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The therapeutic effect of mesenchymal stromal cells (MSCs) has been described for a variety of disorders, including those affecting musculoskeletal tissues. In this context, the literature reports several data about the regenerative effectiveness of MSCs derived from bone marrow, adipose tissue, and an amniotic membrane (BMSCs, ASCs, and hAMSCs, respectively), either when expanded or when acting as clinical-grade biologic pillars of products used at the point of care. To date, there is no evidence about the superiority of one source over the others from a clinical perspective. Therefore, a reliable characterization of the tissue-specific MSC types is mandatory to identify the most effective treatment, especially when tailored to the target disease. Because molecular characterization is a crucial parameter for cell definition, the need for reliable normalizers as housekeeping genes (HKGs) is essential. In this report, the stability levels of five commonly used HKGs (ACTB, EF1A, GAPDH, RPLP0, and TBP) were sifted into BMSCs, ASCs, and hAMSCs. Adult and fetal/neonatal MSCs showed opposite HKG stability rankings. Moreover, by analyzing MSC types side-by-side, comparison-specific HKGs emerged. The effect of less performant HKG normalization was also demonstrated in genes coding for factors potentially involved in and predicting MSC therapeutic activity for osteoarthritis as a model musculoskeletal disorder, where the choice of the most appropriate normalizer had a higher impact on the donors rather than cell populations when compared side-by-side. In conclusion, this work confirms HKG source-specificity for MSCs and suggests the need for cell-type specific normalizers for cell source or condition-tailored gene expression studies.

Purpose: To report the case of an extremely large overhanging bleb, extending from superior fornix to limbus, in a 57-year-old poorly controlled diabetic, six years after trabeculectomy for an uncontrolled primary open angle glaucoma (POAG) with recurrence, months after complete excision. Methods: An overhanging bleb is defined as a filtering cicatrix which has been massaged downward over the cornea by eyelid action. It has been linked to anti-metabolite use during glaucoma filtering surgery. Despite being functional, these blebs result in patient discomfort ranging from foreign body sensation and lacrimation to dysphotopsia.A 57-year-old male presented with complaints of reduced vision, foreign body sensation, watering, and difficulty in eye closure in the left eye (OS) for past 6 months. He had undergone trabeculectomy with mitomycin C 6 years ago for advanced primary open-angle glaucoma with no follow-up beyond the initial one 4 weeks post-surgery. Results: At presentation, he had a giant multi-loculated, cystic filtering bleb (15 mm x 8 mm x 4-5 mm), which was carefully excised. Amniotic membrane was used as an anti-fibrotic as well to cover the defect. Seven months after surgery, there was recurrence of this overhanging cystic bleb when it was again excised with debulking of the conjunctiva done and cryotherapy applied to its margins.Conclusion: Although multiloculated cystic overhanging blebs have been documented before, such a large (posterior extent till fornix), thick-walled multiloculated bleb with histopathological evidence of chronic inflammatory process has not been reported prior.

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.

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.

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.

Purpose: To report the 1-year results of human amniotic membrane patch implantation for optic disc pit maculopathy. Design: A prospective, consecutive, interventional study. Methods: Eleven eyes of 11 patients affected by optic disc pit maculopathy associated with subretinal/intraretinal fluid were included in this single-institution study. A 25-gauge pars plana vitrectomy was performed in all cases, with an implant of a human amniotic membrane patch into the optic disc pit and air was injected as endotamponade. The primary study outcome was the subretinal and intraretinal fluid reabsorption. Secondary outcomes were visual acuity improvement and postoperative complications. Results: Mean central retinal thickness gradually diminished from 512 ± 137 µm to 243 ± 19 µm, at the 12-month follow-up. The mean visual acuity improved from 20/80 at baseline to 20/32 at the 12-month follow-up. Complete fluid resorption occurred in 9 of 11 (81.8%) eyes and there was partial resorption in 2 eyes (18%). No subretinal fluid recurrence was observed during the 12-month follow-up. No intraoperative or postoperative complications were reported during the follow-ups. The amniotic membrane patch remained detectable inside the pit for the entire follow-up time. Conclusion: An amniotic membrane plug may be effective for improving optic disc pit maculopathy. All cases had an anatomical improvement and encouraging visual acuity recovery.

BackgroundThe application of the amniotic membrane could have a favourable effect on tissue repair and regeneration. We report the first case of implant of an amniotic membrane in a patient affected by myo-cutaneous dehiscence, after a radical surgical treatment for vulvar cancer. MethodsWe describe a case of a 74-years-old patient affected by vulvar cancer. After radiotherapy, the patient underwent to an anterior pelvic exenteration with uretero-ileo-cutaneostomy by Wallace, bilateral pelvic lymphadenectomy, omental biopsies, omental flap, bilateral inguinal lymphadenectomy, resection of ulcerated left inguinal lesion, reconstruction with left gracilis muscle flap and locoregional V-Y advancement flap. The patient developed a myo-cutaneous dehiscence. Two months after the surgery, following an accurate curettage of the wound and negative pressure therapy, a patch of human amniotic membrane was implanted. ResultsThe surgical procedure was easy, feasible and did not require long operating room times. No intraoperative or postoperative complications occurred. The results obtained were encouraging with a marked improvement in the surgical wound. Conclusionthe use of amniotic membranes was safely and easily performed to promote the healing of complicated surgical wounds.

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.

Background: Implantation of the amniotic membrane and their derivatives can have a beneficial effect on tissue repair and regeneration. We report for the first time the implant of an amniotic membrane in a patient affected by cryptoglandular anal fistula. Methods: A patch of human amniotic membrane was implanted in a female patient affected by an anterior transphincteric fistula. Following an accurate curettage of the anal fistula, the cryopreserved amniotic membrane was thawed and then washed in the operating room; one side of the membrane was transfixed with a resorbable suture thus creating an implantable fusiform patch. The membrane was subsequently implanted into the fistula tract from the external to the internal opening. The inner and outer parts of the membrane were then sutured to the internal and external fistula openings. Results: No intraoperative or postoperative complications occurred. The patient was discharged one day after the procedure after an uneventful hospitalization. At the 1-week, 1-and 3-month follow-up visits no pain (VAS 0) was referred by the patient and no inflammation was evident at the level of the previous external fistula opening. Conclusions: The implant of human amniotic membrane in a patient affected by cryptoglandular anal fistula was safely and easily performed. Moreover, future studies to assess the efficacy in the long-term follow-up are needed.

Background: Toxic epidermal necrolysis (TEN) is a rare life-threatening disease that mainly affects the skin and mucous membranes, resulting from a toxic delayed-type hypersensitivity (DTH) reaction (type IV reaction) to the presence of foreign antigens such as drugs. The clinical symptoms are caused by pathophysiological processes leading to massive apoptosis of keratinocytes in the dermo-epidermal junction. This results in the formation of a bulla and subsequent separation of the entire epidermis with the exposure of the dermis. The current approach in the local therapy of TEN prefers the use of biological dressings, which helps provide several critical requirements for defect healing; in particular, it helps in the acceleration of the spontaneous wound closure (re-epithelialization) of the skin defect and the reduction of the risk of development of various complications and infections, such as the risk of pathological scar maturation. This paper is a case report of the use of a lyophilized amniotic membrane (AM) for accelerating wound healing in a patient with TEN. Case Presentation: We report a case of an 8-year-old girl transferred to our center with a histologically confirmed diagnosis of TEN. Despite the application of immunosuppressive therapy consisting of corticosteroids and intravenous immunoglobulins, we have observed disease progression and exfoliation of up to 60% of the total body surface area (TBSA). In the facial area, which is cosmetically privileged, we decided to use the lyophilized amniotic membrane (Amnioderm (R)) to cover up approximately 2% of the TBSA. Within 2 days after the application, we observed accelerated reepithelialisation, with rapid wound closure. We have not observed any side effects nor infections during the subsequent phases of wound healing. Skin defects in non-facial areas of the body were treated with synthetic dressings. When compared to the areas covered with the lyophilized AM, the healing process was prolonged. Conclusio

Background: Toxic epidermal necrolysis (TEN) is a rare life-threatening disease that mainly affects the skin and mucous membranes, resulting from a toxic delayed-type hypersensitivity (DTH) reaction (type IV reaction) to the presence of foreign antigens such as drugs. The clinical symptoms are caused by pathophysiological processes leading to massive apoptosis of keratinocytes in the dermo-epidermal junction. This results in the formation of a bulla and subsequent separation of the entire epidermis with the exposure of the dermis. The current approach in the local therapy of TEN prefers the use of biological dressings, which helps provide several critical requirements for defect healing; in particular, it helps in the acceleration of the spontaneous wound closure (re-epithelialization) of the skin defect and the reduction of the risk of development of various complications and infections, such as the risk of pathological scar maturation. This paper is a case report of the use of a lyophilized amniotic membrane (AM) for accelerating wound healing in a patient with TEN. Case Presentation: We report a case of an 8-year-old girl transferred to our center with a histologically confirmed diagnosis of TEN. Despite the application of immunosuppressive therapy consisting of corticosteroids and intravenous immunoglobulins, we have observed disease progression and exfoliation of up to 60% of the total body surface area (TBSA). In the facial area, which is cosmetically privileged, we decided to use the lyophilized amniotic membrane (Amnioderm (R)) to cover up approximately 2% of the TBSA. Within 2 days after the application, we observed accelerated reepithelialisation, with rapid wound closure. We have not observed any side effects nor infections during the subsequent phases of wound healing. Skin defects in non-facial areas of the body were treated with synthetic dressings. When compared to the areas covered with the lyophilized AM, the healing process was prolonged. Conclusio

Background: Toxic epidermal necrolysis (TEN) is a rare life-threatening disease that mainly affects the skin and mucous membranes, resulting from a toxic delayed-type hypersensitivity (DTH) reaction (type IV reaction) to the presence of foreign antigens such as drugs. The clinical symptoms are caused by pathophysiological processes leading to massive apoptosis of keratinocytes in the dermo-epidermal junction. This results in the formation of a bulla and subsequent separation of the entire epidermis with the exposure of the dermis. The current approach in the local therapy of TEN prefers the use of biological dressings, which helps provide several critical requirements for defect healing; in particular, it helps in the acceleration of the spontaneous wound closure (re-epithelialization) of the skin defect and the reduction of the risk of development of various complications and infections, such as the risk of pathological scar maturation. This paper is a case report of the use of a lyophilized amniotic membrane (AM) for accelerating wound healing in a patient with TEN. Case Presentation: We report a case of an 8-year-old girl transferred to our center with a histologically confirmed diagnosis of TEN. Despite the application of immunosuppressive therapy consisting of corticosteroids and intravenous immunoglobulins, we have observed disease progression and exfoliation of up to 60% of the total body surface area (TBSA). In the facial area, which is cosmetically privileged, we decided to use the lyophilized amniotic membrane (Amnioderm (R)) to cover up approximately 2% of the TBSA. Within 2 days after the application, we observed accelerated reepithelialisation, with rapid wound closure. We have not observed any side effects nor infections during the subsequent phases of wound healing. Skin defects in non-facial areas of the body were treated with synthetic dressings. When compared to the areas covered with the lyophilized AM, the healing process was prolonged. Conclusio

Background: It is now well established that factors (free or in extracellular vesicles) secreted by mesenchymal stromal cells (MSC) are important mediators of MSC regenerative actions. Herein we produced the secretome (conditioned medium, CM) from MSC isolated from the amniotic membrane (hAMSC) and CM from the intact amniotic membrane (hAM, no manipulation or enzymatic digestion) in order to potentially identify an effective, easy and less expensive secretome to produce for potential applications in regenerative medicine. Given that immunomodulation is a key mechanism of action through which hAMSC contributes to tissue regeneration, we used a comprehensive panel of in vitro immunomodulatory tests to compare the CMs.Methods: Amniotic membranes were either cut into fragments or used for hAMSC isolation. CMs from hAMSC at passages 0 and 2 were collected after a standard 5-day culture while CM from hAM was collected after a 2- and 5-day culture. Immunomodulation was assessed in terms of PBMC and T-cell proliferation, T-cell subset polarization, T-regulatory cell induction, cell cytotoxicity and monocyte differentiation toward antigen-presenting cells. Furthermore, we performed a comparison between CM obtained from single donors and pooled CM. We also assessed the impact of lyophilization on the immunomodulatory properties of CM.Results: We demonstrate that CM from hAM has comparable immunomodulatory properties to CM from hAMSC at passages 0 and 2. Furthermore, we demonstrate that pooled CMs have similar effects when compared to CM from single donors used separately. Finally, we demonstrate that lyophilization does not alter the in vitro immunomodulatory properties of CM from hAM and hAMSC.Conclusions: The results presented herein support the possibility to produce secretome from intact hAM and open the prospect to highly improve the scalability of the GMP production process while reducing the costs and time related to the process of cell isolation and expansion. Moreove

Aim: Mesenchymal stromal cells (MSCs) emerged as a promising therapeutic option for osteoarthritis (OA) management, in particular those isolated from adipose tissue (hASCs) and amniotic membrane (hAMSCs). The cartilage protective and immunomodulatory features of hASCs and hAMSCs are ascribed to secreted factors, including extracellular vesicles (EVs) and embedded miRNAs. The purpose of this study was to compare EVs and shuttled miRNAs from both MSC types and discuss them in the frame of OA pathological tissues. Methods: Human hASCs and hAMSCs were analyzed by flow cytometry. EVs were analyzed by flow cytometry, nanoparticle tracking analysis, and electron microscopy. High-throughput qRT-PCR miRNA data available in the literature were compared. Abundant miRNAs and their experimentally validated targets were associated with those reported to drive OA pathology at cartilage, synovia, and macrophage levels. Four tools (Genorm, Normfinder, BestKeeper, and Delta Ct) were used to identify EVs stable reference genes. Results: EVs did not show phenotypical or dimensional differences between the two sources, with hAMSCs releasing more particles. In total, 307 EV miRNAs were identified, with 306 shared. Several of the most abundant miRNAs target OA-driving factors and are involved in cartilage and synovia protective mechanisms, with hAMSC-EVs’ preponderance for M2 anti-inflammatory macrophage commitment. miR-34a-5p emerged as the most stable reference gene. Conclusion: Both hASCs and hAMSCs release EVs enriched in joint-protective and anti-inflammatory miRNAs, supporting their use for treatment of joint diseases. Future comparative clinical studies would be needed to test whether hAMSCs’ higher EV secretion and enhanced M2 macrophage polarizing miRNA cargo allow for potentially increased OA therapeutic features.

Amniotic membrane grafts have some therapeutic potential for wounds healing. Early application of amniotic membrane turned out as beneficial in healing ulcers, burns, and dermal injuries. Since the second half of the 20th century, the autotransplants of amniotic/chorion tissue have been also used for the treatment of chronic neuropathic wounds, cornea surface injuries, pterygium and conjunctivochalasis, and dental and neurosurgical applications. The aim of this publication is to prepare a coherent overview of amniotic membrane derivatives use in the field of wound healing and also its efficacy. In total 60 publications and 39 posters from 2000-2020 were examined. In these examined publications of case studies with known study results was an assemblage of 1141 patients, and from this assemblage 977 were successfully cured. In case of posters, the assemblage is 570 patients and 513 successfully cured. From the investigated data it is clear that the treatment efficacy is very high-86% and 90%, respectively. Based on this information the use of the amniotic membrane for chronic wounds can be considered highly effective.

Background: It is now well established that factors (free or in extracellular vesicles) secreted by mesenchymal stromal cells (MSC) are important mediators of MSC regenerative actions. Herein we produced the secretome (conditioned medium, CM) from MSC isolated from the amniotic membrane (hAMSC) and CM from the intact amniotic membrane (hAM, no manipulation or enzymatic digestion) in order to potentially identify an effective, easy and less expensive secretome to produce for potential applications in regenerative medicine. Given that immunomodulation is a key mechanism of action through which hAMSC contributes to tissue regeneration, we used a comprehensive panel of in vitro immunomodulatory tests to compare the CMs. Methods: Amniotic membranes were either cut into fragments or used for hAMSC isolation. CMs from hAMSC at passages 0 and 2 were collected after a standard 5-day culture while CM from hAM was collected after a 2- and 5-day culture. Immunomodulation was assessed in terms of PBMC and T-cell proliferation, T-cell subset polarization, T-regulatory cell induction, cell cytotoxicity and monocyte differentiation toward antigen-presenting cells. Furthermore, we performed a comparison between CM obtained from single donors and pooled CM. We also assessed the impact of lyophilization on the immunomodulatory properties of CM. Results: We demonstrate that CM from hAM has comparable immunomodulatory properties to CM from hAMSC at passages 0 and 2. Furthermore, we demonstrate that pooled CMs have similar effects when compared to CM from single donors used separately. Finally, we demonstrate that lyophilization does not alter the in vitro immunomodulatory properties of CM from hAM and hAMSC. Conclusions: The results presented herein support the possibility to produce secretome from intact hAM and open the prospect to highly improve the scalability of the GMP production process while reducing the costs and time related to the process of cell isolation and expansion. More

Amniotic membrane grafts have some therapeutic potential for wounds healing. Early application of amniotic membrane turned out as beneficial in healing ulcers, burns, and dermal injuries. Since the second half of the 20th century, the autotransplants of amniotic/chorion tissue have been also used for the treatment of chronic neuropathic wounds, cornea surface injuries, pterygium and conjunctivochalasis, and dental and neurosurgical applications. The aim of this publication is to prepare a coherent overview of amniotic membrane derivatives use in the field of wound healing and also its efficacy. In total 60 publications and 39 posters from 2000-2020 were examined. In these examined publications of case studies with known study results was an assemblage of 1141 patients, and from this assemblage 977 were successfully cured. In case of posters, the assemblage is 570 patients and 513 successfully cured. From the investigated data it is clear that the treatment efficacy is very high-86% and 90%, respectively. Based on this information the use of the amniotic membrane for chronic wounds can be considered highly effective., info:eu-repo/semantics/openAccess

Amniotic membrane grafts have some therapeutic potential for wounds healing. Early application of amniotic membrane turned out as beneficial in healing ulcers, burns, and dermal injuries. Since the second half of the 20th century, the autotransplants of amniotic/chorion tissue have been also used for the treatment of chronic neuropathic wounds, cornea surface injuries, pterygium and conjunctivochalasis, and dental and neurosurgical applications. The aim of this publication is to prepare a coherent overview of amniotic membrane derivatives use in the field of wound healing and also its efficacy. In total 60 publications and 39 posters from 2000-2020 were examined. In these examined publications of case studies with known study results was an assemblage of 1141 patients, and from this assemblage 977 were successfully cured. In case of posters, the assemblage is 570 patients and 513 successfully cured. From the investigated data it is clear that the treatment efficacy is very high-86% and 90%, respectively. Based on this information the use of the amniotic membrane for chronic wounds can be considered highly effective., info:eu-repo/semantics/openAccess

Amniotic membrane grafts have some therapeutic potential for wounds healing. Early application of amniotic membrane turned out as beneficial in healing ulcers, burns, and dermal injuries. Since the second half of the 20th century, the autotransplants of amniotic/chorion tissue have been also used for the treatment of chronic neuropathic wounds, cornea surface injuries, pterygium and conjunctivochalasis, and dental and neurosurgical applications. The aim of this publication is to prepare a coherent overview of amniotic membrane derivatives use in the field of wound healing and also its efficacy. In total 60 publications and 39 posters from 2000-2020 were examined. In these examined publications of case studies with known study results was an assemblage of 1141 patients, and from this assemblage 977 were successfully cured. In case of posters, the assemblage is 570 patients and 513 successfully cured. From the investigated data it is clear that the treatment efficacy is very high-86% and 90%, respectively. Based on this information the use of the amniotic membrane for chronic wounds can be considered highly effective., info:eu-repo/semantics/openAccess

For decades, the unique regenerative properties of the human amniotic membrane (hAM) have been successfully utilized in ophthalmology. As a directly applied biomaterial, the hAM should be available in a ready to use manner in clinical settings. However, an extended period of time is obligatory for performing quality and safety tests. Hence, the low temperature storage of the hAM is a virtually inevitable step in the chain from donor retrieval to patient application. At the same time, the impact of subzero temperatures carries an increased risk of irreversible alterations of the structure and composition of biological objects. In the present study, we performed a comprehensive analysis of the hAM as a medicinal product; this is intended for a novel strategy of application in ophthalmology requiring a GMP production protocol including double freezing– thawing cycles. We compared clinically relevant parameters, such as levels of growth factors and extracellular matrix proteins content, morphology, ultrastructure and mechanical properties, before and after one and two freezing cycles. It was found that epidermal growth factor (EGF), transforming growth factor beta 1 (TGF-ß1), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), hyaluronic acid, and laminin could be detected in all studied conditions without significant differences. Additionally, histological and ultrastructure analysis, as well as transparency and mechanical tests, demonstrated that properties of the hAM required to support therapeutic efficacy in ophthalmology are not impaired by dual freezing. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.

The objective of the present research work was to develop a new formulation of amniotic membrane extract (EMA)-loaded deformable liposomes. This pharmacological alternative supposses a less aggressive technology than a placental transplant for the treatment of neurotrophic keratopathy. Firstly, the total protein extraction method was developed, followed by protein quantification using the bicinchoninic acid (BCA) method. Liposome formulations were made using the TLE (Thin Layer Evaporation) technique. To obtain the desired composition of the vesicles, a screening study was realized using the design of experiments and a Taguchi L9 matrix, whose experiments evaluated the size, polydispersity index (PDI), surface charge and encapsulation efficiency (EE). After selecting the suitable conditions for protein extraction, the results of the screening study showed that all the factors evaluated affected the physicochemical characteristics of vesicles. The optimization stage allowed to select as composition for subsequent studies: sodium deoxycholate (NaDC) as a deformable agent, stearylamine as positively-charged agent in an amount of 0.06 mmol and a 75% dilution of the EMA. This study offers new possibilities to improve the treatment of neurotrophic keratopathy through the use of non-invasive formulations., El objetivo del presente trabajo de investigación fue desarrollar nuevas formulaciones de liposomas deformables de extracto de membrana amniótica (EMA), menos agresivas que un trasplante de placenta para el tratamiento de la queratopatía neurotrófica. Para ello, en primer lugar, se puso a punto el método de extracción de las proteínas totales, seguido de la cuantificación de proteínas utilizando el método del ácido bicinconínico (BCA). Las formulaciones de liposomas se elaboraron mediante la técnica TLE (Thin Layer Evaporation). Para obtener la composición deseada de las vesículas, se realizó un estudio de cribado usando el diseño experimental con una matriz de Taguchi L9, de cuyos experimentos se evaluaron el tamaño, índice de polidispersión (PDI), carga superficial y eficacia de encapsulación (EE). Tras establecer las condiciones de extracción de las proteínas, los resultados del estudio de cribado mostraron que todos los factores evaluados afectaron a las características fisicoquímicas. La etapa de optimización permitió seleccionar como composición para los estudios posteriores: desoxicolato sódico (NaDC) como agente deformable, estearilamina como agente de carga en una cantidad de 0.06 mmol y una dilución al 75% del EMA. Este estudio ofrece nuevas posibilidades para mejorar el tratamiento de la queratopatía neurotrófica (QN) mediante el uso de formulaciones no invasivas.

Our objective was to determine the safety and efficacy of amniotic membrane (AM) treatment for epithelialization of complex diabetic foot ulcers, this tissue is obtained from healthy donors after scheduled caesarean section and negative serology for infectious diseases. Eighteen patients with diabetic macro and micro-angiopathy and foot deformations who presented diabetic foot ulcers with more than eight weeks of evolution were treated. The median age was 62 years. The Spanish Agency for Med-icines and Health Products (AEMPS) gave the authori-zation as compassionate use in all patients. The therapy was applied weekly on the wound bed. The mean treat-ment time before AM application was 24 and half months. The average area of the wounds was 10.56 cm2 (0.52 - 42.5). The mean time for epithelialization with AM was 5 months (1.5-13) with a mean of 11.22 applied membranes (4-40). No patient developed adverse events during treatment. Our results show that cryo-preserved human MA treatment is a safe and effective treatment in patients with complex diabetic foot ul-cers., Nuestro obetivo fue determinar la seguridad y la eficacia del tratamiento con membrana amniótica (MA) para la epitelización de úlceras complejas de pie diabético, este tejido se obtiene de donantes sanas tras cesárea programada y serología negativa para enfermedades in-fecciosas. Se trataron a 18 pacientes con macro y micro-angiopatía diabética y deformaciones en los pies que presentaban úlceras de pie diabético con más de 8 semanas de evolución. La mediana de edad fue de 62 años. La Agencia Española de Medicamentos y Produc-tos Sanitarios (AEMPS) dio la autorización como uso compasivo en todos los pacientes. La terapia se aplicó semanalmente sobre el lecho de la herida. El tiempo me-dio de tratamiento antes de la aplicación de MA fue de 24 meses y medio. El área promedio de las heridas fue de 10,56 cm2 (0,52 – 42,5). El tiempo medio de epiteli-zación con MA fue de 5 meses (1,5-13) con una media de 11,22 membranas aplicadas (4-40). Ningún paciente desarrolló acontecimientos adversos durante el trata-miento. Nuestros resultados muestran que el tratamiento con MA humana criopreservada es un tratamiento se-guro y efectivo en pacientes con úlceras complejas de pie diabético.

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The failed macular hole is a full-thickness defect involving the fovea that fails to close despite 1 or more surgeries. While many surgical options have been proposed to manage it, none of these guarantee complete anatomical success and satisfactory visual recovery. We report postoperative outcomes on 36 patients affected by failed macular hole, treated with a human amniotic membrane plug transplant. Follow-ups were performed with a standard ophthalmological examination and with advanced multimodal diagnostic imaging. Anatomical closure was achieved at 3 months in all patients. Mean best-corrected visual acuity improved statistically significantly at 6 months (p < 0.05). Through microperimetric tests, we assessed a partial recovery of the macular sensitivity on the edges of the plug. Analyzing SD-OCT images, we reported a tissutal ingrowth above the plug, and its segmentation into layers, mimicking normal retinal architecture. OCT-Angiography images non invasively analysed the retinal parafoveal capillary microvasculature; the elaboration of Adaptive Optics images showed the presence of photoreceptors at the edges of the plug. This work demonstrates not only the complete anatomical success of our technique, but also remarkable functional results, and opens the door to a greater understanding of modifications induced by the presence of a human amniotic membrane plug.

Purpose: Age-related macular degeneration (AMD) is the leading cause of legal blindness in adults 65 years of age and older. Choroidal neovascularization (CNV) can complicate AMD and lead to severe visual acuity reduction. Despite the several treatments available, if the retinal pigment epithelium is damaged, we have to cope with the impossibility of restoring acceptable visual acuity using only medical treatments. Design: Prospective, consecutive, interventional study. Participants: Eleven patients affected by AMD, 6 patients affected by CNV, and 5 patients affected by geographic atrophy. Methods: All patients underwent a pars plana vitrectomy with subretinal implantation of human amniotic membrane (hAM) to induce photoreceptor regeneration and partial visual acuity restoration. Main Outcome Measures: Primary study outcome was visual acuity improvement. Secondary outcomes were multimodal imaging results. Results: Mean preoperative best-corrected visual acuity (BCVA) was 20/2000 (2 logarithm of the minimum angle of resolution [logMAR]), and all the patients showed a BCVA of counting fingers or less. Mean final BCVA was 20/400 (1.31 logMAR), ranging from 20/2000 to 20/100 (2–0.7 logMAR). OCT angiography was used to measure retinal vascularization in the treated eye compared with the fellow eye. A high correlation between BCVA and deep vascular density was evidenced. Adaptive optics findings, obtained over the retinal area where the highest functionality was observed, were evaluated using microperimetry. The images showed possible photoreceptor presence over the hAM membrane. Conclusions: This work supports the feasibility and safety of the hAM to promote partial retinal function restoration 6 months after surgery with visual acuity improvement. The advanced diagnostics help to understand the interaction between the hAM and photoreceptors and suggest that photoreceptor regeneration may occur.

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Background: Photoaging causes wrinkles, lentigines, keratoses, dyspigmentation, telangiectasia, decreased elasticity, rough texture, and pale color. Facial wrinkles and mottled skin can negatively affect perceived attractiveness, self-esteem, personality, and level of acceptance. Stem cells are important cells in the skin, and they are the source for continuous regeneration of the epidermis. It can be extracted from many sources, including amnion. Amnion membrane stem cell (AMSC) can be cultured and secretes metabolite product, and it can rejuvenate aging skin. Laser-Assisted Drug Delivery (LADD) involves selective destruction of the layers of the epidermis and dermis to allow penetration and absorption of topical drugs as well as large molecular weight drugs such as stem cells. Purpose: To evaluate the long-term effects of topical administration of AMSC metabolite products (AMSC -MP) and fractional CO2 laser on the clinical improvement of photoaging compared to previous skin conditions. Methods: This was an observational study which was aimed to evaluate the long-term effects of clinical improvement after topical therapy of AMSC-MP and fractional CO2 laser in patients with photoaging. Results: The results of the analysis showed a decrease in wrinkles, dark spots, skin tone, and pores. Conclusion: Based on Janus-II® assessment, there was improvement degrees of wrinkles, dark spots, and pores in patients with photoaging who received fractional CO2laser fractional for 12 weeks. There were no side effects found in this study.

Recently, the use of the human amniotic membrane (hAM) has been extended to treat retinal disorders, such as macular holes that failed to close and retinal tears. The hAM has demonstrated the induction of a recovery process of the external retinal layers involving the external limiting membrane (ELM) and the ellipsoid zone (EZ). After that, the application of the hAM for retinal pathologies was extended to large macular tears, high myopic retinal detachment associated with MH, paravascular tears, serous macular detachment associated with optic pit, complicated retinal detachment and advanced age-related macular degeneration (AMD). The hAM has shown a potential in repairing retinal tissue through a regeneration process. This review aims to highlight the use of the hAM in various vitreo-retinal surgical fields, and to confront it with other cutting-edge surgical techniques used to treat challenging vitreo-retinal pathologies.

Mesenchymal stromal cells (MSC) from the amniotic membrane of human term placenta (hAMSC), and the conditioned medium generated from their culture (CM-hAMSC) offer significant tools for their use in regenerative medicine mainly due to their immunomodulatory properties. Interestingly, hAMSC and their CM have been successfully exploited in preclinical disease models of inflammatory and autoimmune diseases where depletion or modulation of B cells have been indicated as an effective treatment, such as inflammatory bowel disease, lung fibrosis, would healing, collagen-induced arthritis, and multiple sclerosis. While the interactions between hAMSC or CM-hAMSC and T lymphocytes, monocytes, dendritic cells, and macrophages has been extensively explored, how they affect B lymphocytes remains unclear. Considering that B cells are key players in the adaptive immune response and are a central component of different diseases, in this study we investigated the in vitro properties of hAMSC and CM-hAMSC on B cells. We provide evidence that both hAMSC and CM-hAMSC strongly suppressed CpG-activated B-cell proliferation. Moreover, CM-hAMSC blocked B-cell differentiation, with an increase of the proportion of mature B cells, and a reduction of antibody secreting cell formation. We observed the strong inhibition of B cell terminal differentiation into CD138+ plasma cells, as further shown by a significant decrease of the expression of interferon regulatory factor 4 (IRF-4), PR/SET domain 1(PRDM1), and X-box binding protein 1 (XBP-1) genes. Our results point out that the mechanism by which CM-hAMSC impacts B cell proliferation and differentiation is mediated by secreted factors, and prostanoids are partially involved in these actions. Factors contained in the CM-hAMSC decreased the CpG-uptake sensors (CD205, CD14, and TLR9), suggesting that B cell stimulation was affected early on. CM-hAMSC also decreased the expression of interleukin-1 receptor-associated kinase (IRAK)-4, consequently i

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

Background: Photoaging causes wrinkles, lentigines, keratoses, dyspigmentation, telangiectasia, decreased elasticity, rough texture, and pale color. Facial wrinkles and mottled skin can negatively affect perceived attractiveness, self-esteem, personality, and level of acceptance. Stem cells are important cells in the skin, and they are the source for continuous regeneration of the epidermis. It can be extracted from many sources, including amnion. Amnion membrane stem cell (AMSC) can be cultured and secretes metabolite product, and it can rejuvenate aging skin. Laser-Assisted Drug Delivery (LADD) involves selective destruction of the layers of the epidermis and dermis to allow penetration and absorption of topical drugs as well as large molecular weight drugs such as stem cells. Purpose: To evaluate the long-term effects of topical administration of AMSC metabolite products (AMSC -MP) and fractional CO2 laser on the clinical improvement of photoaging compared to previous skin conditions. Methods: This was an observational study which was aimed to evaluate the long-term effects of clinical improvement after topical therapy of AMSC-MP and fractional CO2 laser in patients with photoaging. Results: The results of the analysis showed a decrease in wrinkles, dark spots, skin tone, and pores. Conclusion: Based on Janus-II® assessment, there was improvement degrees of wrinkles, dark spots, and pores in patients with photoaging who received fractional CO2laser fractional for 12 weeks. There were no side effects found in this study.

Placenta-derived mesenchymal stromal cells (MSC) have attracted more attention for their immune modulatory properties and poor immunogenicity, which makes them suitable for allogeneic transplantation. Although MSC isolated from different areas of the placenta share several features, they also present significant biological differences, which might point to distinct clinical applications. Hence, we compared cells from full term placenta distinguishing them on the basis of their origin, either maternal or fetal. We used cells developed by Pluristem LTD: PLacenta expanded mesenchymal-like adherent stromal cells (PLX), maternal-derived cells (PLX-PAD), fetal-derived cells (PLX-R18), and amniotic membrane-derived MSC (hAMSC). We compared immune modulatory properties evaluating effects on T-lymphocyte proliferation, expression of cytotoxicity markers, T-helper and T-regulatory cell polarization, and monocyte differentiation toward antigen presenting cells (APC). Furthermore, we investigated cell immunogenicity. We show that MSCs and MSC-like cells from both fetal and maternal sources present immune modulatory properties versus lymphoid (T cells) and myeloid (APC) cells, whereby fetal-derived cells (PLX-R18 and hAMSC) have a stronger capacity to modulate immune cell proliferation and differentiation. Our results emphasize the importance of understanding the cell origin and characteristics in order to obtain a desired result, such as modulation of the inflammatory response that is critical in fostering regenerative processes.

While limbal epithelial cells are used for treating ocular surface wounds, the therapeutic potential of mesenchymal cells cultivated from the limbal stroma (LMSC) is less clear. We have therefore examined the effects of LMSC when applied to acute ocular surface wounds. LMSC derived from male rabbits (RLMSC) were applied to the ocular surface of female rabbits immediately following removal of the corneal and limbal epithelium. Human amniotic membrane (HAM) was used as the vehicle for implanting the RLMSC. The effects of RLMSC were examined when applied alone (n = 3) and in conjunction with a stratified culture of human limbal epithelial cells (HLE) grown on the opposing surface of the HAM (n = 3). Outcomes were monitored over 3 months in comparison with animals receiving no treatment (n = 3) or treatment with HLE alone on HAM (n = 3). Animals treated with RLMSC (n = 6) displayed faster re-epithelialization (∼90% versus 70% healing after 12 weeks), with best results being observed when RLMSC were pre-cultivated and implanted in the presence of HLE (p < 0.01; 90% healing by 7 weeks). While all animals displayed conjunctival cells on the corneal surface (by presence of goblet cells and/or keratin 13 expression) and corneal neovascularization, evidence of corneal epithelial regeneration was observed in animals that received RLMSC in the presence of HLE (by staining for keratin 3 and the absence of goblet cells). Conversely, corneal neovascularization was significantly greater when RLMSC were applied in the absence of HLE (<0.05; 90% of cornea compared with 20–30% in other cohorts). Nevertheless, neither human nuclear antigen nor rabbit Y chromosome were detected within the regenerated epithelium. Our results demonstrate that while cultured LMSC encourage corneal re-epithelialization, healing is improved by the pre-cultivation and implantation of these mesenchymal cells in the presence of limbal epithelial cells.

Since their discovery and characterization, mesenchymal stromal cells (MSC) have been a topic of great interest in regenerative medicine. Over the last 10 years, detailed studies investigated the properties of MSC from perinatal tissues and have indicated that these cells may represent important tools for restoring tissue damage or promoting regeneration and repair of the tissue microenvironment. At first, perinatal tissue-derived MSC drew attention due to their potential differentiation capacities suggested by their early embryological origin. It is nowadays accepted that perinatal tissue-derived MSC are promising for a wide range of regenerative medicine applications because of their unique immune modulatory properties, rather than their differentiation ability. As a matter of fact, the activation and function of various cells of the innate and adaptive immune systems are suppressed and modulated by MSC from different perinatal tissues, such as human term placenta. However, the mechanisms by which they act on immune cells to facilitate tissue repair during pathological processes remain to be thoroughly elucidated to develop safe and efficient therapeutic approaches. In addition to immune modulatory ability, several other peculiar characteristics of placenta MSC, less explored and/or more debated, are being investigated. These include an understanding of the anti-microbial properties and the role of placental MSC in tumor progression. Moreover, a thorough investigation on preparation methods, bioactive factors, mechanisms of action of the cell secretome, and the development of potency assays to predict clinical efficacy of placenta MSC and their products, are necessary to provide a solid basis for their clinical application.

RIVM rapport:Het RIVM heeft een verkenning uitgevoerd naar nieuwe ontwikkelingen en mogelijke risico's rondom het gebruik van het zogeheten amnionmembraan, zowel in Nederland als daarbuiten. Het membraan wordt al decennia als transplantaat gebruikt in de oogheelkunde om beschadigd hoornvlies te laten herstellen. Het amnionmembraan is een zeer dun vlies dat uit een placenta wordt gehaald. In Nederland wordt het in beperkte mate gebruikt. Vooral in de internationale literatuur zijn nieuwe ontwikkelingen rondom het amnionmembraan beschreven. Zo wordt onderzocht of beschadigde weefsels kunnen herstellen door stamcellen uit gezond vergelijkbaar weefsel te halen en op amnionmembraan te laten groeien. Het membraan kan daarna met stamcellen in het beschadigde weefsel worden geplaatst. Een andere ontwikkeling die wordt onderzocht is of stamcellen uit het amnionmembraan kunnen worden gebruikt om beschadigingen in verschillende weefsels te herstellen, zoals in spieren. In Nederland wordt het amnionmembraan voornamelijk ingezet om schade aan het hoornvlies van het oog te bedekken. In de literatuur zijn een beperkt aantal risico's bij het gebruik van het amnionmembraan beschreven, zoals pijn. Voor zover bekend zijn er in Nederland geen complicaties gemeld bij behandelingen met het amnionmembraan., The National Institute for Public Health and the Environment (RIVM) has carried out an exploratory study into new developments and possible risks associated with the use of the so-called amniotic membrane, both in the Netherlands and elsewhere. For decades, the membrane has been used in ophthalmology as a graft to allow damaged corneas to heal. The amniotic membrane is a very thin membrane that is taken from the placenta. Its use is limited in the Netherlands. New developments concerning the amniotic membrane have mainly been described in the international literature. Research has been carried out into repairing damaged tissues by using stem cells obtained from comparable healthy tissue and growing them on amniotic membrane. The membrane with stem cells can then be placed on the damaged tissue. Another development currently being researched is the possibility of using stem cells from the amniotic membrane to repair damage to other tissues, such as muscles. In the Netherlands, amniotic membrane is mainly used to cover damage to the cornea of the eye. The literature reports only a limited number of risks, such as pain, associated with the use of amniotic membrane. As far as is known, in the Netherlands no complications have been reported concerning treatment with amniotic membrane.

Het RIVM heeft een verkenning uitgevoerd naar nieuwe ontwikkelingen en mogelijke risico's rondom het gebruik van het zogeheten amnionmembraan, zowel in Nederland als daarbuiten. Het membraan wordt al decennia als transplantaat gebruikt in de oogheelkunde om beschadigd hoornvlies te laten herstellen. Het amnionmembraan is een zeer dun vlies dat uit een placenta wordt gehaald. In Nederland wordt het in beperkte mate gebruikt. Vooral in de internationale literatuur zijn nieuwe ontwikkelingen rondom het amnionmembraan beschreven. Zo wordt onderzocht of beschadigde weefsels kunnen herstellen door stamcellen uit gezond vergelijkbaar weefsel te halen en op amnionmembraan te laten groeien. Het membraan kan daarna met stamcellen in het beschadigde weefsel worden geplaatst. Een andere ontwikkeling die wordt onderzocht is of stamcellen uit het amnionmembraan kunnen worden gebruikt om beschadigingen in verschillende weefsels te herstellen, zoals in spieren. In Nederland wordt het amnionmembraan voornamelijk ingezet om schade aan het hoornvlies van het oog te bedekken. In de literatuur zijn een beperkt aantal risico's bij het gebruik van het amnionmembraan beschreven, zoals pijn. Voor zover bekend zijn er in Nederland geen complicaties gemeld bij behandelingen met het amnionmembraan., The National Institute for Public Health and the Environment (RIVM) has carried out an exploratory study into new developments and possible risks associated with the use of the so-called amniotic membrane, both in the Netherlands and elsewhere. For decades, the membrane has been used in ophthalmology as a graft to allow damaged corneas to heal. The amniotic membrane is a very thin membrane that is taken from the placenta. Its use is limited in the Netherlands. New developments concerning the amniotic membrane have mainly been described in the international literature. Research has been carried out into repairing damaged tissues by using stem cells obtained from comparable healthy tissue and growing them on amniotic membrane. The membrane with stem cells can then be placed on the damaged tissue. Another development currently being researched is the possibility of using stem cells from the amniotic membrane to repair damage to other tissues, such as muscles. In the Netherlands, amniotic membrane is mainly used to cover damage to the cornea of the eye. The literature reports only a limited number of risks, such as pain, associated with the use of amniotic membrane. As far as is known, in the Netherlands no complications have been reported concerning treatment with amniotic membrane.

To evaluate the safety and efficacy of the surgical use of plasma rich in growth factors fibrin membrane (mPRGF) in different ocular surface pathologies.Fifteen patients with different corneal and conjunctival diseases were included in the study. Patients were grouped according to the use of mPRGF as graft (corneal and/or conjunctival) or dressing; they were also grouped according to the surgical subgroup of intervention (persistent corneal ulcer [PCU], keratoplasty, superficial keratectomy, corneal perforation, and pterygium). Best corrected visual acuity, intraocular pressure (IOP), inflammation control time (ICT), mPRGF AT (PRGF membrane absorption time), and the healing time of the epithelial defect (HTED) were evaluated throughout the clinical follow-up time. Safety assessment was also performed reporting all adverse events.mPRGF showed a total closure of the defect in 13 of 15 patients (86.7%) and a partial closure in 2 patients (13.3%). The mean follow-up time was 11.14.2 (4.8-22.8) months, the mean ICT was 2.5 +/- 1.1 (1.0-4.0) months, the mean mPRGF AT was 12.4 +/- 2.0 (10.0-16.0) days, and for the global HTED the mean was 2.9 +/- 1.2 (1-4.8) months. Results showed an improvement in BCVA in all patients, with an overall improvement of 2.9 in Vision Lines. The BCVA significantly improved (P<.05) in the groups of corneal graft and dressing. In the PCU subgroup (6 patients), the healing time of epithelial defect was significantly reduced (P<.05) in patients treated only with the mPRGF in comparison to those which mPRGF therapy was associated to the amniotic membrane. The IOP remained stable (P>.05) throughout the clinical follow-up time. No adverse events were reported after mPRGF use.The mPRGF is effective and safe as coadjuvant treatment in surgeries related with ocular surface disorders, being an alternative to the use of amniotic membrane. The mPRGF accelerates tissue regeneration after ocular surface surgery thus minimizing inflammation and fibrosis.

RIVM rapport:Het RIVM heeft een verkenning uitgevoerd naar nieuwe ontwikkelingen en mogelijke risico's rondom het gebruik van het zogeheten amnionmembraan, zowel in Nederland als daarbuiten. Het membraan wordt al decennia als transplantaat gebruikt in de oogheelkunde om beschadigd hoornvlies te laten herstellen. Het amnionmembraan is een zeer dun vlies dat uit een placenta wordt gehaald. In Nederland wordt het in beperkte mate gebruikt. Vooral in de internationale literatuur zijn nieuwe ontwikkelingen rondom het amnionmembraan beschreven. Zo wordt onderzocht of beschadigde weefsels kunnen herstellen door stamcellen uit gezond vergelijkbaar weefsel te halen en op amnionmembraan te laten groeien. Het membraan kan daarna met stamcellen in het beschadigde weefsel worden geplaatst. Een andere ontwikkeling die wordt onderzocht is of stamcellen uit het amnionmembraan kunnen worden gebruikt om beschadigingen in verschillende weefsels te herstellen, zoals in spieren. In Nederland wordt het amnionmembraan voornamelijk ingezet om schade aan het hoornvlies van het oog te bedekken. In de literatuur zijn een beperkt aantal risico's bij het gebruik van het amnionmembraan beschreven, zoals pijn. Voor zover bekend zijn er in Nederland geen complicaties gemeld bij behandelingen met het amnionmembraan., The National Institute for Public Health and the Environment (RIVM) has carried out an exploratory study into new developments and possible risks associated with the use of the so-called amniotic membrane, both in the Netherlands and elsewhere. For decades, the membrane has been used in ophthalmology as a graft to allow damaged corneas to heal. The amniotic membrane is a very thin membrane that is taken from the placenta. Its use is limited in the Netherlands. New developments concerning the amniotic membrane have mainly been described in the international literature. Research has been carried out into repairing damaged tissues by using stem cells obtained from comparable healthy tissue and growing them on amniotic membrane. The membrane with stem cells can then be placed on the damaged tissue. Another development currently being researched is the possibility of using stem cells from the amniotic membrane to repair damage to other tissues, such as muscles. In the Netherlands, amniotic membrane is mainly used to cover damage to the cornea of the eye. The literature reports only a limited number of risks, such as pain, associated with the use of amniotic membrane. As far as is known, in the Netherlands no complications have been reported concerning treatment with amniotic membrane.

To evaluate the safety and efficacy of the surgical use of plasma rich in growth factors fibrin membrane (mPRGF) in different ocular surface pathologies.Fifteen patients with different corneal and conjunctival diseases were included in the study. Patients were grouped according to the use of mPRGF as graft (corneal and/or conjunctival) or dressing; they were also grouped according to the surgical subgroup of intervention (persistent corneal ulcer [PCU], keratoplasty, superficial keratectomy, corneal perforation, and pterygium). Best corrected visual acuity, intraocular pressure (IOP), inflammation control time (ICT), mPRGF AT (PRGF membrane absorption time), and the healing time of the epithelial defect (HTED) were evaluated throughout the clinical follow-up time. Safety assessment was also performed reporting all adverse events.mPRGF showed a total closure of the defect in 13 of 15 patients (86.7%) and a partial closure in 2 patients (13.3%). The mean follow-up time was 11.14.2 (4.8-22.8) months, the mean ICT was 2.5 +/- 1.1 (1.0-4.0) months, the mean mPRGF AT was 12.4 +/- 2.0 (10.0-16.0) days, and for the global HTED the mean was 2.9 +/- 1.2 (1-4.8) months. Results showed an improvement in BCVA in all patients, with an overall improvement of 2.9 in Vision Lines. The BCVA significantly improved (P<.05) in the groups of corneal graft and dressing. In the PCU subgroup (6 patients), the healing time of epithelial defect was significantly reduced (P<.05) in patients treated only with the mPRGF in comparison to those which mPRGF therapy was associated to the amniotic membrane. The IOP remained stable (P>.05) throughout the clinical follow-up time. No adverse events were reported after mPRGF use.The mPRGF is effective and safe as coadjuvant treatment in surgeries related with ocular surface disorders, being an alternative to the use of amniotic membrane. The mPRGF accelerates tissue regeneration after ocular surface surgery thus minimizing inflammation and fibrosis.

RIVM rapport:Het RIVM heeft een verkenning uitgevoerd naar nieuwe ontwikkelingen en mogelijke risico's rondom het gebruik van het zogeheten amnionmembraan, zowel in Nederland als daarbuiten. Het membraan wordt al decennia als transplantaat gebruikt in de oogheelkunde om beschadigd hoornvlies te laten herstellen. Het amnionmembraan is een zeer dun vlies dat uit een placenta wordt gehaald. In Nederland wordt het in beperkte mate gebruikt. Vooral in de internationale literatuur zijn nieuwe ontwikkelingen rondom het amnionmembraan beschreven. Zo wordt onderzocht of beschadigde weefsels kunnen herstellen door stamcellen uit gezond vergelijkbaar weefsel te halen en op amnionmembraan te laten groeien. Het membraan kan daarna met stamcellen in het beschadigde weefsel worden geplaatst. Een andere ontwikkeling die wordt onderzocht is of stamcellen uit het amnionmembraan kunnen worden gebruikt om beschadigingen in verschillende weefsels te herstellen, zoals in spieren. In Nederland wordt het amnionmembraan voornamelijk ingezet om schade aan het hoornvlies van het oog te bedekken. In de literatuur zijn een beperkt aantal risico's bij het gebruik van het amnionmembraan beschreven, zoals pijn. Voor zover bekend zijn er in Nederland geen complicaties gemeld bij behandelingen met het amnionmembraan., The National Institute for Public Health and the Environment (RIVM) has carried out an exploratory study into new developments and possible risks associated with the use of the so-called amniotic membrane, both in the Netherlands and elsewhere. For decades, the membrane has been used in ophthalmology as a graft to allow damaged corneas to heal. The amniotic membrane is a very thin membrane that is taken from the placenta. Its use is limited in the Netherlands. New developments concerning the amniotic membrane have mainly been described in the international literature. Research has been carried out into repairing damaged tissues by using stem cells obtained from comparable healthy tissue and growing them on amniotic membrane. The membrane with stem cells can then be placed on the damaged tissue. Another development currently being researched is the possibility of using stem cells from the amniotic membrane to repair damage to other tissues, such as muscles. In the Netherlands, amniotic membrane is mainly used to cover damage to the cornea of the eye. The literature reports only a limited number of risks, such as pain, associated with the use of amniotic membrane. As far as is known, in the Netherlands no complications have been reported concerning treatment with amniotic membrane.

To evaluate the safety and efficacy of the surgical use of plasma rich in growth factors fibrin membrane (mPRGF) in different ocular surface pathologies.Fifteen patients with different corneal and conjunctival diseases were included in the study. Patients were grouped according to the use of mPRGF as graft (corneal and/or conjunctival) or dressing; they were also grouped according to the surgical subgroup of intervention (persistent corneal ulcer [PCU], keratoplasty, superficial keratectomy, corneal perforation, and pterygium). Best corrected visual acuity, intraocular pressure (IOP), inflammation control time (ICT), mPRGF AT (PRGF membrane absorption time), and the healing time of the epithelial defect (HTED) were evaluated throughout the clinical follow-up time. Safety assessment was also performed reporting all adverse events.mPRGF showed a total closure of the defect in 13 of 15 patients (86.7%) and a partial closure in 2 patients (13.3%). The mean follow-up time was 11.14.2 (4.8-22.8) months, the mean ICT was 2.5 +/- 1.1 (1.0-4.0) months, the mean mPRGF AT was 12.4 +/- 2.0 (10.0-16.0) days, and for the global HTED the mean was 2.9 +/- 1.2 (1-4.8) months. Results showed an improvement in BCVA in all patients, with an overall improvement of 2.9 in Vision Lines. The BCVA significantly improved (P<.05) in the groups of corneal graft and dressing. In the PCU subgroup (6 patients), the healing time of epithelial defect was significantly reduced (P<.05) in patients treated only with the mPRGF in comparison to those which mPRGF therapy was associated to the amniotic membrane. The IOP remained stable (P>.05) throughout the clinical follow-up time. No adverse events were reported after mPRGF use.The mPRGF is effective and safe as coadjuvant treatment in surgeries related with ocular surface disorders, being an alternative to the use of amniotic membrane. The mPRGF accelerates tissue regeneration after ocular surface surgery thus minimizing inflammation and fibrosis.

Among the many cell types useful in developing therapeutic treatments, human amniotic cells from placenta have been proposed as valid candidates. Both human amniotic epithelial and mesenchymal stromal cells, and the conditioned medium generated from their culture, exert multiple immunosuppressive activities. Indeed, they inhibit T and B cell proliferation, suppress inflammatory properties of monocytes, macrophages, dendritic cells, neutrophils, and natural killer cells, while promoting induction of cells with regulatory functions such as regulatory T cells and anti-inflammatory M2 macrophages. These properties have laid the foundation for their use for the treatment of inflammatory-based diseases, and encouraging results have been obtained in different preclinical disease models where exacerbated inflammation is present. Moreover, an immune-privileged status of amniotic cells has been often highlighted. However, even if long-term engraftment of amniotic cells has been reported into immunocompetent animals, only few cells survive after infusion. Furthermore, amniotic cells have been shown to be able to induce immune responses in vivo and, under specific culture conditions, they can stimulate T cell proliferation in vitro. Although immunosuppressive properties are a widely recognized characteristic of amniotic cells, immunogenic and stimulatory activities appear to be less reported, sporadic events. In order to improve therapeutic outcome, the mechanisms responsible for the suppressive versus stimulatory activity need to be carefully addressed. In this review, both the immunosuppressive and immunostimulatory activity of amniotic cells will be discussed.

Mesenchymal stromal cells from the human amniotic membrane (i.e., human amniotic mesenchymal stromal cells [hAMSCs]) of term placenta are increasingly attracting attention for their applications in regenerative medicine. Osteochondral defects represent a major clinical problem with lifelong chronic pain and compromised quality of life. Great promise for osteochondral regeneration is held in hydrogel-based constructs that have a flexible composition and mimic the physiological structure of cartilage. Cell loading within a hydrogel represents an advantage for regenerative purposes, but the encapsulation steps can modify cell properties. As pectin gels have also been explored as cell vehicles on 3D scaffolds, the aim of this study was to explore the possibility to include hAMSCs in pectin gel. Immobilization of hAMSCs into pectin gels could expand their application in cell-based bioengineering strategies. hAMSCs were analyzed for their viability and recovery from the pectin gel and for their ability to differentiate toward the osteogenic lineage and to maintain their immunological characteristics. When treated with a purposely designed pectin/hydroxyapatite gel biocomposite, hAMSCs retained their ability to differentiate toward the osteogenic lineage, did not induce an immune response, and retained their ability to reduce T cell proliferation. Taken together, these results suggest that hAMSCs could be used in combination to pectin gels for the study of novel osteochondral regeneration strategies.

When used as scaffolds for cell therapies, biomaterials often present basic handling and logistical problems for scientists and surgeons alike. The quest for an appropriate mounting device for biomaterials is therefore a significant and common problem. In this review we provide a detailed overview of the factors to consider when choosing an appropriate mounting device including those experienced during cell culture, quality assurance and surgery. By way of example, we draw upon our combined experience in developing epithelial cell therapies for the treatment of eye diseases. We discuss commercially available options for achieving required goals and provide a detailed analysis of four experimental designs developed within our respective laboratories within Australia, the United Kingdom and Belgium.

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] Introduction: Localized trauma-derived breakdown of the hyaline articular cartilage may progress toward osteoarthritis, a degenerative condition characterized by total loss of articular cartilage and joint function. Tissue engineering technologies encompass several promising approaches with high therapeutic potential for the treatment of these focal defects. However, most of the research in tissue engineering is focused on potential materials and structural cues, while little attention is directed to the most appropriate source of cells endowing these materials. In this study, using human amniotic membrane (HAM) as scaffold, we defined a novel static in vitro model for cartilage repair. In combination with HAM, four different cell types, human chondrocytes, human bone marrow-derived mesenchymal stromal cells (hBMSCs), human amniotic epithelial cells, and human amniotic mesenchymal stromal cells (hAMSCs) were assessed determining their therapeutic potential. Material and Methods: A chondral lesion was drilled in human cartilage biopsies simulating a focal defect. A pellet of different cell types was implanted inside the lesion and covered with HAM. The biopsies were maintained for 8 weeks in culture. Chondrogenic differentiation in the defect was analyzed by histology and immunohistochemistry. Results: HAM scaffold showed good integration and adhesion to the native cartilage in all groups. Although all cell types showed the capacity of filling the focal defect, hBMSCs and hAMSCs demonstrated higher levels of new matrix synthesis. However, only the hAMSCs-containing group presented a significant cytoplasmic content of type II collagen when compared with chondrocytes. More collagen type I was identified in the new synthesized tissue of hBMSCs. In accordance, hBMSCs and hAMSCs showed better International Cartilage Research Society scoring although without statistical significance. Conclusion: HAM is a useful material for articular cartilage repair in vitro wh

[Abstract] Introduction: Localized trauma-derived breakdown of the hyaline articular cartilage may progress toward osteoarthritis, a degenerative condition characterized by total loss of articular cartilage and joint function. Tissue engineering technologies encompass several promising approaches with high therapeutic potential for the treatment of these focal defects. However, most of the research in tissue engineering is focused on potential materials and structural cues, while little attention is directed to the most appropriate source of cells endowing these materials. In this study, using human amniotic membrane (HAM) as scaffold, we defined a novel static in vitro model for cartilage repair. In combination with HAM, four different cell types, human chondrocytes, human bone marrow-derived mesenchymal stromal cells (hBMSCs), human amniotic epithelial cells, and human amniotic mesenchymal stromal cells (hAMSCs) were assessed determining their therapeutic potential. Material and Methods: A chondral lesion was drilled in human cartilage biopsies simulating a focal defect. A pellet of different cell types was implanted inside the lesion and covered with HAM. The biopsies were maintained for 8 weeks in culture. Chondrogenic differentiation in the defect was analyzed by histology and immunohistochemistry. Results: HAM scaffold showed good integration and adhesion to the native cartilage in all groups. Although all cell types showed the capacity of filling the focal defect, hBMSCs and hAMSCs demonstrated higher levels of new matrix synthesis. However, only the hAMSCs-containing group presented a significant cytoplasmic content of type II collagen when compared with chondrocytes. More collagen type I was identified in the new synthesized tissue of hBMSCs. In accordance, hBMSCs and hAMSCs showed better International Cartilage Research Society scoring although without statistical significance. Conclusion: HAM is a useful material for articular cartilage repair in vitro wh

Availability of limbus stem cells (LSC) is very limited considering the number of donors SCL very less as compared to the needs, so it is necessary LSC production in vitro. The success of in vitro culture of LSC is strongly influenced by environmental conditions, one of which is the use of the extracellular matrix. Selection of the appropriate type of matrix in the LSC culture, can increase proliferation and facilitate the application of transplantation. This study aimed to get activities fibronectin (FN) as extracellular matrix to produce LSC. Research conducted at the Center for Biomedical and Basic Technology of Health. In this study, production of SPL rats performed in vitro using the method of explants on a petri dish with FN as the extracellular matrix. SPL proliferation rate that with and without FN analyzed by calculating the population doublings (PD), PD/day and the population doubling time (PDT). SPL characterization was performed by quantifiying RNA of CD90 gene, p63, ABCG2 and Krt12 as a marker SPL. The results showed that the PD,PD/day PDT in the SPL with and without FN respectively are 2.13 and 2.11, 0.44 and 0.42 , 57.65 and 61.46 (p> 0.05). While the RNA quntitative of CD90 , p63, ABCG2 and Krt12 SPL on with and without of FN respectively are 17.70 and 19.75, 17.08 and 18.42, 15.23 and 19.09, 17.42 and 18.85 (p> 0.05). This shows that the FN isn't effective as an extracellular matrix in in vitro cultureSPL.Abstract Availability of limbus stem cells (LSC) is very limited considering the number of donors SCL very less as compared to the needs, so it is necessary LSC production in vitro. The success of in vitro culture of LSC is strongly influenced by environmental conditions, one of which is the use of the extracellular matrix. Selection of the appropriate type of matrix in the LSC culture, can increase proliferation and facilitate the application of transplantation. This study aimed to get activities fibronectin (FN) as extracellular matrix to p