Dynamics of Microbiome Changes in the Endometrium and Uterine Cervix during Embryo Implantation: A Comparative Analysis.

BACKGROUND The microbiome is the collection of all micro-organisms and their genes, which naturally live in and on the body. The cervical and endometrial bacterial microbiome has previously been reported to affect fertility and influence the outcomes of assisted reproductive therapy (ART), including embryo transfer. This study aimed to evaluate the cervical and endometrial bacterial microbiome in 177 women treated for infertility before, during, and after embryo implantation, and the outcomes. MATERIAL AND METHODS Cervical and endometrial swabs were collected from 177 women diagnosed with infertility at 3 time points: (1) during the initial examination, (2) during implantation, (3) 10-14 days after implantation. Next-generation sequencing (NGS) was used to analyze the bacterial microbiome. Taxonomic identification was performed with the Usearch algorithm. RESULTS There was a significant change in the number of patients with Escherichia coli depending on the collection time. For the first swab collection, there were significant negative relationships between the percentage of Gardnerella vaginalis and Lactobacillus spp. For the second collection, there was a negative relationship between Lactobacillus helveticus and Lactobacillus jensenii. For the third collection, negative relationships were found between Escherichia coli and Lactobacillus spp. A similar distribution of the bacterial microbiome was observed in all 3 swab collections. CONCLUSIONS Lactobacillus spp. were the main bacteria identified in the cervix and endometrium, present before, during, and after successful embryo transfer. E. coli and G. vaginalis reduced the protective effect of Lactobacilli before, during, and after embryo transfer.

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.

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).

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.

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.

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.

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.