comparing the biomechanical properties to amniotic membrane (AM), the most frequently applied matrix for ocular surface reconstruction today, the decellularised conjunctiva was more extensible and elastic but exhibited less tensile strength. The in vivo application in a rabbit model proofed significantly enhanced transplant stability and less suture losses comparing PDC and HDC to AM while none of the matrices induced considerable inflammation. Ten days after implantation, all PDC, 4 of 6 HDC but none of the AM transplants were completely integrated into the recipient conjunctiva with a partially multi-layered epithelium. Altogether, decellularised conjunctivas of porcine and human origin were superior to AM for conjunctival reconstruction after xenogeneic application in vivo . Statement of Significance Conjunctival integrity is essential for a healthy ocular surface and clear vision. Its reconstruction is required in case of immunological diseases, after trauma, chemical or thermal burns or surgery involving the conjunctiva. Due to limitations of currently used substitute tissues such as amniotic membrane, there is a need for the development of new matrices for conjunctival reconstruction. Decellularised tissues are frequently applied clinically for tissue engineering. The present study identifies porcine and human decellularised conjunctiva as biocompatible and well tolerated scaffolds with superior integration into the recipient conjunctiva compared to amniotic membrane. Decellularised conjunctiva depicts a promising substitute for conjunctival reconstruction in ophthalmology. [ABSTRACT FROM AUTHOR]

Background Dissection of brain surface adhesions during recurrent glioma surgery carries a risk of injury to cortical vessels and important surface vessels. We present our experience with the use of BioD film, a biocompatible amniotic membrane implant, to help prevent postoperative adhesions. We describe a novel method for preventing postoperative adhesions after high-grade glioma surgery using BioD film. Methods Amniotic sac implants were laid on the brain surface after resection of gliomas located near major surface arteries (sylvian fissure) and major veins (parasagittal convexity). Seven cases involved reoperation for tumor recurrence. Results In all 7 of the cases requiring reoperation, a new arachnoid-like surface layer was formed without any dural adhesions. The newly formed layer allowed for easy and simple dissection and mobilization of surface vessels while avoiding any trauma to the cortex. Conclusions Amniotic sac implants have a promising role in preventing most surgical brain adhesions associated with recurrent glioma surgery, reducing the risks of cortical vessel and tissue injury. [ABSTRACT FROM AUTHOR]

Placenta-derived mesenchymal stromal cells (MSC) have often been considered to linger behind their equivalents from other tissues, such as MSC from bone marrow, in many aspects including their therapeutic potential in regenerative medicine. Nowadays however, it is clear that certain aspects make placental MSC attractive as a cellular therapy, such as their lack of ethical concerns and ease of isolation from human term placenta, a material long regarded as biological waste. Moreover, placental MSC virtually lack expression of human leukocyte antigens and co-stimulatory molecules, making them very attractive for transplantation in allogeneic settings. In the context of cancer, cell therapy remains an area of intense investigation whereby MSC have been shown to play opposing roles, and placental MSC are no exception. In this review, we will discuss dichotomy of placental MSC that underscores the challenges in understanding their therapeutic potential in oncology. [ABSTRACT FROM AUTHOR]

Wound healing usually follows a predictable sequence and prognosis of events. Its evolutionary process is the result of a complicated interaction between patient-related factors, the wound, the treatment used and the skills and knowledge of the professionals who treat them. Only through a meticulous initial assessment of the wound is it possible to identify the factors that contribute to its complexity. The challenge for professionals will be to implement efficient therapies at the right time and in the most cost-efficient way in order to reduce associated problems, treat the symptoms and expectations of the patients and achieve adequate wound healing whenever possible. This is particularly evident in big chronic wounds with considerable tissue loss, which become senescent in the process of inflammation or proliferation losing the ability to epithelialize. Generally, chronic wounds do not respond to current treatments, therefore they need special interventions. AM is a tissue of particular interest as a biological dressing and it has well-documented reepithelialization effects which are in part related to its capacity to synthesize and release biological active factors. Our studies have demonstrated that amniotic membrane (AM) is able to induce epithelialization in chronic wounds that were unable to epithelialize. AM induces several signaling pathways that are involved in cell migration and/or proliferation. Additionally, AM is able to selectively antagonize the anti-proliferative effect of transforming growth factor-ß (TGF-β) by modifying the genetic program that TGF-β induces on keratinocytes. The combined effect of AM on keratinocytes, promoting cell proliferation/migration and antagonizing the effect of TGF-β is the perfect combination, allowing chronic wounds to move out of their non-healing state and progress into epithelialization. [ABSTRACT FROM AUTHOR]

Cardiovascular disease (CD) is a major burden for Western society. Regenerative medicine has provided encouraging results, yet it has not addressed the focal defects causing CD and mainly related to the inefficient repair programme of the heart. In this scenario, stem cells have been broadly investigated and their paracrine effect proposed as a possible working strategy to boost endogenous mechanisms of repair and regeneration from within the cardiac tissue. The scientific community is now focusing on identifying the most effective stem cell secretome, as the whole of bioactive factors and extracellular vesicles secreted by stem cells and endowed with regenerative potential. Indeed, the adult stem cell-paracrine potential for cardiac regeneration have been widely analyzed with positive outcome. Nevertheless, low yield, invasive sampling and controversial self-renewal may limit adult stem cell application. On the contrary, fetal and perinatal stem cells, which can be easily isolated from leftover sample via prenatal screening during gestation or as clinical waste material after birth, can offer an ideal alternative. These broadly multipotent immature progenitors share features with both adult and embryonic stem cells, show high self-renewal, but they are not tumorigenic neither cause any ethical concern. While fetal and perinatal stem cells demonstrated to improve cardiac function when injected in the injured heart, the comprehensive characterization of their secretome for future applications is still at its infancy. In this review, we will discuss the paracrine potential of the fetal and perinatal stem cell secretome to provide cardiac repair and resurge the dormant mechanisms of cardiac regeneration for future therapy. [ABSTRACT FROM AUTHOR]

The most important factors in the non-optimal healing of diabetic wounds are the lack of a suitable scaffold in the wound site for the migration and replacement of cells, as well as the lack of blood supply and effective growth factors in the wound site. Herein we investigated whether a bioengineered micro-porous three-dimensional decellularized amniotic membrane-scaffold (DAMS) in combination with adipose-derived stem cells (ASCs) could promote healing in ischemic wounds in diabetic type 1 rat. The diabetic animals were randomly divided into non-treated (untreated group), engraftment by DAMS (DAMS group), transplanted by ASCs (ASC group), and DAMS in combination with ASCs (DAMS+ASC group). Stereological, immunohistochemical, molecular, and tensiometrical assessments were performed on post-surgical days 7, 14, and 21. We found that the rate of wound closure, the volumes of new epidermis and dermis, the numerical density of fibroblasts and blood vessels, the numbers of proliferating cells and collagen deposition as well as biomechanical properties of the healed wounds were significantly higher in the treatment groups in comparison to the untreated group, and were the highest in DAMS+ASC ones. The transcripts for TGF-β and VEGF genes were significantly upregulated in all treatment regimens compared to the untreated group and were the highest for DAMS+ASC group. This is while expression of TNF-α and IL-1β as well as cell density of neutrophils decreased more significantly in DAMS+ASC group as compared with other groups. Overall, it was found that using both DAMS engraftment and ASC transplantation has more impact on diabetic wound healing. • The DAMS and ASCs promote healing. • The DAMS and ASCs increase the amount of collagen. • The DAMS and ASCs regulate the inflammatory process. • The stereological characterizes is higher when the DAMS and ASCs are used. • The DAMS and ASCs upregulated regenerative genes. • The DAMS and ASCs increase biomechanical characteristics. [ABSTRACT FROM AUTHOR]

Several strategies have been proposed to enhance wound healing results. Along with other forms of wound dressing, the human amniotic membrane (HAM) has long been regarded as a biological wound dressing that decreases infection and enhances healing. This study investigates the feasibility and effectiveness of wound healing using decellularized HAM (dAM) and stromal HAM (sAM) in combination with adipose-derived human mesenchymal stem cells (AdMSCs). The dAM and sAM sides of HAM were employed as wound dressing scaffolds, and AdMSCs were seeded on top of either dAM or sAM. Sixty healthy Wistar rats were randomly divided into three groups: untreated wound, dAM/AdMSCs group, and sAM/AdMSCs group. The gene expression of VEGF and COL-I was measured in vitro. Wound healing was examined after wounding on days 3, 7, 14, and 21. The expression level of VEGF was significantly higher in sAM/AdMSCs than dAM/AdMSCs (P ≤ 0.05), but there was no significant difference in COL-I expression (P ≥ 0.05). In vivo research revealed that on day 14, wounds treated with sAM/AdMSCs had more vascularization than wounds treated with dAM/AdMSCs (P ≤ 0.01) and untreated wound groups on days 7 (P ≤ 0.05) and 14 (P ≤ 0.0001), respectively. On days 14 (P < 0.05 for sAM/AdMSCs, P < 0.01 for dAM/AdMSCs), and 21 (P < 0.05 for sAM/AdMSCs, P < 0.01 for dAM/AdMSCs), the collagen deposition in the wound bed was significantly thicker in the sAM/AdMSCs and dAM/AdMSCs groups compared to untreated wounds. The study demonstrated that the combination of sAM and AdMSCs promotes wound healing by enhancing angiogenesis and collagen remodeling. [Display omitted] [ABSTRACT FROM AUTHOR]

Background aims. Human amniotic mesenchymal stromal cells (hAMSCs) are a potent and attractive stem cell source for use in regenerative medicine. However, the safe uses of therapeutic-grade MSCs are equally as important as the efficiency of MSCs. To provide efficient, clinic-compliant (safe for therapeutic use) MSCs, hAMSC lines that completely eliminate the use of animal products and have been characterized for carcinogenicity and biosafety are required. Methods. Here, we have efficiently generated 10 hAMSC lines under human umbilical cord blood serum (hUCS)-supplemented medium (xeno-free culture) and fetal bovine serum (FBS)-supplemented medium (standard culture) and investigated carcinogenicity and immunosuppressive properties in the resultant hAMSC lines. All hAMSC lines were examined for efficiency (growth kinetics, cryopreservation, telomere length, phenotypic characterization, differentiation potential), carcinogenicity (proto-oncogene and tumor suppressor gene and epigenomic stability) and safety (immunosuppressive properties). Results. Stem cell characteristics between the xeno-free hAMSC lines and the cell lines generated using the standard culture system showed no differences. Xeno-free hAMSC lines displayed normal growth proliferation potential, morphological, karyotypic, phenotypic differentiation properties and telomere lengths. Additionally, they retained normal immunosuppressive effects. As a marker of carcinogenicity and biosafety, proto-oncogenes expression levels showed no differences in xeno-free hAMSCs, and we detected no SNP mutations on hotspot codons of the P53 tumor suppressor gene and stable epigenomic imprinting in xeno-free hAMSC lines. Conclusions. Xeno-free hAMSC lines retain essential stem cell characteristics, with a high degree of certainty for meeting biosafety and carcinogenicity standards for a xeno-free system supplemented with allogenic hUCS. The cell lines are suitable and valuable for therapeutic purposes. [ABSTRACT FROM AUTHOR]

Background. Mesenchymal stromal cells (MSCs) are being investigated as a potential alternative for cellular therapy. This study was designed to compare the biological characteristics of MSCs isolated from amniotic membrane (A-MSCs), chorionic membrane (C-MSCs), placental decidua (D-MSCs) and umbilical cord (UC-MSCs) to ascertain whether any one of these sources is superior to the others for cellular therapy purposes. Methods. MSCs were isolated from amniotic membrane, chorionic membrane, umbilical cord and placental decidua. Immunophenotype, differentiation ability, cell size, cell complexity, polarity index and growth kinetics of MSCs isolated from these four sources were analyzed. Results. MSCs were successfully isolated from all four sources. Surface marker profile and differentiation ability were consistent with human MSCs. C-MSCs in suspension were the smallest cells, whereas UC-MSCs presented the greatest length and least width. A-MSCs had the lowest polarity index and UC-MSCs, as more elongated cells, the highest. C-MSCs, D-MSCs and UCMSCs exhibited similar growth capacity until passage 8 (P8); C-MSCs presented better lifespan, whereas insignificant proliferation was observed in A-MSCs. Discussion. Neonatal and maternal tissues can serve as sources of multipotent stem cells. Some characteristics of MSCs obtained from four neonatal tissues were compared and differences were observed. Amniotic membrane was the least useful source of MSCs, whereas chorionic membrane and umbilical cord were considered good options for future use in cell therapy because of the known advantages of immature cells. [ABSTRACT FROM AUTHOR]

Amniotic membranes (AM) have shown its great potential in reconstructive surgery due to their regenerative capacity. However, AM is regarded to be relatively weak when applied for load-bearing purposes. This study aims to produce an AM-based scaffold that can withstand the mechanical loads applied in vesicovaginal fistula repair. Different strategies are investigated to improve the mechanical characteristics of AM. Single and multilayered AM, and composite constructs of AM with electrospun poly-4-hydroxybutyrate (P4HB) or bovine pericardial tissue combined with the use of fibrin glue, were mechanically tested in this study. Suture retention strength and mechanical characteristics (tensile stress, elongation, tangent modulus and maximum load) were assessed by uniaxial testing. The effect of degradation of the composite constructs on the mechanical characteristics was determined by uniaxial testing after 4 and 8 weeks. Single and multilayered AM could not provide the mechanical requirements needed for surgical implantation (>2N load). AM was combined successfully with electrospun P4HB and bovine pericardium with the use of fibrin glue and were able to exceed the 2N load. The composite constructs with AM showed sufficient mechanical characteristics for surgical implantation. Electrospun P4HB combined with AM seemed the most promising candidate since the mechanical characteristics of P4HB can be further modified to meet the requirements of the application site and the degradation of the P4HB allows a gradual transfer of load. Eventhough the scaffold is intended for fistula repair, it can potentially be applied in surgical reconstruction of other hollow organs by modifying the mechanical characteristics. [ABSTRACT FROM AUTHOR]

Sutures are a common way to attach scaffolds in patients. For tubular cardiac scaffolds, the ‘suture retention strength’ is commonly used to evaluate the resistance of a scaffold against the pull-out of a suture. In order to make this quantity accessible for ophthalmological scaffolds the test procedure has been modified in a novel way. Polycaprolactone (PCL) films of different thicknesses and an amniotic membrane (AM) were used for the experiments. Circular samples with a radius of 7 mm were taken and a suture was passed through each sample and tied to a loop. The sample was clamped in a tensile tester and a bolt was passed through the loop. The suture was then pulled with a constant deformation rate until pull-out occurred. The suture retention strength, the deformation at the suture retention strength, and the deformation at rupture were determined for each sample. The presented modified suture retention test allows to measure the relevant parameters of samples on the scale of ophthalmological scaffolds in a reproducible way. A comparison between the first data on PCL and AM has been made. [ABSTRACT FROM AUTHOR]

Highlights • HAM can be a useful, potential scaffold for treatment of infected, irregular, deep wounds due to the promotion of epithelial regeneration, elastic and antibacterial characteristics. • Clinical case of chronic wound with an entero-cutaneous fistula was successfully treated with HAM dressings after 8 weeks. • Histological analysis showed partial re-epithelialisation and muscle cells recovery 4 weeks after the treatment. • The preliminary results support the initiation of prospective trial and registry according to IDEAL. Abstract Introduction Infected wounds are difficult to treat and there are no standardized protocols. Presentation of case We report a case of infected postoperative wound and entero-cutaneous fistula in a 83 years-old woman. An innovative treatment protocol for Human amniotic membrane (HAM)-assisted dressing of infected wound as the Idea Stage following the IDEAL recommendations is presented. The development of amnion preparation and the involved treatment steps are described. No adverse events and no graft rejection have been detected. Discussion Favorable results confirm the technical simplicity, safety and efficacy of this procedure. HAM has been shown to promote wound healing and to have antibacterial characteristics, which was supported by the presented case. Conclusion We are able to report a successful treatment of an infected wound caused by entero-cutaneous fistula with HAM dressing. Following the IDEAL recommendations, consecutive prospective cohort trials are justified. [ABSTRACT FROM AUTHOR]

The human amniotic membrane (hAM) surrounds and protects the fetus. It mainly consists of a thick basement membrane and an avascular stromal matrix. The structural, physical, and biological properties of AM make it a wonderful choice to use in regenerative medicine applications. This review discusses recent applications of hAM in skin transplantation and wound healing, ophthalmology, orthopedics, cell culture matrix, cell delivery system, cardiac injury, male and female reproductive systems, and liver. Moreover, an overview is presented on the advantages and disadvantages of using hAM and some methods to systematically refine and standardize hAM preparation. [Display omitted] [ABSTRACT FROM AUTHOR]

Background/purpose: Periodontal disease is a chronic inflammatory process that potentially leads to alveolar bone destruction and tooth loss. Tissue engineering combined with stem cell therapy is a potential effective treatment for periodontal bone loss. Amniotic membrane (AM) is a potential scaffold enriched with multiple growth factors. It has the effects of anti-inflammation, antiangiogenesis, and immunosuppression. Herein, we used adipose-derived stem cells (ADSCs) and an AM co-cultured system to study bone regeneration in a rat periodontal defect model in vivo.Methods: Human ADSCs were isolated from the infrapatellar fat pad, and characterized by flow cytometry, reverse transcription-polymerase chain reaction, and multipotent differentiation assays. The co-culture system was applied in the periodontal two-wall osseous defect in a rat model, and computed tomography was used to measure the effect.Results: Human ADSCs isolated from the infrapatellar fat pad showed spindle-like morphology. Flow cytometry results demonstrated that ADSCs expressed a high level of CD90 and CD105, but not CD31, CD34, and CD45. ADSCs strongly expressed stemness genes, including SOX2, OCT4, NANOG, and KLF4 on different passages. Furthermore, ADSCs were able to differentiate into osteogenic, chondrogenic, and adipogenic cells. In the periodontal osseous defect rat model, ADSCs and the AM co-culture system significantly increased bone regeneration.Conclusion: This study provides the basis for using ADSCs with an AM co-culture system as stem cell therapy and scaffold transplantation in clinical periodontology. [ABSTRACT FROM AUTHOR]

Objective Management of traumatic facial soft tissue injury poses a major challenge for surgeons due to aesthetic concerns. Human amniotic membrane (AM) and collagen membrane (CM), biologic materials, have attained importance in various clinical fields, especially in wound healing. Though both materials have their own unique properties, there is a need to compare and evaluate the efficacy of AM and CM as a wound dressing material for post-traumatic soft tissue injuries of the maxillofacial region. Methods A study was conducted in Department of Oral And Maxillofacial Surgery, at our institution which encompassed all the patients (total of 20 patients) with facial soft tissue injuries (abrasions), due to road traffic accidents from January 2014 to March 2014. They were randomly divided into two groups, with 10 patients treated with AM and 10 patients treated with CM. The groups were compared according to the wound compliance, time taken for granulation tissue formation and scarring. Results It was observed that the pain score was much higher in Collagen group than in Amnion group and complete adherence of the membrane to the wound was detected in both the groups. A statistically significant result in favour of AM was found with regard to average time taken for granulation tissue formation ( P = 0.036) and the resultant scarring. Incidence of wound infection was reported in two cases in Collagen group. Conclusion When compared to Collagen, AM had good adhesive, antibacterial, anti-immunogenic and anti-scarring property. Its low cost of procurement and storage makes it an ideal wound dressing material. [ABSTRACT FROM AUTHOR]

Oral mucositis is a reaction to chemoradiation therapy during cancer treatment. The aim of this study was to investigate the use of amniotic membrane as a biological dressing for oral mucositis lesions in rats. Sixty Wistar rats were divided into three groups ( n = 20): control, 5-fluoruracil (5-FU), 5-fluoruracil + amniotic membrane (5-FU + AM). Each group was subdivided ( n = 5) according to the time interval to sacrifice (3, 7, 14, and 21 days). Histology (haematoxylin–eosin staining) and immunocytochemistry (anti-rat antibodies CD4, CD8, VEGF, and PCNA) were evaluated. Immunocytochemistry results were analyzed using one-way ANOVA and Tukey tests. The amniotic membrane (5FU + AM) played an important role in cell proliferation (PCNA 3 days 27.08 ± 4.65, 7 days 27.90 ± 3.34) and especially in neovascularization (VEGF 3 days 23.00 ± 1.40, 7 days 26.00 ± 0.95) for all time intervals, when compared to 5-FU (PCNA 3 days 23.12 ± 1.61, 7 days 37.21 ± 1.20; VEGF 3 days 17.05 ± 1.51, 7 days 8.45 ± 1.35) and control (PCNA 3 days 29.99 ± 0.92, 7 days 16.33 ± 2.88; VEGF 3 days 13.65 ± 0.55, 7 days 15.70 ± 1.39). It was biocompatible, showing significant differences compared to the other groups in CD4 ( F = 40.72; P = 0.001) and CD8 ( F = 69.99, P = 0.001) staining together, only during the inflammation phase (7 days). Amniotic membrane presented biocompatibility and stimulated cell proliferation and neovascularization, functioning as a promising biological dressing. [ABSTRACT FROM AUTHOR]

The purpose of this study was to investigate the carbodiimide cross-linking of amniotic membrane (AM) in the presence of amino acid bridges. The biological tissues were treated with glycine, lysine, or glutamic acid and chemically cross-linked to examine the role of amino acid types in collagenous biomaterial processing. Results of zeta potential measurements showed that the use of uncharged, positively and negatively charged amino acids dictates the charge state of membrane surface. Tensile strength and water content measurements demonstrated that the addition of lysine molecules to the cross-linking system can increase the cross-linking efficiency and dehydration degree while the introduction of glutamic acid in the AM samples decreases the number of cross-links per unit mass of chemically modified tissue collagen. The differences in the cross-linking density further determined the thermal and biological stability by differential scanning calorimetry and in vitro degradation tests. As demonstrated in matrix permeability studies, the improved formation of covalent cross-linkages imposed by lysine facilitated construction of stronger cross-linking structures. In contrast, the added glycine molecules were insufficient to enhance the resistances of the proteinaceous matrices to thermal denaturation and enzymatic degradation. The cytocompatibility of these biological tissue membranes was evaluated by using human corneal epithelial cell cultures. Results of cell viability, metabolic activity, and pro-inflammatory gene expression level showed that the AM materials cross-linked with carbodiimide in the presence of different types of amino acids are well tolerated without evidence of detrimental effect on cell growth. In addition, the amino acid treated and carbodiimide cross-linked AM implants had good biocompatibility in the anterior chamber of the rabbit eye model. Our findings suggest that amino acid type is a very important engineering parameter to mediate carbodiimide cross-linking of AM collagen. [ABSTRACT FROM AUTHOR]

Human amniotic membrane-derived stromal cells (hAMSC) are candidates for cell-based therapies. We examined the characteristics of hAMSC including the interaction between hAMSC and breast cancer cells, MCF-7, and MDA-MB-231. Human amniotic membrane–derived stromal cells showed typical MSC properties, including fibroblast-like morphology, surface antigen expression, and mesodermal differentiation. To investigate cell–cell interaction via secreted molecules, we cultured breast cancer cells in hAMSC-conditioned medium (hAMSC-CM) and analyzed their proliferation, migration, and secretome profiles. MCF-7 and MDA-MB-231 cells exposed to hAMSC-CM showed increased proliferation and migration. However, in hAMSC-CM, MCF-7 cells proliferated significantly faster than MDA-MB-231 cells. When cultured in hAMSC-CM, MCF-7 cells migrated faster than MDA-MB-231 cells. Two cell types showed different profiles of secreted factors. MCF-7 cells expressed much amounts of IL-8, GRO, and MCP-1 in hAMSC-CM. Human amniotic membrane–derived stromal cells interact with breast cancer cells through secreted molecules. Factors secreted by hAMSCs promote the proliferation and migration of MCF-7 breast cancer cells. For much safe cell-based therapies using hAMSC, it is necessary to study carefully about interaction between hAMSC and cancer cells. [ABSTRACT FROM AUTHOR]

Introduction Human term placenta has attracted increasing attention as an alternative source of stem cells for regenerative medicine since it is accessible without ethical objections. The amniotic membrane (AM) contains at least two stem cell types from different embryological origins: ectodermal amniotic epithelial stem cells, and mesodermal mesenchymal stromal cells. Among the second group we studied the characteristics of amniotic mesenchymal cells (AMC) versus the ones enriched for the commonly used surface marker c-Kit (amniotic progenitor/stem cells-ASC), a stem cell factor receptor with crucial functions in a variety of biological systems and presents in early progenitors of different origin, as been already demonstrated in the enriched chorionic stem cells. Methods After isolation, cells from the amniotic membranes (amniotic cells-AC) were selected for c-Kit (ASC) and compared these cells with c-Kit unselected (AMC), evaluating the expression of other stem cell markers (Oct-4, Tra-1-81, SSEA-4), CD271 and Slug. Results Immunofluorescence analysis showed that ASC cells exhibited greater stem cell marker expression and included more CD271 and Slug positive cells. This was consistent with the interpretation that c-Kit enriched AC show greater stemness capacity compared to c-Kit unselected AMC. Discussion AMC and ASC can both differentiate into various cell types including adipogenic, osteogenic, chondrogenic, neurogenic and hepatic lineages, but the enrichment in c-Kit improved stemness and differentiation potential of ASC. [ABSTRACT FROM AUTHOR]

In the present study, we developed photo-cross-linked amniotic membrane (AM) as a limbal stem cell niche. After ultraviolet (UV) irradiation for varying time periods, the biological tissues were studied by determinations of cross-linking structure, degradability, and nutrient permeation ability. Our results showed that the number of cross-links per unit mass of AM significantly increased with increasing illumination time from 5 to 50 min. However, the cross-link formation was inhibited by longer irradiation time (i.e., 150 min), probably due to the scission of tissue collagen chains through irradiation. The biological stability and matrix permeability of photo-cross-linked AM materials strongly depended on their cross-linking densities affected by the UV irradiation. In vitro biocompatibility studies including cell viability and pro-inflammatory gene expression analyses demonstrated that, irrespective of the irradiation time employed, the physically cross-linked biological tissues exhibited negligible cytotoxicity and similar interleukin-6 (IL-6) mRNA levels. The data clearly indicate that these AM matrices do not cause potential harm to the corneal epithelial cells. After the growth of limbal epithelial cells (LECs) on AM substrates, Western blot analyses were conducted to examine the expression of ABCG2. It was found that the ability of UV-irradiated AM to maintain the undifferentiated precursor cell phenotype was significantly enhanced with increasing extent of photo-cross-linking. In summary, the UV irradiation time may have a profound influence on the fabrication of photo-cross-linked AM matrices for LEC cultivation. [ABSTRACT FROM AUTHOR]

Introduction There is evidence that premature rupture of the fetal membrane at term/preterm is a result of stretch and tissue weakening due to enhanced prostaglandin E 2 (PGE 2 ) production. However, the effect of tensile strain on inflammatory mediators and the stretch sensitive protein connexin-43 (Cx43) has not been examined. We determined whether the inflammatory environment influenced tissue composition and response of the tissue to tensile strain. Methods Human amniotic membranes isolated from the cervix ( C AM) or placenta regions ( P AM) were examined by second harmonic generation to identify collagen orientation and subjected to tensile testing to failure. In separate experiments, specimens were subjected to cyclic tensile strain (2%, 1 Hz) for 24 h. Specimens were examined for Cx43 by immunofluorescence confocal microscopy and expression of COX-2 and Cx43 by RT-qPCR. PGE 2 , collagen, elastin and glycosaminoglycan (GAG) levels were analysed by biochemical assay. Results Values for tensile strength were significantly higher in P AM than C AM with mechanical parameters dependent on collagen orientation. Gene expression for Cx43 and COX-2 was enhanced by tensile strain leading to increased PGE 2 release and GAG levels in P AM and C AM when compared to unstrained controls. In contrast, collagen and elastin content was reduced by tensile strain in P AM and C AM. Discussion Fibre orientation has a significant effect on amniotic strength. Tensile strain increased Cx43/COX-2 expression and PGE 2 release resulting in tissue softening mediated by enhanced GAG levels and a reduction in collagen/elastin content. Conclusion A combination of inflammatory and mechanical factors may disrupt amniotic membrane biomechanics and matrix composition. [ABSTRACT FROM AUTHOR]