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 CO ),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.

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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.

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.

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.

To understand the molecular mechanism controlling in vitro plant morphogenesis, a culture system enabling induction of alternative morphogenic pathways (somatic embryogenesis, SE; shoot organogenesis, ORG) in a well defined population of somatic cells is needed. Arabidopsis is the most useful model plant for genomic studies, but a system in which SE or ORG can be induced alternatively in the same type of explant has not been proposed. Immature zygotic embryos (IZEs) of Arabidopsis provide the only explants with embryogenic potential, and have been recommended for studying mechanisms of SE induced in vitro. This study was aimed at defining culture conditions promoting induction of alternative morphogenic pathways: shoot ORG in IZE explants. The established protocol involves pretreatment of IZE explants with liquid auxin-rich callus induction (CIM) medium, followed by subculture on solid cytokinin-rich shoot induction medium (SIM). The method enables efficient shoot induction in Columbia (Col-0) and Wassilewskija (Ws), genotypes commonly used in molecular studies. During 3 weeks of culture up to 90% of Col-0 and 70% of Ws explants regenerated shoots via an indirect morphogenic pathway. We analyzed the qRT-PCR expression patterns of the LEC (LEC1, LEC2 and FUS3) genes, the key regulators of Arabidopsis embryogenesis, in the IZE explants induced to promote shoot ORG. The sharp decline of LEC expression on SIM medium confirmed that culture of Arabidopsis IZE explants enables experimental manipulation of the morphogenic response of somatic cells. A scheme illustrating various in vitro morphogenic responses of IZEs in relation to hormonal treatment is presented.K Ke ey y w wo or rd ds s: : Arabidopsis, immature zygotic embryos, in vitro morphogenesis, shoot regeneration.

Radiation sterilization eliminates microbiological infections but causes the degradation of the cell factor. The negative result of microbiological examination for tissue transplants is one of the conditions for approval for distribution in patients. The study attempts to verify impact of the presence of microbes onto material for transplant loss. In the 2011-2015 period, we analyzed 293 donors of skin and amnion. Microbiological sampling was performed. The total of 21 strains of bacteria, molds and fungi was identified in collected tissue. The widest spectrum of strains was found in skin (17), followed by amnia (8). The total number of positive findings was 147 and was again highest in skin (129), while the number of positive findings in amnia was 18 only. The general percentage of fungal infections was very low. The presence of fungal strains was only observed in allogeneic skin (2%). Large number of microorganisms isolated from the skin before sterilization was observed, so it seems impossible to use allogeneic intravital skin. However, the intravital application of allogeneic amnion obtained from cesarean section remains to be considered.

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.