Amnion as an Innovative Antiseptic Carrier: A Comparison of the Efficacy of Allogeneic and Xenogeneic Transplantations in the Context of Burn Therapy.

Background and Objectives: The amniotic membrane is widely used in the treatment of chronic wounds, in toxic epidermal necrolysis (TEN), and in the treatment of burns. In our clinical practice, we use amniotic dressings on shallow skin wounds caused by burns. Counteracting infections is an important aspect of working with burn wounds. Therefore, the main goals of this work are to demonstrate the usefulness of amniotic membrane soaked in antiseptics for the prevention of wound infections and to compare the antibacterial efficacy of selected variants of allogeneic and xenogeneic amniotic membrane grafts soaked in specific antiseptic agents. Materials and Methods: The studied material consisted of human and pig placenta. The human and animal amnions were divided in two parts. The first part consisted of amniotic discs placed on rigid mesh discs and preparing the fresh amnion. The second part of the amnion was frozen at a temperature of -80 °C for 24 h. Then, it was radio-sterilized with a dose of 35 kGy. The amniotic discs were placed on rigid mesh to prepare the radiation-sterilized amnion. The amniotic discs were placed in a 12-well plate and immersed in 3 mL of the appropriate antiseptic solutions: Prontosan, Braunol, Borasol, Microdacyn, Octenilin, Sutrisept, and NaCl as a control. The amniotic discs were incubated in antiseptics for 3 h. The microbiological tests were conducted by placing the antiseptic-infused amniotic discs on microbiological media inoculated with hospital strains. Results: The largest average zone of growth inhibition was observed in dressings soaked with Sutrisept, Braunol, and Prontosan. The greatest inhibition of bacterial growth was achieved for radiation-sterilized porcine amnion impregnated with Braunol and Sutrisept, as well as for radiation-sterilized human amnion impregnated with Braunol. Conclusions: Human and porcine amniotic membrane is effective in carrying antiseptics. Radiation-sterilized amnion seems to inhibit the growth of microorganisms better than fresh amnion.

The urgent need to achieve an optimal strategic stock of human allogeneic skin graft materials in case of a mass disaster in Poland.

A burn is a sudden injury which immediate or long-term consequences may be life-threatening for the patient. A mass disaster event may involve large numbers of severely burned patients. Patients of this type typically have a limited area of healthy, unburned skin from which an autologous split thickness skin graft could be collected. In a clinical situation of this type, it is necessary to use a particular skin substitute. Non-viable allogeneic human skin graft materials might be considered as the most suitable skin substitutes in the treatment of such patients. At present, Poland does not have a sufficient supply of human allogeneic skin graft materials to meet the needs arising from a sudden and unforeseen mass disaster. This study involved an analysis of selected mass disasters. From this an estimate was made from a verified casualty profile of the necessary minimum stock of human allogeneic skin graft materials. An insufficient amount of skin results from an inadequate number of skin donors, which in turn results from the current tissue donation system. Therefore, a proposal has been made for the organizational, legal and systemic changes required to improve the situation in Polish transplantology, with particular emphasis on skin donation. In order to achieve a strategic stock of human skin grafts, a tissue collecting transplantation team should be organized. The rights and obligations of the non-physician transplant team member should be extended. Proposals have been made for awareness campaigns (adverts, posters etc.) and educational schemes (educational video, lectures during transplant coordinator training, etc.). Finally, a proposal has been made for possible methods to deal with the logistic management of the allogeneic skin stock. The required, essential stock of human allogeneic skin in the event of a mass disaster has been estimated at 600,000 cm2.

Tissue Engineering in Skin Substitute.

Thermal injuries may cause significant damage to large areas of the skin. Extensive and deep burn wounds require specialized therapy. The optimal method in the strategy of treating extensive, full thickness burns (III°) is the use of autologous split thickness skin grafts STSG (Busuioc et al. Rom J Morphol Embryol 4:1061-1067, 2012; Kitala D, Kawecki M, Klama-Baryla A, Labus W, Kraut M, Glik J, Ryszkiel I, Kawecki MP, Nowak M. Allogeneic vs. Autologous Skin Grafts in the Therapy of Patients with Burn Injuries: A Restrospective, Open-label Clinical Study with Pair Matching. Adv Clin Exp Med. 2016 Sep-Oct;25(5):923-929.; Glik J, Kawecki M, Kitala D, Klama-Baryla A, Labus W, Grabowski M, Durdzinska A, Nowak M, Misiuga M, Kasperczyk A. A new option for definitive burn wound closure - pair matching type of retrospective case-control study of hand burns in the hospitalized patients group in the Dr Stanislaw Sakiel Center for Burn Treatment between 2009 and 2015. Int Wound J. 2017 Feb 21. https://doi.org/10.1111/iwj.12720 . [Epub ahead of print]; Prim et al. May 24Wound Repair Regen., 2017; Grossova et al. Mar 31Ann Burns Fire Disasters 30:5-8, 2017). The main limitation of that method is the inadequate amount of healthy, undamaged skin (donor sites), which could be harvested and used as a graft. Moreover, donor sites are an additional wounds that require analgesic therapy, leave scars during the healing process and they are highly susceptible to infection (1-6). It must be emphasized that in terms of the treatment of severe, deep and extensive burns, and there should be no doubt that the search for a biocompatible skin substitute that would be able to replace autologous STSG is an absolute priority. The above-mentioned necessitates the search for new treatment methods of severe burn wounds. Such methods could consider the preparation and application of bioengineered, natural skin substitutes. At present, as the clinical standard considered by the physicians may be use of available biological skin substitutes, e.g., human allogeneic skin, in vitro cultured skin cells, acellular dermal matrix ADM and revitalized ADMs, etc. (Busuioc et al. Rom J Morphol Embryol 4:1061-1067, 2012; Kitala D, Kawecki M, Klama-Baryla A, Labus W, Kraut M, Glik J, Ryszkiel I, Kawecki MP, Nowak M. Allogeneic vs. Autologous Skin Grafts in the Therapy of Patients with Burn Injuries: A Restrospective, Open-label Clinical Study with Pair Matching. Adv Clin Exp Med. 2016 Sep-Oct;25(5):923-929.; Glik J, Kawecki M, Kitala D, Klama-Baryla A, Labus W, Grabowski M, Durdzinska A, Nowak M, Misiuga M, Kasperczyk A. A new option for definitive burn wound closure - pair matching type of retrospective case-control study of hand burns in the hospitalised patients group in the Dr Stanislaw Sakiel Center for Burn Treatment between 2009 and 2015. Int Wound J. 2017 Feb 21. https://doi.org/10.1111/iwj.12720 . [Epub ahead of print]; Prim et al. May 24Wound Repair Regen., 2017; Grossova et al. Mar 31Ann Burns Fire Disasters 30:5-8, 2017; Labus et al. FebJ Biomed Mater Res B Appl Biomater 106:726-733, 2018).

Transfer of fibroblast sheets cultured on thermoresponsive dishes with membranes.

In cell or tissue engineering, it is essential to develop a support for cell-to-cell adhesion, which leads to the generation of cell sheets connected by extracellular matrix. Such supports must be hydrophobic and should result in a detachable cell sheet. A thermoresponsive support that enables the cultured cell sheet to detach using only a change in temperature could be an interesting alternative in regenerative medicine. The aim of this study was to evaluate plates covered with thermoresponsive polymers as supports for the formation of fibroblast sheets and to develop a damage-free procedure for cell sheet transfer with the use of membranes as transfer tools. Human skin fibroblasts were seeded on supports coated with a thermoresponsive polymer: commercial UpCell™ dishes (NUNC™) coated with thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and dishes coated with thermoresponsive poly(tri(ethylene glycol) monoethyl ether methacrylate) (P(TEGMA-EE)). Confluent fibroblast sheets were effectively cultured and harvested from both commercial PNIPAM-coated dishes and laboratory P(TEGMA-EE)-coated dishes. To transfer a detached cell sheet, two membranes, Immobilon-P(®) and SUPRATHEL(®), were examined. The use of SUPRATHEL for relocating the cell sheets opens a new possibility for the clinical treatment of wounds. This study established the background for implementing thermoresponsive supports for transplanting in vitro cultured fibroblasts.

Influence of electron beam irradiation on extracellular matrix of the human allogeneic skin grafts.

The nonviable allogeneic human skin grafts might be considered as the most suitable skin substitutes in the treatment of extensive and deep burns. However, in accordance to biological security such grafts require the final sterilization prior to clinical application. The aim of the study was to verify the influence of electron beam irradiation of three selected doses: 18, 25, and 35 kGy on the extracellular matrix of human skin. Prior to sterilization, the microbiological tests were conducted and revealed contamination in all examined cases. Individual groups were subjected to single electron beam radiation sterilization at proposed doses and then subjected to microbiological tests again. The results of microbiological testing performed for all irradiation doses used were negative. Only in the control group was a growth of microorganisms observed. The FTIR spectrometry tests were conducted followed by the histological evaluation and mechanical tests. In addition, cost analysis of radiation sterilization of individual doses was performed. The results of spectroscopic analysis, mechanical tests, and histological staining showed no significant changes in composition and characteristics of tested tissues after their irradiation, in comparison to control samples. The cost analysis has shown that irradiation with 18 kGy is the most cost-effective and 35 kGy is the least favorable. However, according to biological risk reduction, the recommended sterilization dose is 35 kGy, despite the higher price compared to the other doses tested.

The use of biostatic human amnion and platelet-rich plasma in topical treatment of toxic epidermal necrolysis-A case report.

BACKGROUND: Toxic epidermal necrolysis (TEN) (also known as Lyell syndrome) and Stevens-Johnson syndrome (SJS) are life-threatening mucocutaneous blistering diseases. They are characterized by generalized blisters and epidermal inflammation, most likely resulting from the administration or interaction of medicines. AIMS: To report potential new method in the treatment of TEN. PATIENTS/METHODS: This article presents a case report of a 35-year-old man suffering from TEN covering about 95% of his body surface. Lesions occurred in the patient during antiepileptic therapy, after taking simultaneously amoxicillin (with clavulanic acid) and naproxen followed by lamotrigine treatment. Standard general treatment was performed. Intravenous feeding was necessary. Due to acute respiratory failure, the patient required mechanical ventilation. Two methods were combined in topical treatment: application of platelet-rich plasma (PRP) and a simultaneous biostatic human amnion transplant. RESULTS: In the presented case, the combination of both methods contributed to a significant acceleration of wound healing. After the application of PRP and biostatic amnion transplantation, the healing of wounds on the back and posterior surfaces of the legs was completed after six days. The surgical treatment most probably contributed to a significant acceleration of wound healing. CONCLUSION: The case report shows that topical TEN/SJS treatment with biostatic human amnion and PRP has a positive clinical effect and may be a new method of treatment of TEN.

The Causes for the Loss of Cellular Material at the Qualification Stage and the Influence of the Patient's Flora on Culture Infection.

There are several causes leading to the loss of cellular material earmarked for transplantation. This paper aims to evaluate the number of lost donors and lost cells in the culture by means of verifying both the results of the qualification tests and the presence of microorganisms in the cellular material. The analysis involved 86 donors hospitalized for thermal burns, from whom cells were harvested for keratinocyte and fibroblast cultures in the years 2011 to 2015. All potential donors underwent qualification tests: Anti-HIV-1,2; HBsAg; Anti-HCV-Ab; HBc, and a specific test for syphilis. In the case of skin fragments collected for culturing, the microbiological tests included the carrying fluid, the medium after 1 change, and the medium during culturing and before transplantation. Skin donors for cell cultures were assigned to the groups based on if the skin was collected up to 7 days following the burn or later. On average, 12% of the disqualifications were reported among donors for cell culturing. The most frequent cause of donor disqualification (54% of all disqualifications) was a positive HBc(+). The occurrence of fungal infections detected in the cellular material was over 30%. Establishing the culture after day 7 following the injury immediately increases the risk of infection by 25% in comparison to those cultures established before or on day 7 following the injury. Proper disinfection of donor place is crucial, but sometimes insufficient for maintenance sterility in cell culture. The risk of infection increases 25% after 7 following the injury.

Is Transgenic Porcine Skin as Good as Allogeneic Skin for Regenerative Medicine? Comparison of Chosen Properties of Xeno- and Allogeneic Material.

BACKGROUND: Burn treatment is associated with the need of dressing large cutaneous defects. There is a need of alternative search for the allogeneic skin as a source of grafting for a clinical use. Such sources include animals. For many years, porcine skin was used as a biological dressing for wounds or donor's fields, or residual fields after skin grafting. Current studies aim to minimize immunogenicity, inter alia, through the decellularization process. MATERIALS AND METHODS: The decellularization methods and porcine skin resettlement of human keratinocytes and fibroblasts were evaluated. The mechanical properties of the dressings and their influence on the viability, apoptosis, population doubling, and cell cycle of keratinocytes and fibroblasts were examined. The inheritance of cell antigens responsible for histocompatibility on the human keratinocyte and fibroblast surface in the cultures incubated with examined variants of dressings from porcine skin were analyzed. RESULTS: The most effective acellularization method is trypsinization. Morphology of the cell remained proper and stable during the whole experiment. In both fibroblast and keratinocyte cultures, the highest number of apoptotic cells was observed when samples were incubated with allogeneic skin. In the keratinocyte cultures, the highest number of live cells was observed when incubated with porcine transgenic acellular dermal matrix. The acellular matrices influence the increase of population doubling of keratinocytes in the cultures. CONCLUSION: For routine acellurization, trypsinization was chosen as the most effective method with preservation of tissue properties.

Infrared Thermal Imaging as a Method of Improving Skin Graft Qualification Procedure and Skin Graft Survivability.

BACKGROUND: Appropriate wound classification (evaluation of skin damage depth) is a key element in the first days after the burn injury. In this study, we used a thermovision system to evaluate the degree of burn wound shortly after injury and the regenerative process of engraftment of autologous split-thickness skin grafts (STSGs). The results were referenced to wound assessment made by a physician. METHODS: Twenty-one patients with STSGs were included in the study. The analysis of the dependence between the assessment of burn depth obtained with the use of numeric models (thermovision examination) and clinical assessment by physicians was conducted. Computed tomography angiography (CTA) was performed. RESULTS: The study demonstrated that thermovision images allowed physicians a deeper assessment of burns. Decrease in the temperature difference was observed in subsequent measurements. Temperature increase during normal graft healing was observed. Graft vascularization was confirmed by CTA. CONCLUSION: Thermovision may be useful in assessment of the clinical condition of burn wounds and monitoring of wound healing progress.

Application of Amniotic Stem Cells on an Acellular Dermal Matrix Scaffold in a Burned Patient: A Case Report.

BACKGROUND: Advances in science have allowed newly created medicinal products based on gene therapy, somatic cell therapy, and tissue engineering to be used in the treatment of human diseases. December 2008 legislation aims to ensure they are both safe for patients and available throughout the European Union. Amniotic stem cells are designated by the European Medicines Agency Committee for Advanced Therapies as an advanced therapy medicinal product; therefore, production must be in accordance with Regulation (EC) No. 1394/2007 of the European Council of 13 November 2007 on advanced therapy medicinal products. OBJECTIVES: This article details preliminary results of innovative amniotic stem cell transplant (supported by an acellular dermal matrix [ADM] produced in-house with Suprathel wound and burn dressing) in a burn patient and compares them with results in a patient treated with allogeneic skin. MATERIAL AND METHODS: Amniotic stem cells were applied to a 40-year-old patient with IIb°/III° thermal burns of 36% of total body surface area and III°/IV° of 1%. Wound healing was assessed by histologic examination and the Bates-Jensen scale. Reduction in pain perception was verified by 10-point visual analog scale. Hospitalization time was compared to length of stay for patients treated with standard therapy. RESULTS: The patient was discharged from hospital on the 12th day after surgery with complete wound healing (almost 2 times shorter than the control). CONCLUSIONS: The use of amniotic stem cells and ADM may be the optimal method for burn treatment.

Porcine Transgenic, Acellular Material as an Alternative for Human Skin.

Although new therapeutic approaches for burn treatment have made progress, there is still need for efficient coverage of donor fields. Promising dressing for skin graft donor site should be biocompatible, attach easily to the wound bed, remain in place until donor site has renewed, and decrease morbidity at the site. Porcine skin may be applied as a dressing for severe burns. Therefore pig skin xenografts can be used also as donor field coverage. In the Burn Treatment Centre, we used gauze soaked in Vaseline to secure donor fields. The aim of the study was to check if transgenic porcine skin is better than standard in donor site coverage used in our center. We showed that dressing reduces pain experienced by patients. The dressing leads to a reduction of hospitalization time by an average of 8 days. The dressing is as safe as the gold standard. Securing the donor field reduces the risk of colonization of the wound in the second smear after application by 60%. The disadvantage of the dressing is the inability to absorb blood; the use of hemostatic ointments in combination with the skin of transgenic pigs should be considered in the future.

Amniotic and Umbilical Cord of Transgenic Pigs as an Alternative Source of Stem Cells.

BACKGROUND: The placenta is worthy of consideration as a source of tissues for transplantation. Porcine material preparation allows significantly more grafts to be obtained than human material. Amniotic cells are a very practicable stem cell type and easy to obtain with great potential, compared with, for instance, adipose-derived stem cells. The aim of this paper was to verify if porcine transgenic amnion and umbilical cord could be as effective for xenotransplantation of stem cells as human material. METHODS: Oxytocin was administered to the sows on the day the material was obtained. The obtained material was divided by weight into 4 relatively equal parts, which were transferred into 1 of 4 containers. The containers had different transport media and a concentration of antibiotics. After cell isolation by homogenization, the number of live, dead, and apoptotic cells was assessed. RESULTS: Maintaining the sterility when obtaining material from breeding pigs was the biggest problem. Transport medium, despite the addition of antibiotics, was in most cases infected. Tests revealed that porcine cells have a tendency to leave tissue material and adhere to plastic as human cells do. Porcine cells are also fibroblast like, which can suggest that these cells might be tested for the presence of mesenchymal stem cells. However, some differences are visible in all parameters, which can result from contamination of material or improper transport medium. There were significant differences between viability and apoptotic cell number in human and transgenic pig cells isolated from both sources---amniotic membrane and umbilical cord.

Amniotic Stem Cells Cultured on Thermoresponsive Polymers Allow Obtaining a Full Cell Sheet.

Amniotic stem cells promote adhesion and migration of epithelial cells. Obtaining a full sheet containing amniotic stem cells seems to be the best solution for the treatment of burn wounds. The main advantage of this method is obtaining a full sheet of cells by lowering the temperature below the transition temperature, which does not affect extracellular matrix. The purpose of this work was to produce a skin substitute-a full sheet consisting of amniotic mesenchymal stem cells-and compare with well-known fibroblast sheet. Amniotic membrane cells revealed better tendency to full sheet detachment than fibroblasts. Confluence after 24 hours was always higher on polymer-coated dishes than on normal polypropylene dishes. Also viability was higher than on the control culture dish, while the number of apoptotic cells was always highest on polypropylene (control). Ile-Lys-Val-ala-Val (IKVAV) 0.28 addition to poly (poly [ethylene glycol] ethyl methacrylate) (PTEGMA) caused best cell confluence and highest percentage of cells in mitosis phase of cell cycle, but also worst cell detachment was observed in both cell types on PTEGMA IKVAV 0.28. Viability of cells transferred in cell sheet form onto a new culture dish was higher than when detached as suspension enzymatically. Additionally, percentage of apoptotic cells transferred in cell sheet form onto a new culture dish was always lower than when detached as suspension enzymatically. Culturing of PTEGMA, PTEGMA IKVAV 0.28 and PTEGMA IKVAV 0.14 have a stimulating effect on number of cells in mitosis in amniotic cell culture even after cell sheet transfer onto a new dish, whereas such effect with fibroblast was not observed.

A new approach to the production of a biovital skin graft based on human acellular dermal matrix produced in-house, in vitro revitalized internally by human fibroblasts and keratinocytes on the surface.

Patients with extensive and deep burns who do not have enough donor sites for autologous skin grafts require alternative treatment methods. Tissue engineering is a useful tool to solve this problem. The aim of this study was to find the optimal method for the production of a biovital skin substitute based on acellular dermal matrix (ADM) and in vitro cultured fibroblasts and keratinocytes. In this work, nine methods of ADM production were assessed. The proposed methods are based on the use of the following enzymes: Dispase II, collagenase I/ethylenediaminetetraacetic acid (EDTA), collagenase II/EDTA, and mechanical perforation using DermaRoller and mesh dermatome. The obtained ADMs were examined (both on the side of the basement membrane and on the "cut-off" side) by means of scanning electron microscopy, immunohistochemistry tests and strength tests. ADM was revitalized with human fibroblasts and keratinocytes. The ability of in-depth revitalization of cultured fibroblasts and their ability to secrete collagen IV was examined. The obtained results indicate that the optimal method of production of live skin substitutes is the colonization of autologous fibroblasts and keratinocytes on the scaffold obtained using two-step incubation method: Trypsin/EDTA and dispase II.

Heterogeneous Mixture of Amniotic Cells is Likely a Better Source of Stem Cells than Adipose Tissue.

Stem cells are increasingly being used in the course of burn treatment. As several different types of stem cells are available for the purposes, it is important to chose the most efficient and the most practicable stem cell type. The aim of this study was to compare the potential of heterogeneous amnion cell mixture with the presently used standard therapy, the adipose tissue-derived stem cells. The placenta was collected during a Cesarean section procedure. Adipose tissue tissue-derived cells were isolated using the Cytori's Celution® System. Cells were tested for fulfillment of the minimum criteria for stem cells. The efficiency of cell cultures was tested by an analysis of population doubling, cell proliferation, cell cycle and cell migration. Amniotic cells presented a higher ability for differentiation to chondrocytes and osteocytes than adipose-derived regenerative cells but a lower ability for differentiation toward adipocytes. Additionally, in vitro experiments have demonstrated a higher applicability of amniotic cells than adipose tissue-derived stem cells. Amniotic cells show several advantages: easy access to placenta, low costs and a lack of ethical dilemmas related to stem cell harvesting. The main disadvantage is, however, their availability, as isogenic treatment would only be possible for women around children-bearing age, unless personalized banks for amniotic cells would be established.

Does the transplantation of keratinocytes really reduce the risk of death? Survival analysis of patients hospitalized at the Dr Stanislaw Sakiel Centre for Burns Treatment in 2008-2015.

BACKGROUND: Keratinocyte transplantation is an adjuvant procedure in the extensive burn therapy method. However, it must be taken into consideration that clinical results of keratinocyte transplantation are ambiguous and progress achieved in this method is still being verified, especially due to the high cost of cultured epithelial autograft (CEA) transplants. OBJECTIVES: The aim of this study was to verify the impact of cultured keratinocyte application on patients' survival. This study included a group of patients with the highest chance for a successful outcome of the therapy and excluded patients with no compelling reason to apply for such an expensive therapy. MATERIAL AND METHODS: This study included all the patients with burns diagnosed between January 1, 2008 and January 1, 2016, who were treated with cultured skin cells. Patients' age and gender, percentage of total body surface area (TBSA) affected, percentage of burn depth of the 3rd/4th degree, number of days between admission and surgery, and need for rehabilitation were analyzed. RESULTS: The cultured cell application did not significantly affect the risk of death (p > 0.05). CONCLUSIONS: Keratinocytes should be applied as an adjunctive method for the treatment of burns with at least 40% TBSA affected, but with a maximal burn depth of the 2nd degree. In the group of patients below 50 years of age, a higher number of transplants with a cell population above 20 million/mL and a significantly lower mortality rate were observed, which means that in the mentioned age group, this graft was more effective. It has been suggested that patients older than 50 years of age with burns deeper than of the 2nd degree should be treated with more advanced methods like, e.g., the application of stem cells.

Amniotic cells share clusters of differentiation of fibroblasts and keratinocytes, influencing their ability to proliferate and aid in wound healing while impairing their angiogenesis capability.

An alternative to cultured skin cell grafts usage in burn treatment is the graft of allogenic stem cells. We verified whether amniotic stem cells are better than the present therapeutic standard: grafts of autologous keratinocytes and fibroblasts along with autologous adipose-derived stem cells, and whether amniotic stem cells can support the growth of autologous keratinocytes and fibroblasts in the culture. The study was performed on the material from 18 amnia. Skin cells were obtained from 3 patients. In order to assess the influence of stem cells on keratinocytes and fibroblasts, the following experiments were performed: impact on viability and cell cycle, wound healing capability, angiogenesis capability, influence on the proliferation speed and capability to differentiate into skin cells. We demonstrated that human amniotic membrane-derived mesenchymal stem cells (hAMMSCs) share amniotic proteins with skin cells. Amniotic stem cells may replace skin fibroblasts in grafts due to the close similarity in their surface antigens, with significantly larger proliferative potential and ability to stimulate wound healing. It was shown that adding amniotic cells to both keratinocytes and fibroblast cultures accelerates directional migration by ≥ 40%. We confirmed in this study the influence of amniotic cells on the proliferation and cell cycle of fibroblasts and keratinocytes. Amniotic stem cells can be successfully used not only as a first choice graft but also to replace 3T3 line cells, supporting the proliferation of the cells during the culturing, as well as a supplementary graft supporting an autologous graft of keratinocytes and fibroblasts.

Isolation, culturing and preparation for transplantation of amniotic mesenchymal stem cells Repetitive and reproducible laboratory, technical protocol.

To date, different methods of isolation of amniotic stem cells have been developed. Our previous studies have demonstrated that there are significant differences in viability and efficiency of the isolation and culture process depending on the enzyme and medium used. The aim of this study was to present efficient protocol, which can be used within good manufacturing practise conditions. Amniotic membranes were obtained from ten woman 31-39 years old who signed informed constent. GMP regulations are applicable. The described protocol aims to obtain a clinically significant cell yield (>1*108). The cells may be maintained in the growth phase even for 2 months. The mesenchymal cells constitute about 75-95% of the cells in primary culture. Supervisory authorities require repetitive and reproducible laboratory protocol for stem cells culture. Presented protocol allow achieving clinically significant cell yield (>1*108) in 4-5 weeks. Cells can be transplanted as suspension or cell sheet.

A review of decellurization methods caused by an urgent need for quality control of cell-free extracellular matrix' scaffolds and their role in regenerative medicine.

The natural extracellular matrix (ECM),thanks to its specific properties (e.g., collagenous lattice, a reservoir of growth factors, ECM-cell anchoring areas, an optimal pH and CO2 ),ensures an optimal microenvironment for homeostatic and regenerative cell development. In the context of regenerative medicine, ECM is a lair for residual and infiltrative cells. The aim of the clinical usage of cell-free ECM scaffolds is the enhancement of tissue regeneration with possible minimization of an adverse host reaction on allogeneic or xenogeneic biomaterial. Thus, the objective of decellularization is to obtain acellular grafts characterized by optimal biological properties, such as a lack of remaining cellular elements (e.g., cell membrane phospholipids and proteins, nucleic acids, mitochondria), lack of immunogenicity, lack of calcification promotion and lack of cytotoxicity (e.g., in unrinsed detergents). Furthermore, cell-free ECM scaffolds should present the optimal mechanical and structural properties that may ensure the biocompatibility of the graft. The maintenance of the ultrastructure composition of the ECM is one of the most important goals of decellularization. All physical, chemical, and biological methods proposed (used separately or in combination to extract cells from tissues/organs) are not 100% effective in cell removal and always cause a disruption of the ECM texture, as well as a probable loss of important structure components. Although cell-free ECM scaffolds are generally classified as medical devices, there are no widely accepted or legally defined criteria for quality control/evaluation methods of obtained matrices. Such criteria must be provided. Some of them have been proposed in this manuscript. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 909-923, 2018.

Atomic force microscopy in the production of a biovital skin graft based on human acellular dermal matrix produced in-house and in vitro cultured human fibroblasts.

The most efficient method in III° burn treatment is the use of the autologous split thickness skin grafts that were donated from undamaged body area. The main limitation of this method is lack of suitable donor sites. Tissue engineering is a useful tool to solve this problem. The goal of this study was to find the most efficient way of producing biovital skin substitute based on in house produced acellular dermal matrix ADM and in vitro cultured fibroblasts. Sixty samples of sterilized human allogeneic skin (that came from 10 different donors) were used to examine the influence of decellularizing substances on extracellular matrix and clinical usefulness of the test samples of allogeneic human dermis. Six groups of acellular dermal matrix were studied: ADM-1 control group, ADM-2 research group (24 h incubation in 0.05% trypsin/EDTA solution), ADM-3 research group (24 h incubation in 0.025% trypsin/EDTA solution), ADM-4 research group (24 h incubation in 0.05% trypsin/EDTA solution and 4 h incubation in 0,1% SDS), ADM-5 research group (24 h incubation in 0.025% trypsin/EDTA solution and 4 h incubation in 0,1% SDS), and ADM-6 research group (24 h incubation in 0,1% SDS). Obtained ADMs were examined histochemically and by atomic force microscopy (AFM). ADMs were settled by human fibroblasts. The number of cultured cells and their vitality were measured. The obtained results indicated that the optimal method for production of living skin substitutes is colonization of autologous fibroblasts on the scaffold prepared by the incubation of human allogeneic dermis in 0.05% trypsin/EDTA. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 726-733, 2018.