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.

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.

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.

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.

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.

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 Centre for Burn Treatment between 2009 and 2015.

Nearly 80% of all burns include the hands of affected individuals. Skin grafting is the gold standard in burns treatment, but in the case of the burn wound bed, it may require the necessity of utilising skin substitutes to facilitate closure. The aim of this study is to assess the impact of a porcine-derived wound dressing (Oasis™) for application to hand burns compared to a synthetic dressing (Suprathel™). Comparative assessments were made, including the time to heal, quality of healing and pain intensity. A retrospective, unblinded, matching pair case-control of hand burns was performed. A control group of 24 patients was treated with Suprathel dressing, and a study group of six patients underwent application of the Oasis dressing. The wound healing process was evaluated by taking histopathological specimens and also utilising the Bates-Jensen Wound Assessment Tool. A 10-cm Visual Analogue Scale (VAS) was used for pain assessment. Other parameters measured included dressing loss because of infection and the need of rehabilitation. The progress of wound healing on the fourth day in the study group was 30%. A decrease in the level of pain was recorded on the fourth day after surgery. There was a decrease of 5% in the risk of rehabilitation in the treatment group.

The Isolation and Production of the Ready-to-Use Product (the Amniotic Stem Cell Culture) in Accordance with Good Manufacturing Practice Regulations.

According to the Committee for Advanced Therapies, amniotic stem cells were classified as an advanced therapy medicinal product. This work aims to standardize the isolation of amniotic stem cells and the selection of the optimal time of transplantation and cell application methods in burn patients according to the guidelines of the Good Manufacturing Practice. The placenta used in the study was sourced during a Cesarean section. The remnants of the amnion preparation were placed in a sterile container and transferred to a class B environment, where the primary cultures began. The highest average number of cells was obtained by tissue homogenization and culture growth on the AmnioGrow medium. The isolation of the pure monoculture should be performed using the antibodies against CD105. On the basis of an analysis of population doubling, the aging of a population, the cells' viability, and the severity of injury, the cells should be used between passages 3 and 6. Significant differences were found in the number and viability of cells that were transferred as a full sheet, depending on the transfer method. To sum up, amniotic cells are a promising source in the treatment of burns and can be used as a hospital exemption.

Own Experience From The Use Of A Substitute Of An Allogeneic Acellular Dermal Matrix Revitalized With In Vitro Cultured Skin Cells In Clinical Practice.

As a result of the removal of cells from human allogeneic dermis, a collagen scaffold is obtained, which can be populated de novo with autologous/allogeneic skin cells and transplanted onto the area of skin loss. The optimal method for production of acellular dermal matrices (ADM) has been selected. Three female patients (a mean age of 54 years) were subjected to the transplantation of either autologous or allogeneic keratinocytes and fibroblasts into the holes of acellular dermal matrix (ADM) mesh graft. The method for burn wound treatment based on the use of a viable dermal-epidermal skin substitute (based on ADM and in vitro cultured fibroblasts and keratinocytes) may be the optimal method of burn treatment.