Autologous mesenchymal stem cells application in post-burn scars treatment: a preliminary study.

Mesenchymal stem cells (MSCs) are multi-potent cells characterized by long term self-renewal and by potential for differentiation into cells of different mesenchymal tissue types such as fibroblasts, osteocytes, chondrocytes, and adipocytes. Their unique properties offer broad therapeutic potentials. Bone marrow has been used as the most common MSCs source, but it is gradually going to be replaced by adipose tissue which showed to contain more MSCs per unit than the bone marrow and clinical application of MSCs procured from the adipose tissue have been demonstrating at least similar results. Post-burn scars result frequently in severe both functional and aesthetic impairments in restitution and rehabilitation periods of the burn disease. Despite extensive research in the last decades, the exact mechanisms of scar formation remains unclear. The development of post-burn scars is influenced by multiple factors such as initial depth of the burn, methods of burn wound therapy, duration of the open wound until final wound closure, burn wound infection, genetic predisposition, and many others in both acute and rehabilitation periods. The aim of this study was to point out versatility of the implementation of this method with respect to different types of scars (atrophic scars, hypertrophic scars, keloids). Autologous adipose tissue derived MSCs were applied to post-burn scars in all 8 patients undergoing surgical scar reconstructions at the Department of Burns and Reconstructive Surgery of the University Hospital in Bratislava. The study was approved by Ethical Committee of Ruzinov Hospital. The procedures used for scar reconstructions included dermabrasion, scar excisions, contractures corrections and local plasties combined by lipografting of lipoaspirate containing parenchymal adipocytes and stromal vascular fraction including MSCs, or application of separated autologous MSCs isolated from lipoaspirates. Based on desired result one of these MSCs application methods was selected depending on characteristics of reconstructed scar and required volume of transferred fat. Isolation of MSCs following procurement was provided by the Central Tissue Bank cell culture laboratory which is one of the parts of the burn department. The average time of scars duration was 79 months, ranging from 6 to 216 months. The postburns scars were assessed clinically according to Vancouver Scar Scale (VSS) prior to surgery, including photo documentation, and re-evaluated after 6 months following MSCs application. As the results have shown, the average VSS score before treatment was 7.88 points ranging from 4 to 11 points. The average VSS 6 months after surgical procedure and MSCs application was 2.34 points ranging from 1 to 4 points. According to the results obtained, the favourable effect of adipose tissue derived autologous MSCs application on scar remodelling following surgical reconstruction of post-burn scars could be promising.

Deep frozen amniotic membrane used as a scaffold and/or carrier for different cell types.

Amniotic membrane is a biological material widely used in plastic and reconstructive surgery and in ophthalmology. Due to its excellent biocompatibility and strength we tried to use it as a scaffold for the in vitro cultivation of different cell types, especially keratinocytes and limbal stem cells. It was possible to cultivate limbal stem cells and keratinocytes without using 3T3 mouse fibroblast feeder cells on deep frozen amniotic membranes. The amniotic membrane can also be used as a carrier for suspensions of different types of cells, allowing a substantial reduction of the cultivation time needed to prepare cell cultures for clinical application to burn patients. Our results show that the amniotic membrane seems not only to be an excellent carrier for human keratinocytes and corneal limbal stem cells, but also for other cell types, including dermal fibroblasts, adipose tissue-derived mesenchymal stem cells and chondrocytes.

Delayed post mastectomy breast reconstructions with allogeneic acellular dermal matrix prepared by a new decellularizationmethod.

Acellular dermal matrix (ADM) is a tissue graft of allogeneic origin from post-mortem tissue donors prepared by an innovative decellularization process. The newly developed non-toxic and low cost decellularization process of cadaver origin dermis included ADM in breast reconstruction procedures proved to help coverage of the lower-pole of breast expanders or implants. As the results have shown, it did help to eliminate autologous dermis donor site morbidity along with shortening the operation time by avoiding elevation of additional muscle or fascia during the operation. Main aims of this article include histology evaluation of allogeneic acellular dermal matrix prepared by a new decellularization method and presentation of clinical results of its use. A total of 22 patients underwent 26 ADM based breast reconstructions. The mean patient's follow up was 12.6 months. Average total size of ADM used for one breast was 273 cm2. Post-operative complications occurred in 3 patients including one expander infection, one expander extrusion and one expander pocket disfiguration. Microscopic analysis of tissue samples has confirmed incorporation of the acellular dermal matrices into the surrounding connective tissue without any noticeable immune reaction. In a majority of the ADM samples we found pseudocapsullar formation on implant side of samples without acute or chronic inflammatory cells. The use of ADM prepared by new preparation method in expansive post mastectomy breast reconstruction was associated by a relatively low complication rate resulting in good outcomes.

Stem cell regenerative potential for plastic and reconstructive surgery.

Stem cells represent heterogeneous population of undifferentiated cells with unique characteristics of long term self renewal and plasticity. Moreover, they are capable of active migration to diseased tissues, secretion of different bioactive molecules, and they have immunosuppressive potential as well. They occur in all tissues through life and are involved in process of embryogenesis and regeneration. During last decades stem cells attracted significant attention in each field of medicine, including plastic and reconstructive surgery. The main goal of the present review article is to present and discuss the potential of stem cells and to provide information about their safe utilization in chronic wounds and fistulae healing, scar management, breast reconstruction, as well as in bone, tendon and peripheral nerve regeneration.

Cytotoxicity testing of a polyurethane nanofiber membrane modified with chitosan/ß-cyclodextrin/berberine suitable for wound dressing application: evaluation of biocompatibility.

In this study we evaluated the biocompatibility of a modified polyurethane nanofiber membrane on a polypropylene spunbond substrate. This material was treated with plasma using diffuse coplanar surface barrier discharge, and subsequent modification was done by continuous spraying of a biologically active chitosan solution (CHIT) containing an inclusion complex of ß-cyclodextrin (ß-CD) encapsulating berberine (BRB). Biocompatibility was evaluated using several in vitro assays. Human dermal fibroblasts (HDFs) and 3T3 murine fibroblasts were used as biological models. The results of these assays showed that a polyurethane nanofiber membrane modified by CHIT/ß-CD/BRB appears to be non-toxic and biocompatible; potentially, it could be used as a wound dressing after further testing.

Induced pluripotent stem cells and their implication for regenerative medicine.

In 2006 Yamanaka's group showed that stem cells with properties similar to embryonic stem cells could be generated from mouse fibroblasts by introducing four genes. These cells were termed induced pluripotent stem cells (iPSCs). Because iPSCs avoid many of ethical concerns associated with the use of embryonic material, they have great potential in cell-based regenerative medicine. They are suitable also for other various purposes, including disease modelling, personalized cell therapy, drug or toxicity screening and basic research. Moreover, in the future, there might become possible to generate organs for human transplantation. Despite these progresses, several studies have raised the concern for genetic and epigenetic abnormalities of iPSCs that could contribute to immunogenicity of some cells differentiated from iPSCs. Recent methodological improvements are increasing the ease and efficacy of reprogramming, and reducing the genomic modification. However, to minimize or eliminate genetic alternations in the derived iPSC line creation, factor-free human iPSCs are necessary. In this review we discuss recent possibilities of using iPSCs for clinical applications and new advances in field of their reprogramming methods. The main goal of present article was to review the current knowledge about iPSCs and to discuss their potential for regenerative medicine.

Mesenchymal stem cells for chronic wounds therapy.

Wound healing is a complex process that involves interaction of soluble mediators, extracellular matrix and infiltrating blood cells. Chronic and non-healing skin defects contribute significantly to morbidity and mortality of many patients. Recently, despite the current medical progress, the chronic and non-healing wounds still represent a serious medical problem. In many cases, conventional therapeutic approaches, such as dermal substitutes and growth factor therapy failed and do not produce the expected results, patients are exposed to a high risk of infection, sepsis or amputation. For that reason clinicians and researchers are forced to searching for alternative methods to induce healing process which may result into complete wound closure. Mesenchymal stem cells (MSCs) represent a unique tool of tissue engineering and regenerative medicine and a promising therapeutic strategy. Due to their unique biological properties, MSCs seem to be the perspective modality method for these patients. Many preclinical and clinical studies suggest the possibility of using these cells in tissue regeneration, healing acute and chronic wounds and scar remodelling. The objective of the present review is to summarize the current information and preclinical data about MSCs, their biological characteristics and mode of action during regenerative and healing processes, as well as their clinical application in chronic wounds treatment.

Ultra-structural morphology of long-term cultivated white adipose tissue-derived stem cells.

White adipose tissue was long perceived as a passive lipid storage depot but it is now considered as an active and important endocrine organ. It also harbours not only adipocytes and vascular cells but also a wide array of immunologically active cells, including macrophages and lymphocytes, which may induce obesity-related inflammation. Recently, adipose tissue has been reported as a source of adult mesenchymal stem cells with wide use in regenerative medicine and tissue engineering. Their relatively non-complicated procurement and collection (often performed as liposuction during aesthetic surgery) and grand plasticity support this idea even more. We focused our research on exploring the issues of isolation and long-term cultivation of mesenchymal stem cells obtained from adipose tissue. Ultra-structural morphology of the cells cultivated in vitro has been studied and analysed in several cultivation time periods and following serial passages--up to 30 passages. In the first passages they had ultra-structural characteristics of cells with high proteosynthetic activity. Within the cytoplasm, big number of small lipid droplets and between them, sparsely placed, small and inconspicuous, electron-dense, lamellar bodies, which resembled myelin figures were observed. The cells from the later passages contained high number of lamellar electron-dense structures, which filled out almost the entire cytoplasm. In between, mitochondria were often found. These bodies were sometimes small and resembled myelin figures, but several of them reached huge dimensions (more than 1 µm) and their lamellar structure was not distinguishable. We did not have an answer to the question about their function, but they probably represented the evidence of active metabolism of lipids present in the cytoplasm of these cells or represented residual bodies, which arise after the breakdown of cellular organelles, notably mitochondria during long-term cultivation.

Cytotoxicity testing of scaffolds potentially suitable for the preparation of three-dimensional skin substitutes.

The preparation and study of three-dimensional functional skin substitutes has been the focus of intense research for several decades. Dermal substitutes are now commonly used in medical practice for a variety of applications. Here, we assess the toxicity of seven selected acellular dermal matrix materials to establish their potential for use in future three-dimensional skin substitute studies. The cytotoxicity of acellular dermis (of Allo- and Xenograft origin) prepared in our lab and biomaterials based on collagen and hyaluronic acid (Coladerm H and Coladerm H-L) were compared to that seen in three commercially available products (Xe-Derma, AlloDerm and Xeno-Impl). Murine fibroblasts NIH-3T3 and human dermal fibroblasts were used in cytotoxicity tests, with any resultant cytotoxic effects caused by the seven tested dermal scaffolds visualised using an inverted microscope system and confirmed in parallel using colorimetric MTT cell proliferation assays. While most of the dermal substitutes did not demonstrate a cytotoxic effect on our two cell types, Xeno and Xeno-Impl scaffolds clearly did. The cytotoxic effect of acellular Xeno dermal matrix could essentially be removed through a regime of multiple washes, but we were unable to remove the cytotoxic effect of Xeno-Impl. Thus, Xeno-Impl alone has been excluded from our future work on preparation of 3D skin substitutes.

[Currently available skin substitutes].

The current trend of burn wound care has shifted to more holistic approach of improvement in the long-term form and function of the healed burn wounds and quality of life. Autologous split or full-thickness skin graft are the best definitive burn wound coverage, but it is constrained by the limited available sources, especially in major burns. Donor site morbidities in term of additional wounds and scarring are also of concern of the autograft application. This has demanded the emergence of various skin substitutes in the management of acute burn injury as well as post burn reconstructions. This paper reviews currently available skin substitutes, produced in not for-profit skin banks as well as commercially available. They are divided according to type of material included, as biological, biosynthetic and synthetic and named respectively.

Amniotic membrane application in surgical treatment of conjunctival tumors.

The amniotic membrane (AM) has special properties, making it ideal for clinical applications in various surgical fields like ophthalmology. It is used more frequently to cover conjunctival and corneal defects. In our retrospective study we have been combined 68 patients with epibulbar conjunctival tumors they have been surgically treated in the period of 2011-2021. Seven (10.3%) patients have been treated with AM application after surgical removal of the tumor. 54 (79%) cases were malignant, and 14 (21%) were benign. In the analyzed dataset the males had just slightly higher chance of malignancy than females, 80% versus 78.3%. For the significancy calculation the Fisher exact test was used and the result proved no significancy (p = 0.99). Six patients with AM application were malignant. The observed difference in the number of quadrants of the bulbar conjunctiva infiltrated versus significant malignancy with p = 0.050 calculated by Fisher Exact test and with p = 0.023 calculated by Likelihood-ratio test. The results of our study indicate that AM grafts are an effective alternative to cover defects after removal of epibulbar lesions due to their anti-inflammatory properties because the conjunctiva must be preserved, and especially the most important application is in malignant epibulbar conjunctival tumors.

Effect of Annealing on the Surface Hardness of High-Fluence Nitrogen Ion-Implanted Titanium.

Commercially pure titanium grade II was kinetically nitrided by implanting nitrogen ions with a fluence in the range of (1-9)·1017 cm-2 and ion energy of 90 keV. Post-implantation annealing in the temperature stability range of TiN (up to 600 °C) shows hardness degradation for titanium implanted with high fluences above 6·1017 cm-2, leading to nitrogen oversaturation. Temperature-induced redistribution of interstitially located nitrogen in the oversaturated lattice has been found to be the predominant hardness degradation mechanism. The impact of the annealing temperature on a change in surface hardness related to the applied fluence of implanted nitrogen has been demonstrated.

Experience gained during the long term cultivation of keratinocytes for treatment of burns patients.

Both allogenic and autologous cultured skin cells have been used clinically on burn patients. In vitro cultivation of human keratinocytes has been routinely provided by the Central Tissue Bank in Bratislava since 1996, with an average annual production of around 7,000 cm(2). Keratinocytes have been cultivated using a version of the original by Rheinwald and Green (Cell 6:317-330, 1975) methodology which has been modified over time in our laboratory as we gained more experience with this serial passage system. We have observed that the growth of cultured keratinocytes depends on several important factors, including the timing of skin sample procurement, the method of skin sample procurement, the general condition of the patient, the quality and composition of the culture media and, to a lesser extent, the age of the patient. We aim to share our experience with other cell cultivation facilities.

[Employment of current results of tissue engineering in the development of skin substitutes]. / Vyuzívanie súcasných poznatkov tkanivového inzinierstva pri príprave kozných náhrad.

Substitution of skin, particularly in extensive burns, is one of the key points for patients mortality reduction. In addition to the use of allogeneic and autologous skin substitutes, new developments in tissue engineering would enable the use biosynthetic and combined skin substitutes, which could mimic the structure and functions of normal skin. Several such types of substitutes like cultured allogeneic and autologous keratinocytes, allogeneic/autologous composites, acellular matrices, matrices based on biological substances such as collagen/hyaluronic acid, and matrices seeded by different cell types (keratinocytes, dermal fibroblasts, stem cells) already exist. Recent development in skin substitutes research aims gradually to establish a fully functional skin substitute which could mimic skin not only by its structure, but which could be capable to assure also its revascularization, reinnervations, and replacement of skin appendages (hair follicles, sebaceous glands etc.) as well. Creation of such a skin substitute will require collaboration of a wide range of research specialists including molecular biology, material sciences, genetic and tissue engineering, computer sciences, and, of course, clinical specialists in the field of plastic surgery and burn medicine. Recent advances in this field are promising and give hope that in the near future such a fully functional skin substitute would become a reality. This article aims to give information on the available skin substitutes at the present time.

Impact of Antibiotics Associated with the Development of Toxic Epidermal Necrolysis on Early and Late-Onset Infectious Complications.

Toxic epidermal necrolysis (TEN) is a rare disease, which predominantly manifests as damage to the skin and mucosa. Antibiotics count among the most common triggers of this hypersensitive reaction. Patients with TEN are highly susceptible to infectious complications due to the loss of protective barriers and immunosuppressant therapy. The aim of this study was to investigate the potential relationship between antibiotics used before the development of TEN and early and late-onset infectious complications in TEN patients. In this European multicentric retrospective study (Central European Lyell syndrome: therapeutic evaluation (CELESTE)), records showed that 18 patients with TEN used antibiotics (mostly aminopenicillins) before the disease development (group 1), while in 21 patients, TEN was triggered by another factor (group 2). The incidence of late-onset infectious complications (5 or more days after the transfer to the hospital) caused by Gram-positive bacteria (especially by Enterococcus faecalis/faecium) was significantly higher in group 1 than in group 2 (82.4% vs. 35.0%, p = 0.007/p corr = 0.014) while no statistically significant difference was observed between groups of patients with infection caused by Gram-negative bacteria, yeasts, and filamentous fungi (p > 0.05). Patients with post-antibiotic development of TEN are critically predisposed to late-onset infectious complications caused by Gram-positive bacteria, which may result from the dissemination of these bacteria from the primary focus.

Bromelain-based enzymatic debridement as a treatment of choice in high-risk patient with deep facial burns, a case report.

INTRODUCTION: Deep facial burns are often combined with inhalation injury that could lead to patient destabilization. Accurate timing of surgical debridement of deep burns in a critical patient is the real medical art. Especially in patients with deep burned face and hands, in whom early debridement promises better functional and aesthetic results. CASE PRESENTATION: A fifty-three-year-old woman sustained burns of 16% TBSA including face area. The treatment of the burn injury was complicated by severe inhalation trauma, which led to patient destabilization shortly after admission. Standard surgical debridement was risky at the time. We used a new enzymatic agent for early burn eschar necrolysis instead. All the debrided areas were temporarily covered with porcine xenografts. The facial burns healed spontaneously without the need for a skin transplant. Definitive surgery treatment of full-thickness burns was postponed until the patient´s stabilization. DISCUSSION: The new enzymatic debridement is minimally invasive and can be applied bedside without the need for general anesthesia. All advantages of the new enzymatic debridement had led to extend its use at the face area, although it was not tested in this area during pre-registration studies. Especially in facial area high selectivity and significant reduction of skin grafting expect a better aesthetic and functional outcome. CONCLUSION: Bromelain-based enzymatic debridement proved to be safe and effective on the face in a very high-risk patient with unstable circulation and severe inhalation injury as an alternative to tangential excision.

Induction of metalloproteinase 9 secretion from human keratinocytes by pleuran (beta-glucan from Pleurotus ostreatus).

Glucan preparations, primarily modified water-soluble glucans, are involved in the activation of the body's natural defense mechanisms and in the acceleration of the skin's wound-healing processes. Pleuran, an insoluble beta-D-glucan in hydrogel form, offers a natural alternative to more common chemically derivated soluble beta-D-glucans. Pleuran was applied to human keratinocyte primary cultures, and after 24 h of incubation the release of matrix metalloproteinase 9 (MMP-9) and metalloproteinase 2 (MMP-2) by stimulated keratinocytes was detected using gelatine zymography. There was a concentration-dependent increase in pro-MMP-9 release after treatment with pleuran over the concentration range of 2 to 200 microg/ml, but pro-MMP-2 was detected at a constant level. Moreover, the active forms of both MMPs were not detectable, indicating that in vitro autoactivation of these zymogens did not occur. The results indicate that pleuran is a potent keratinocyte stimulator of proMMP-9 release, which implies its application in dermatological therapies.

What happens to an acellular dermal matrix after implantation in the human body? A histological and electron microscopic study.

Acellular matrices are used for various purposes and they have been studied extensively for their potential roles in regenerating tissues or organs. The acellular matrix generates physiological cues that mimic the native tissue microenvironment. Acellular dermal matrix (ADM) is a soft connective tissue graft generated by a decellularization process that preserves the intact extracellular skin matrix. Upon implantation, this structure serves as a scaffold for donor-side cells to facilitate subsequent incorporation and revascularization. In breast reconstruction, ADM is used mainly for lower pole coverage and the shaping of a new breast. It helps control the positioning of the implant in the inframammary fold, and prevent the formation of contractile pseudocapsule around the breast implant. In this study, we provide a comprehensive histological description of ADM used for human breast reconstruction over the course of several months following implementation. Using immunohistochemical methods (a panel of 12 antibodies) coupled with optical and transmission electron microscopy, we confirmed that the original acellular dermal matrix became recolonized by fibroblasts and myofibroblasts, and also by various other free cells of the connective tissue (lymphocytes, macrophages and multinucleated giant cells, granulocytes, mast cells) after implantation into the patient's body. Within the implanted ADM, there was a relatively rapid ingrowth of blood vessels. Lymphatic vessels were only detected in one case 9 months after the implantation of the ADM. These results suggest that lymphangiogenesis is a longer process than angiogenesis.

Eschar removal by bromelain based enzymatic debridement (Nexobrid®) in burns: An European consensus.

Early debridement and/or eschar removal is regarded as a significant step in the treatment of deep partial and full thickness burns. It aims to control wound bioburden and allows early wound closure by conservative treatment or skin grafting. Preservation of viable dermis accompanied by early wound closure, is regarded as a necessary step to reduce scar related complication, e.g. functional limitations and/or unaesthetic scar formation. Aside from the classical techniques of surgical excision as tangential excision for eschar removal, hydro-surgery, maggot therapy, laser, enzymatic debridement have been described as additional techniques in the burn surgeon's armamentarium. It is widely accepted that early eschar removal within 72h improves the outcome of burn wound treatment by reducing bacterial wound colonization, infection and length of hospital stay. In contrast, the right technique for eschar removal is still a matter of debate. There is increasing evidence that enzymatic debridement is a powerful tool to remove eschar in burn wounds, reducing blood loss, the need for autologous skin grafting and the number of wounds requiring surgical excision. In order to assess the role and clinical advantages of enzymatic debridement by a mixture of proteolytic enzymes enriched in Bromelain (Nexobrid®) beyond the scope of the literature and in view of users' experience, a European Consensus Meeting was scheduled. The aim was to provide statements for application, based on the mutual experience of applying enzymatic debridement in more than 500 adult and pediatric patients by the consensus panelists. Issues to be addressed were: indications, pain management and anesthesia, timing of application, technique of application, after-intervention care, skin grafting after enzymatic debridement, blood loss, training strategies and learning curve and areas of future research needs. Sixty-eight (68) consensus statements were provided for the use of enzymatic debridement. The degree of consensus was remarkably high, with a unanimous consensus in 88.2% of statements, and lowest degree of consensus of 70% in only 3 statements. This consensus document may serve as preliminary guideline for the use of enzymatic debridement with user-oriented recommendations until further evidence and systematic guidelines are available.

Electrical measurement of the absorption surfaces of tree roots by the earth impedance method: 1. Theory.

Analysis of plant root systems is difficult under field conditions, especially root systems of adult trees, which are large and complex and include fine absorbing roots as well as conducting coarse roots. Although coarse roots can be visualized by several methods, there are technical difficulties with root quantification. The method presented here focuses on the quantification of absorbing root surfaces through an electrical (the modified earth impedance) method. It is based on the experimentally verified fact that an applied electric current flows from the roots to the soil (or vice versa) through the same interfacial areas and predominantly in the same way as water (water solution of minerals or nutrients) flows from the soil to the tree. Based on the different conductivities of tree tissues and soil, the interfacial area, which represents the absorbing root surfaces (or root absorption zones), can be calculated. Only the theoretical description of the method is presented in this paper: the experimental verification of the method under field conditions is presented in the accompanying paper.