bromochloroacetic-acid and Burns

Severe illness or trauma alters the body's metabolic rate. After injury, host-defence protein synthesis and increased energy requirements are satisfied from available protein, usually active muscle tissue. A prolonged hypercatabolic state persists and may lead to increased morbidity and mortality in severely burned patients. Growth hormone (GH) is an anabolic agent shown to decrease some of the deleterious effects of hypermetabolism. This article will review the effects of GH on burn wound repair and gut healing. Studies on GH have shown a significant reduction in wound-healing times in burned patients given GH at a dose of 0.6 IU/kg/day (0.2 mg/kg/day). At this dose, other studies have shown no increase in mortality, and a number of beneficial effects in critically burned children have been demonstrated. Animal studies have suggested that insulin-like growth factor I (IGF-I), stimulated through the GH axis, plays an important role in the reconstitution of intestinal epithelial integrity following mucosal injury. Many encouraging papers report positive results regarding both the efficacy and safety of GH and IGF-I, therefore warranting continued investigation.

Topics: Adolescent; Burns; Child; Child, Preschool; Cicatrix; Collagen; Dose-Response Relationship, Drug; Double-Blind Method; Epithelial Cells; Female; Growth Hormone; Human Growth Hormone; Humans; Insulin-Like Growth Factor I; Intestinal Mucosa; Keratins; Laminin; Male; Wounds and Injuries

Deep partial-thickness burns damage most of the dermis and can cause severe pain, scarring, and mortality if left untreated. This study serves to evaluate the effectiveness of crosslinked keratin-alginate composite sponges as dermal substitutes for deep partial-thickness burns. Crosslinked keratin-alginate sponges were tested for the ability to support human dermal fibroblasts in vitro and to support the closure and healing of partial-thickness burn wounds in

Topics: Alginates; Animals; Bandages, Hydrocolloid; Burns; Cells, Cultured; Dermis; Humans; Hydrogels; Keratins; Male; Materials Testing; Severity of Illness Index; Skin; Swine; Wound Healing

Keratin is a natural material that can be derived from the cortex of human hair. Our group had previously presented a method for the printed, sequential production of three-dimensional (3D) keratin scaffolds. Using a riboflavin-sodium persulfate-hydroquinone (initiator-catalyst-inhibitor) photosensitive solution, we produced 3D keratin-based constructs through ultraviolet crosslinking in a lithography-based 3D printer. In this study, we have used this bioink to produce a keratin-based construct that is capable of delivering small molecules, providing an environment conducive to healing of dermal burn wounds

Topics: Animals; Burns; Hydrogels; Keratins; Models, Animal; Printing, Three-Dimensional; Swine; Wound Healing

This study demonstrates that adipose-derived stem cells from debrided skin (dsASCs) of burn patients can be isolated in sufficient quantities and differentiated into cytokeratin-expressing cells by treating them with all-trans retinoic acid (ATRA) and the peroxisome proliferator-activated receptor-α (PPARα) specific activator fenofibrate. Differentiation of dsASCs with ATRA and a combination of growth factors induced expression of simple epithelial markers (KRT7, KRT8, KRT18, and KRT19), along with low levels of stratified epithelial markers (KRT5, KRT10, KRT13, and KRT14). We have optimized a condition to induce dsASCs differentiation to epithelial cells by treatment with ATRA and fenofibrate alone. Real-time polymerase chain reaction analysis showed a significant increase in transcript levels (>75-fold) for basal (KRT5 and KRT14), suprabasal (KRT10), and cornified envelope markers (involucrin [IVL] and Loricrin [LOR]) with this treatment. Expression of the proteins encoded by these transcripts was confirmed by immunocytochemical analysis. Further, we show that dsASCs differentiated to a skin epithelial cell phenotype through activation of nuclear hormone receptors PPARα and RXRγ. Collectively this study shows that dsASCs can be differentiated to skin epithelial cells, without the requirement for exogenous growth factors. This differentiation protocol using dsASCs in combination with an appropriate biocompatible scaffold can be adapted to develop epithelial skin substitute for burn wound treatment.

Topics: Adipose Tissue; Burns; Cell Differentiation; Cells, Cultured; Epithelial Cells; Humans; Immunohistochemistry; Keratin-18; Keratin-19; Keratin-7; Keratin-8; Keratins; PPAR alpha; Stem Cells; Tretinoin

In the Present-day medicinally applied wound bandages have many drawbacks for, instance, rigidity, non-porosity, low mechanical potency, also an affinity for bandages to stick onto the injury exterior; additionally, a greater part of the bandages did not secure bactericidal activity. Hydrogel derived injury bandages would be supportive to afford a chill feeling with a humidity atmosphere, in addition, to performing as an obstruction to bacteria. To overcome these drawbacks, we have fabricated porous keratin-chitosan/n-ZnO nanocomposite (KCBZNs) bandages via the inclusion of nano-ZnO into the keratin-chitosan hydrogel. The functional group and surface of as-fabricated bandages were characterized using Fourier transform infrared spectroscopy (FT-IR), X-ray diffractometry (XRD), and scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDX) and transmission electron microscopy with selected area diffraction (TEM-SAD). Moreover, mechanical, swelling, bactericidal, bio-compatibility of nanocomposite was assessed to exhibit its efficacy for biological applications. The nanocomposite illustrated increased swelling, and bactericidal activity. Bio-compatibility of the nanocomposite has been investigated in normal human fibroblast cells. Also, the in vivo assessments in SD rats exposed that as-fabricated nanocomposite bandages increased the wound curing with assisted for quicker skin cell construction along with collagen development. Hence, the acquired information strongly supports to utilize of this nanocomposite hydrogels for burn wounds.

Topics: Animals; Anti-Infective Agents; Bandages; Burns; Cell Line; Chitosan; Elastic Modulus; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; Hydrogels; Keratins; Microscopy, Electron, Transmission; Nanocomposites; Porosity; Rats; Rats, Sprague-Dawley; Spectrometry, X-Ray Emission; Spectroscopy, Fourier Transform Infrared; Wound Healing; Zinc Oxide

Autologous cell-spray grafting of non-cultured epidermal cells is an innovative approach for the treatment of severe second-degree burns. After treatment, wounds are covered with dressings that are widely used in wound care management; however, little is known about the effects of wound dressings on individually isolated cells. The sprayed cells have to actively attach, spread, proliferate, and migrate in the wound for successful re-epithelialization, during the healing process. It is expected that exposure to wound dressing material might interfere with cell survival, attachment, and expansion. Two experiments were performed to determine whether some dressing materials have a negative impact during the early phases of wound healing. In one experiment, freshly isolated cells were seeded and cultured for one week in combination with eight different wound dressings used during burn care. Cells, which were seeded and cultured with samples of Adaptic(®), Xeroform(®), EZ Derm(®), and Mepilex(®) did not attach, nor did they survive during the first week. Mepitel(®), N-Terface(®), Polyskin(®), and Biobrane(®) dressing samples had no negative effect on cell attachment and cell growth when compared to the controls. In a second experiment, the same dressings were exposed to pre-cultured cells in order to exclude the effects of attachment and spreading. The results confirm the above findings. This study could be of interest for establishing skin cell grafting therapies in burn medicine and also for wound care in general.

Topics: Antigens, CD34; Apoptosis; Burns; Caspase 3; Cell Culture Techniques; Cell Survival; Cell Transplantation; Cells, Cultured; Coated Materials, Biocompatible; fas Receptor; Gene Expression; Humans; In Vitro Techniques; Integrin alpha6; Keratinocytes; Keratins; Ki-67 Antigen; Membrane Proteins; Microscopy, Phase-Contrast; Occlusive Dressings; Phenols; Re-Epithelialization; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Silicones; Wound Healing

This n=40 cohort study on superficial and partial thickness burns compares novel keratin-based products with the standard products used at our facility. The keratin products are found to facilitate healing with minimal scarring, be well tolerated with minimal pain and itch, be easy to use for the health professional and be cost effective for the health care provider. For these reasons they are being adopted into use at our facility.

Topics: Adolescent; Bandages; Burns; Case-Control Studies; Child; Child, Preschool; Cicatrix; Coated Materials, Biocompatible; Cohort Studies; Female; Humans; Keratins; Male; Petrolatum; Polyesters; Polyethylenes; Re-Epithelialization; Silicones; Trauma Severity Indices

Infection is a leading cause of morbidity and mortality in burn patients. Current therapies include silver-based creams and dressings, which display limited antimicrobial effectiveness and impair healing. The need exists for a topical, point-of-injury antibiotic treatment that provides sustained antimicrobial activity without impeding wound repair. Fitting this description are keratin-based hydrogels, which are fully biocompatible and support the slow-release of antibiotics. Here we develop a porcine model of an infected partial-thickness burn to test the effects of ciprofloxacin-loaded keratin hydrogels on infection and wound healing. Partial-thickness burns were inoculated with either Pseudomonas aeruginosa or Methicillin-resistant Staphylococcus aureus, resulting in infections that persisted for >2 weeks that exceeded 10(5) and 10(6) cfu per gram of tissue, respectively. Compared to silver sulfadiazine, ciprofloxacin-loaded keratin hydrogel treatment significantly reduced the amount of P. aeruginosa and S. aureus in the burn by >99% on days 4, 7, 11, and 15 postinjury. Further, burns treated with ciprofloxacin-loaded keratin hydrogels exhibited similar healing patterns as uninfected burns with regards to reepithelialization, macrophage recruitment, and collagen deposition and remodeling. The ability of keratin hydrogels to deliver antibiotics to fight infection and support healing of partial-thickness burns make them a strong candidate as a first-line burn therapy.

Topics: Animals; Burns; Ciprofloxacin; Disease Models, Animal; Drug Carriers; Female; Hydrogels; Keratins; Methicillin-Resistant Staphylococcus aureus; Pseudomonas aeruginosa; Swine; Wound Healing; Wound Infection

Partial thickness burns can advance to full thickness after initial injury due to inadequate tissue perfusion and increased production of inflammatory cytokines, which has been referred to as burn wound progression. In previous work, we demonstrated that a keratin biomaterial hydrogel appeared to reduce burn wound progression. In the present study, we tested the hypothesis that a modified keratin hydrogel could reduce burn wound progression and speed healing. Standardized burn wounds were created in Yorkshire swine and treated within 30 minutes with keratin hydrogel (modified and unmodified), collagen hydrogel, or silver sulfadiazine (SSD). Digital images of each wound were taken for area measurements immediately prior to cleaning and dressing changes. Wound tissue was collected and assessed histologically at several time points. Wound area showed a significant difference between hydrogels and SSD groups, and rates of reepithelialization at early time points showed an increase when keratin treatment was used compared to both collagen and SSD. A linear regression model predicted a time to wound closure of approximately 25 days for keratin hydrogel while SSD treatment required 35 days. There appeared to be no measurable differences between the modified and unmodified formulations of keratin hydrogels.

Topics: Animals; Burns; Disease Models, Animal; Hydrogels; Keratins; Silver Sulfadiazine; Swine; Wound Healing

Thermal burns typically display an injury pattern dictated by the transfer of the thermal energy into the skin and underlying tissues and creation of three zones of injury represented by a necrotic zone of disrupted cells and tissue, an intermediate zone of injured and dying cells, and a distant zone of stressed cells that will recover with proper treatment. The wound healing capabilities of a keratin biomaterial hydrogel were studied in two pilot studies, one using a chemical burn model in mice and the other a thermal burn model in swine. In both studies, keratin was shown to prevent enlargement of the initial wound area and promote faster wound closure. Interestingly, treating thermally stressed dermal fibroblast in culture demonstrated that soluble keratin was able to maintain cell viability and promote proliferation. Separation of so-called alpha and gamma fractions of the keratin biomaterial had differential effects, with the gamma fraction producing more pronounced cell survival and recovery. These results suggest that the gamma fraction, composed essentially of degraded alpha keratin proteins, may facilitate cell rescue after thermal injury. Treatment of burns with gamma keratin may therefore represent a potential therapy for wounds with an intermediate zone of damaged tissue that has the potential to contribute to spontaneous healing.

Topics: Animals; Biocompatible Materials; Burns; Burns, Chemical; Cells, Cultured; Humans; Hydrogels; Keratins; Materials Testing; Mice; Pilot Projects; Regeneration; Skin Physiological Phenomena; Sus scrofa; Wound Healing

When skin is thermally burned, transfer of heat energy into the skin results in the destruction of cells. Some of these cells are damaged but may be capable of self-repair and survival, thereby contributing to spontaneous healing of the wound. Keratin protein-based biomaterials have been suggested as potential treatments for burn injury. Isolation of cortical proteins from hair fibers results in an acid soluble fraction of keratin proteins referred to as "gamma" keratose. In the present study, treatment with this fraction dissolved in media was able to maintain cell viability after thermal stress in an in vitro model using primary mouse dermal fibroblasts. PCR array analysis demonstrated that gamma keratose treatment may assist in the survival and salvage of thermally stressed cells by maintaining their viability through regulation of cell death pathway-related genes. Gamma keratose may be a promising biomaterial for burn treatment that aids in spontaneous wound healing from viable tissue surrounding the burn.

Topics: Animals; Burns; Cell Survival; Cells, Cultured; Fibroblasts; Gene Expression Regulation; Keratins; Mice; Signal Transduction; Wound Healing

Epithelioid sarcoma (ES) is a rare, aggressive soft tissue tumor with a characteristic predilection for adolescents and young adults, and a tendency to occur on distal extremities. We report a case of ES arising in an 80-year-old woman within a burn scar that histopathologically showed unusual 'angiomatoid' features. The patient presented initially with a solitary nodule on her right wrist arising at the site of a burn scar. Histopathologically, the tumor was composed of a proliferation of relatively bland, epithelioid and spindle cells focally arranged in a nodular pattern around areas of 'geographic' necrosis. In addition, there were prominent foci of hemorrhage and blood-filled spaces as well as tumor cells with intracytoplasmic vacuoles, features suggestive of an angiomatous process. Immunohistochemistry showed positivity of tumor cells for cytokeratins and epithelial membrane antigen (EMA) whereas all vascular markers tested were negative. The overall histopathologic features were consistent with a diagnosis of ES. Follow up showed multiple recurrences arising proximally along the right upper extremity. Our case underlines the clinical and histopathological heterogeneity of ES, emphasizing the unusual occurrence of ES with 'angiomatoid' features in the elderly. In this uncommon setting, this tumor should be especially distinguished from epithelioid hemangioendothelioma and epithelioid angiosarcoma. The significance of development of ES on a healed burn scar is uncertain, but may suggest a possible causal relationship.

Topics: Aged, 80 and over; Angiomatosis; Biomarkers, Tumor; Burns; Cicatrix; Female; Humans; Keratins; Mucin-1; Neoplasm Recurrence, Local; Sarcoma; Soft Tissue Neoplasms; Wrist

To develop a composite material containing human hair keratin (HHK), collagen sponge (inner layer) and poly 2-hydroxyethyl methacrylate (PHEMA) film that allows sustained release of polydatin and test its effect as a biological dressing in promoting burn wound healing in SD rats.. Three HHK materials with fast, moderate, and low degradation rates were mixed at the ratio of 4:3:3 to prepare a reticular structure, which was processed into a composite material with bovine tendon-derived collagen sponge, and further complexed with HEMA film containing PD prepared by polymerization. Degree II burn wound was induced in SD rats by scalding and within postburn day 2-5, the wounds were cleansed and covered with the composite material or with glutaraldehyde-treated porcine skin (positive control). At week 1, 2, 4, 6 and 8 following wound dressing, 6 full-thickness skin samples were harvested from the wounds for histological observation and immunohistochemical detection of collagen and elastic fibers, and the wound healing time and healing rate were recorded.. The prepared collagen sponge film was transparent and porous (50-300 microm in diameter) and allowed sustained PD release into normal saline within 48 h. Compared with the porcine skin, the composite material reduced exudation and maintained ideal moisture of the wound, and significantly shortened the wound healing time (P=0.000). On day 7, 14, and 21 following dressing, the composite material and porcine skin significantly increased the wound healing rate as compared with the negative control group (P=0.000), and on day 14, the composite achieved significantly greater healing rate than the porcine skin (P<0.05).. HHK-collagen sponge-PHEMA/PD composite as a dressing material promotes burn wound healing in rats by allowing in vivo construction of tissue engineered epidermis. PHEMA is feasible for sustained drug delivery in this composite.

Topics: Animals; Biological Dressings; Burns; Cattle; Collagen; Drug Delivery Systems; Drugs, Chinese Herbal; Glucosides; Humans; Keratins; Polyhydroxyethyl Methacrylate; Rats; Rats, Sprague-Dawley; Stilbenes; Swine; Tissue Engineering; Wound Healing

Cultured skin substitutes, consisting of fibroblasts and keratinocytes in a biopolymer matrix, are an adjunctive treatment for full thickness burn wounds. Previous studies revealed that cultured skin substitutes in vitro exhibit a gene expression profile similar to hyperproliferative skin or wounded normal skin. In the present study, we sought to determine whether this hyperproliferative phenotype is maintained after healing of grafted cultured skin in vivo. Immunohistochemistry was used to localize multiple keratin proteins in native human skin, and in cultured skin substitutes in vitro and after grafting to athymic mice. Keratin 6, keratin 16, and keratin 17, which are known to be upregulated during keratinocyte activation and in hyperproliferative epidermis, were highly expressed in cultured skin substitutes in vitro. These proteins were low or undetectable in native human skin, and were reduced in cultured skin after grafting. Conversely, keratin 15, which is downregulated in activated keratinocytes, was not detected in cultured skin substitutes in vitro but was upregulated after grafting to mice. The results confirm previous observations suggesting a hyperproliferative or activated phenotype in cultured skin substitutes in vitro, similar to wounded native skin. After grafting to athymic mice, the expression patterns suggest a normalization of cultured skin substitutes to a phenotype more closely resembling uninjured human skin.

Topics: Adult; Animals; Burns; Cells, Cultured; Female; Fibroblasts; Graft Survival; Humans; Immunohistochemistry; Keratinocytes; Keratins; Mice; Mice, Nude; Phenotype; Skin Transplantation; Skin, Artificial; Treatment Outcome

In hypertrophic scar tissue, no sweet gland and hair follicle exist usually because of the dermal and epidermal damage in extensive thermal skin injury, thus imparing regulation of body temperature. This study was designed to reveal the morphological and distributional characteristics of the sweat glands in normal skin and hypertrophic scar obtained from children and adults, and to study the possible interfering effects of the scar on regeneration of the sweat gland after burn injury.. Biopsies of hypertrophic scar were taken from four children (4 - 10 years) and four adults (35 - 51 years). Normal, uninjured full-thickness skin adjacent to the scar of each patient was used as control. Keratin 19 (K19) was used as the marker for epidermal stem cells and secretory portion of the sweat glands, and keratin 14 (K14) for the tube portion, respectively. Immunohistochemical and histological evaluations were performed.. Histological and immunohistochemical staining of skin tissue sections from both the children and adults showed K19 positive cells in the basement membrane of epidermis of normal skin. These cells were seen only single layer and arranged regularly. The secretory or duct portion of the eccrine sweat glands was situated in the dermis and epidermal layer. However, in the scar tissue, K19 positive cells were scant in the basal layer, and the anatomic location of the secretory portion of sweat glands changed. They were located between the border of the scar and reticular layer of the dermis. These secretory portions of sweat glands were expanded and were organized irregularly. But a few K14 positive cells were scattered in the scar tissues in cyclic form.. There are some residual sweat glands in scar tissues, in which the regeneration process of active sweat glands is present. Possibly the sweat glands could regenerate from adult epidermal stem cells or residual sweat glands in the wound bed after burn injury.

Topics: Adult; Burns; Child; Child, Preschool; Cicatrix, Hypertrophic; Epidermal Cells; Humans; Immunohistochemistry; Keratin-14; Keratins; Middle Aged; Regeneration; Skin; Stem Cells; Sweat Glands

Inappropriate treatment at early stage of wound could result in the formation of pseudoepitheliomatous granuloma (PEG). The correlation of abnormal transdifferentiation of epithelial cells to immunologic cells and the occurrence of PEG lesion was investigated.. Morphological change of epithelial tissue was observed with histopathology in 11 specimens of PEG lesions and 6 specimens of normal skins from PEG edge (PEG-N) from 11 patients with damaged skin. The expression characteristics and distribution of pan-cytokeratin (CKp), IV type collagen, laminin (LM), epithelial cadherin (E-Cad), beta-catenin (beta-Cat), focal adhesion kinase (FAK), stem cell factor (SCF) and its receptor-c-Kit, proliferating cell nuclear antigen(PCNA), and cluster of differentiation-14 (CD14), CD68 and mast cell tryptase (MCT) in PEG were detected with the immunohistochemical and the indirect immunofluorescent double-staining.. In comparison with PEG-N, epithelial tissue take on squamous metaplasia, and stroma was infiltrated with intensive microvessels and inflammatory cells in the PEG lesion. Poor epithelial basal layer constitution, basal polarization, and migration of basal cells to stroma could be observed. In the ultrastructure, the loose intercellular junction of basal cells and the increased nucleus/cytoplasm ratio and intercellular space could be observed, neonatal monocytoid cells and macrophages and mast cells as a exuviate-like manner brooded from cytoplasm of original epithelial cells and basement membrane. protein expression of CKp and E-Cad by basal cells was significantly decreased, and the IV type collagen and LM protein could not be found in basement membrane of identical locus. By contrast, the immunoreactivity of beta-Cat and FAK was apparently increased. In addition, CD14(+) monocytes, CD68(+) macrophages, MCT(+) mast cells and CD68(+)/MCT(+) cells with various size, and these cells of stronger immuno-staining of SCF, c-Kit and PCNA antigen could be found in epithelial tissue and stroma.. Epithelial cells in PEG related to wound are characteristized by transdifferentiation of epithelial cells to immunologic cells, wich may be associated with local infectious and inflammatory reaction, ultimately resulting in enhancement the ratio of beta-Cat/E-Cad signal and activation SCF-c-Kit signal pathway. The phenomena of transdifferentiation epithelial cells in the PEG lesion will help to recognize of the neoplatic immune and trauma repair mechanism.

Topics: Adolescent; Adult; Aged; Antigens, CD; Antigens, Differentiation, Myelomonocytic; beta Catenin; Burns; Cadherins; Cell Differentiation; Child; Child, Preschool; Collagen Type IV; Cytoskeletal Proteins; Epithelial Cells; Female; Fluorescent Antibody Technique, Indirect; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Granuloma; Humans; Immunohistochemistry; Infant; Keratins; Laminin; Lipopolysaccharide Receptors; Male; Middle Aged; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-kit; Serine Endopeptidases; Skin; Stem Cell Factor; Trans-Activators; Tryptases

Keratin is a major protein produced during epithelialization following burn injury and is a useful marker for assessing wound healing. Transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the keratin 5 (K5) promoter (K5GFP mice) were used to monitor keratin expression, and thus, re-epithelialization of burn wounds.. K5GFP transgenic mice were created using conventional techniques, with PCR and Southern blot confirmation of transgene incorporation, followed by selection of the line with the most intense and consistent basal epithelial EGFP expression. Epi-fluorescent microscopy of 24 K5GFP mouse flanks and 10 negative littermate controls was used to characterize EGFP intensity, before wounding and serially for 30 days after administration of a standardized burn wound and excision. Biopsy sections of K5GFP and negative control mice were stained with K5 antibody and imaged with confocal microscopy to characterize the distribution of EGFP and K5 at baseline and after injury and to examine the correlation between K5 expression and EGFP expression during healing.. Green fluorescence intensity increased at the advancing wound margin of burned K5GFP mice, reaching a maximum between days 12 and 15 post-burn and then decreasing as healing completed. K5 and EGFP expression increased in parallel in burned K5GFP mice as demonstrated by confocal microscopy.. EGFP expression correlated with K5 expression during wound healing and therefore serves as a good marker of re-epithelialization. This transgenic model allows noninvasive, real-time assessment of in vivo K5 expression and will be useful in the study of wound healing.

Topics: Animals; Burns; Female; Green Fluorescent Proteins; Keratin-15; Keratin-5; Keratins; Luminescent Proteins; Male; Mice; Mice, Transgenic; Models, Animal; Monitoring, Physiologic; Skin; Wound Healing

Our group has previously shown that the intestinal epithelium exhibits increased postburn barrier permeability and bacterial translocation associated with deranged neutrophil activity. The purpose of this investigation is to explore possible underlying intestinal structural mechanisms, leading to those functional changes with emphasis on (1) neutrophil influx and extravasation in the intestinal lamina propria 1-3 days after burn and (2) enterocyte proliferation, migration, apoptosis, and E-cadherin junctional epithelium levels 3 days after burn.. Freshly isolated ileum specimens were quick frozen, then cut by a cryostat into 30-micron-thick sections. Sections from day 1 postburn rats were immunostained with (1) anti-granulocyte or anti-elastase antibodies to assess neutrophil influx or (2) combined anti-granulocyte and anti-von Willebrand factor double immunolabeling to compare levels of neutrophil extravasation. Sections from day 3 postburn rats were immunostained with (1) bromodeoxyuridine immunohistochemistry 1, 3, 6, or 18 hrs after bromodeoxyuridine injection to assess enterocyte proliferation and migration, (2) cytokeratin-18 M30-immunohistochemistry to compare levels of enterocyte apoptosis, and (3) E-cadherin immunohistochemistry to compare junctional E-cadherin integrity. Ileal myeloperoxidase activity and bacterial translocation of Enterococcus faecalis were assessed biochemically and by E. faecalis-specific bacterial cultures, respectively, in day 3 postburn rats.. : Research laboratories in a medical center and an academic institution.. Male Sprague-Dawley rats given sham treatment or treatment as a burn model with full-thickness skin scald over 30% total body surface area.. We report (1) increased levels of neutrophil influx and extravasation in villi lamina propriae, including elastase-positive cells (postburn day 1), (2) heightened levels of intestinal myeloperoxidase activity (postburn day 3), (3) decreased levels of epithelial cell proliferation, migration, and E-cadherin (postburn day 3), and (4) increased enterocyte apoptosis and E. faecalis bacterial translocation (postburn day 3). Based on these structural and functional abnormalities, we propose a mechanism for burn injury-related intestinal barrier dysfunction that includes increased trans- and para-cellular leakage caused by impaired enterocyte renewal and decreased junctional E-cadherin levels subsequent to increased neutrophil influx and extravasation within the villus lamina propria microenvironment.

Topics: Animals; Bacterial Translocation; Burns; Cadherins; Disease Models, Animal; Enterococcus faecalis; Enterocytes; Ileum; Intestinal Mucosa; Keratins; Male; Neutrophils; Rats; Rats, Sprague-Dawley

To investigate the different location and the expression characteristics of epidermal stem cells in normal adult skin and scar tissue.. Skin tissue specimens were harvested from the corresponding sites from 6 healthy volunteers and from scar tissue of 6 patients 1 year after major deep burn. beta1 integrin and keratin 19 (K19) were employed as the biochemical markers for stem cells and transit amplifying cells identification and keratin 14 (K14) and keratin 10 (K10) as markers for post-mitotic cells and terminally differentiated cells respectively. Integrin and keratin were determined by Elivision two-step immunohistochemistry.. The expression of beta1 integrin and the K19 positive cell count in the epithelial basal layers of scar tissue were evidently decreased and weakened than those in normal adult healthy skin. Furthermore, the positive cells expressing K14 in epidermis of scar tissue were only located in 2 - 3 layers of basal epidermis, and their number was much less than that in normal adult skin. Whereas the cells positively expressing K10 were distributed wider in area than that in normal healthy skin. The epidermal stem cells and transit amplifying cells in scar epidermis were much less in number than that in normal skin. The differentiation process of scar epidermal stem cells was different from that of normal skin. And the proportions of post-mitotic cells and terminally differentiated cells were abnormal.. The results indicated that the self-renewal ability of the scar epidermis was decreased, and the differentiation process of it was in disorder, which may be a reason for the abnormality of structure and function of the epidermis in scar, and a reason for the decreased ability of wound healing of scar tissue.

Topics: Adult; Burns; Cicatrix; Epidermal Cells; Epidermis; Humans; Immunohistochemistry; Integrin beta1; Keratin-10; Keratin-14; Keratins; Middle Aged; Skin; Stem Cells

Fibrin glue is an excellent template for cellular migration and has been shown to be an effective delivery system for cultured autologous keratinocytes. We have investigated whether fibrin glue has any benefit on the percentage of epithelial cover when cultured autologous keratinocytes are sprayed onto a freshly debrided wound bed. Three pigs were used for this study. This provided a total of 18 full thickness, vertically orientated wounds, each 4cm in diameter and isolated in PTFE chambers to prevent re-epithelialisation from the wound margins. Eight wounds were sprayed with cultured autologous keratinocytes suspended in 2ml culture medium and eight wounds were sprayed with cultured autologous keratinocytes suspended in 1ml of the fibrin/aprotinin component of Tisseel fibrin glue (Baxter) mixed with 1ml of culture medium. In the latter group the thrombin component of the fibrin glue kit was applied to the wound bed immediately prior to grafting. The remaining two wounds were used as controls and sprayed with either culture medium or fibrin glue without cells. Epithelial cover was calculated in whole-wound biopsies at 3 weeks using image analysis, histology and immunohistochemistry. The cell suspension in fibrin glue appeared to spread more evenly over the wound surface, with no pooling in the inferior aspect of the wound. However, mean epithelial area at 3 weeks in the fibrin group was 1.6cm(2) per wound compared with 1.8cm(2) for the non-fibrin group, as measured by image analysis of digital photographs. There was no statistically significant difference between the two groups (P=0.802). This surprising result was confirmed by histological analysis of the wound biopsies, with a good correlation between histological and image analysis data (R=0.967). There was no observable difference in the quality of the epithelium on histological and immunohistological analysis of either group.

Topics: Aerosols; Animals; Burns; Cells, Cultured; Collagen Type VII; Epithelium; Female; Fibrin Tissue Adhesive; Image Processing, Computer-Assisted; Keratin-14; Keratinocytes; Keratins; Skin; Skin Transplantation; Swine; Wound Healing

To investigate the distribution of epidermal stem cells (ESCs) in different degrees of burn wounds in scalded rats.. Thirty-two Sprague-Dawley (SD) rats were employed in the study. First degree (I), shallow (shallow II) and deep partial thickness (deep II) and full thickness burn wounds (III) were created on the rat skin. Burn wound samples were harvested at 24 postburn hours (PBHs) from all the wounds and were processed to tissue slices. The tissue slices were stained by immunohistochemistry technique. The expression and distribution of ESCs in different degrees of burn wounds were observed with integrins alpha 2 beta 1 and keratin 10 (K10) as first antibodies.. K10 positive cells were found to distribute in the strata spinosum, granulosum and lucidum in the first degree burn wound (I) with large amounts of integrins alpha 2 beta 1 positive cells in the residual basal layer and skin appendages (hair follicles) in shallow partial thickness burn wound (shallow II degree), and there were less integrins alpha 2 beta 1 positive cells in the remaining skin appendages in deep dermis in deep partial thickness burn wound (deep II degree). Finally, integrins alpha 2 beta 1 positive cells were sparsely found in the III degree burn wound.. The distribution of ESCs in burn wounds was closely related to the depth of burn wound. The residual ESCs might be the origin of burn wound regeneration and reepithelization.

Topics: Animals; Burns; Female; Immunohistochemistry; Integrin alpha2beta1; Keratin-10; Keratins; Male; Rats; Rats, Sprague-Dawley; Stem Cells

RHPS, composed of confluent allogeneic keratinocytes cultured on cell-free pig dermis, stimulates wound healing when applied with the keratinocyte layer facing the wound. So far it has not been clarified whether the confluent keratinocytes implanted 'upside-down' can 'take' or only stimulate healing by producing growth factors. Confluent male keratinocytes were grafted onto donor sites of three female patients. Biopsies were taken on days 4, 6 and 9 after grafting. The fate of donor cells was followed in paraffin sections by FISH for the Y chromosome and by persisting expression of vimentin taken as a marker of cultured keratinocytes. Histological evaluation was complemented by detection of keratin 10 and involucrin. All three donor sites healed within one week. On day 4 the early neoepidermis was multilayered but disordered after transplantation. A large proportion of cells were apparently of donor origin as indicated by the presence of Y chromosomes, irregular morphology, expression of vimentin in the bottom and upper layers of the neoepidermis, and by irregular expression of involucrin and keratin 10 only in the central layer of the neoepidermis. From day 6 onwards, the new epidermis acquired an ordered stratification. Involucrin and keratin 10 renewed normal distribution in suprabasal layers. Concomitantly, vimentin expression was decreasing. The Y chromosome was still found on day 6 but not on day 9. We concluded that confluent allogeneic keratinocytes temporarily 'take' to the wound and contribute to rapid wound closure, being replaced by the patient's epidermal cells after about one week.

Topics: Adult; Animals; Biomarkers; Burns; Cell Survival; Cells, Cultured; Coculture Techniques; Dermis; Female; Graft Survival; Humans; In Situ Hybridization, Fluorescence; Keratin-10; Keratinocytes; Keratins; Male; Protein Precursors; Swine; Tissue Engineering; Transplantation, Homologous; Transplants; Vimentin; Wound Healing; Y Chromosome

Transforming growth factor-beta (TGF-beta) isoforms are multifunctional cytokines that play an important role in wound healing. Transgenic mice overexpressing TGF-beta in the skin under control of epidermal-specific promoters have provided models to study the effects of increased TGF-beta on epidermal cell growth and cutaneous wound repair. To date, most of these studies used transgenic mice that overexpress active TGF-beta in the skin by modulating the latency-associated-peptide to prevent its association with active TGF-beta. The present study is the first to use transgenic mice that overexpress the natural form of latent TGF-beta 1 in the epidermis, driven by the keratin 14 gene promoter to investigate the effects of locally elevated TGF-beta 1 on the healing of partial-thickness burn wounds made on the back of the mice using a CO(2) laser. Using this model, we demonstrated activation of latent TGF-beta after wounding and determined the phenotypes of burn wound healing. We found that introduction of the latent TGF-beta1 gene into keratinocytes markedly increases the release and activation of TGF-beta after burn injury. Elevated local TGF-beta significantly inhibited wound re-epithelialization in heterozygous (42% closed versus 92% in controls, P < 0.05) and homozygous (25% versus 92%, P < 0.01) animals at day 12 after wounding. Interestingly, expression of type I collagen mRNA and hydroxyproline significantly increased in the wounds of transgenic mice, probably as a result of a paracrine effect of the transgene.

Topics: Animals; Burns; Cell Division; Collagen Type I; Epidermis; Gene Expression Regulation; Humans; Hydroxyproline; Immunohistochemistry; Keratin-14; Keratinocytes; Keratins; Lasers; Mice; Mice, Transgenic; Promoter Regions, Genetic; Recombinant Fusion Proteins; RNA, Messenger; Skin; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transgenes; Wound Healing

The reconstruction of epidermal architecture over time in normotrophic and hypertrophic scars in untransplanted, spontaneously healed partial-thickness burns has scarcely been studied, unlike the regeneration of epidermal grafts used to cover burn wounds and the regeneration of the dermis during hypertrophic scarring. The expression of markers of epidermal proliferation, differentiation and activation in normotrophic and hypertrophic scars in spontaneously healed partial-thickness burns was assessed and compared with the expression of these markers in normal control skin of healthy persons to determine whether hypertrophic scarring is associated with abnormalities in the phenotype of keratinocytes. Punch biopsies were taken both of partial-thickness burns after re-epithelialisation and of matched unburned skin. At 4 and 7 months post-burn, biopsies were taken of normotrophic and hypertrophic scars that had developed in these wounds. The biopsies were analysed using immunostaining for markers of keratinocyte proliferation, differentiation and activation (keratins 5, 10, 16 and 17, filaggrin, transglutaminase and CD36). We observed a higher expression of markers for proliferation, differentiation and activation in the epidermis of scars at 1 month post-burn than in normal control skin of healthy persons. There was a striking difference between normotrophic and hypertrophic scars at 4 months post-burn. Keratinocytes in hypertrophic scars displayed a higher level of proliferation, differentiation and activation than did normotrophic scars. At 7 months post-burn all keratinocyte proliferation and differentiation markers showed normal expression, but the activation marker CD36 remained upregulated in both normotrophic and hypertrophic scars. Surprisingly, in matched unburned skin of burn patients, a state of hyperactivation was observed at 1 month. Our results suggest that keratinocytes may be involved in the pathogenesis of hypertrophic scarring.

Topics: Adult; Aged; Burns; CD36 Antigens; Cicatrix; Cicatrix, Hypertrophic; Epidermis; Filaggrin Proteins; Follow-Up Studies; Humans; Immunohistochemistry; Keratins; Middle Aged; Reference Values; Skin; Time Factors

Skin malignancies can originate in burn scars (Marjolin's ulcer). The most common is squamous cell carcinoma, usually appearing years after injury. Split-thickness skin graft donor sites as a source of malignant transformation are far less frequent and demonstrate a shorter interval between surgery and tumor onset. Keratoacanthomas have rarely been reported to arise in such scars. We describe the simultaneous occurrence of keratoacanthomas on a spontaneously healed second-degree burn on the flank and in the scar of a skin graft donor site on the thigh, 4 months after a 40% total body surface area burn.

Topics: Burns; Cicatrix; Dermis; Eosinophils; Epidermis; Fibrosis; Humans; Keratins; Keratoacanthoma; Lymphocytes; Male; Middle Aged; Skin Transplantation

Cultured epithelial autografts (CEA) derived from sole skin were transplanted to full-thickness wounds excised to muscle fascia over a variety of diverse body sites in 12 pediatric patients treated for acute burns or giant congenital nevi. The skin regenerated from the grafts was biopsied from 7 days to 6 years after grafting. The resultant epidermal phenotype was analyzed histologically and by immunohistochemical localization of keratin 9 (K9) as objective evidence of sole-type site-specific differentiation. Expression of K9 was also verified by one-dimensional gel electrophoresis of epidermal cytoskeletal extracts and K9 immunoblot analysis. Grafts prepared from epidermis of axilla; groin or foreskin and transplanted to wounds of comparable depth in an identical manner in the same patients served as controls of postgrafting differentiation. Biopsies of sole skin from amputation specimens from patients of comparable age served as normal positive controls, and biopsies of nonsole skin from patients of comparable age served as normal negative controls. As early as 2 weeks postgrafting, the histologic appearance of sole-derived CEA differed substantively from that of axilla- or groin-derived CEA controls and displayed a phenotype characteristic of sole skin with a thick compact stratum corneum, a thick stratum granulosum, and a distinct stratum lucidum. In sole-derived grafts rete ridges regenerated within 2 months postgrafting, whereas nonsole-derived grafts required 4-6 months for rete ridge regeneration. Once acquired, the sole skin phenotype was maintained long-term by all sole-derived CEA. In vitro, sole-derived keratinocytes synthesized little, if any, K9. However, within 7 days after grafting, K9 synthesis by multiple suprabasal keratinocytes was seen within the epidermis regenerated from sole-derived CEA. Protein of K9 appeared progressively more diffuse throughout the suprabasal layers, attaining a confluent pattern of expression comparable to normal controls of sole skin by 6 to 12 months postgrafting, and the confluent pattern of suprabasal K9 synthesis was maintained long-term. The results demonstrate that site-specific differentiation is an intrinsic property of postnatal human keratinocytes and can be expressed and maintained in a permissive environment in the absence of dermal tissue.

Topics: Adolescent; Biomarkers; Burns; Cell Differentiation; Cells, Cultured; Child; Child, Preschool; Epidermal Cells; Female; Foot; Humans; Infant; Keratinocytes; Keratins; Male; Nevus; Phenotype; Protein Isoforms; Skin Neoplasms; Transplantation, Autologous

Malignant melanoma occurring in burn scars is rare and cases of malignant melanoma and squamous cell carcinoma arising in a burn scar are extremely rare. We report a case of malignant melanoma and squamous cell carcinoma arising in one tumour on a stable thermal burn scar on the right leg of a 55-year-old man after a long latent period of about 50 years. The case was unique in that the malignant melanoma and squamous cell carcinoma occurred synchronously next to each other and produced one tumour. Immunohistochemical stainings with keratin, S-100 protein and HMB 45 clearly distinguished the two kinds of atypical tumour cells. Following the total resection of the original tumour, metastasis of malignant melanoma in the inguinal lymph node was found. This case underlines the possibility that another tumour may co-exist even if pathological observation reveals one kind of tumour.

Topics: Antigens, Neoplasm; Antigens, Surface; Burns; Carcinoma, Squamous Cell; Cicatrix; Humans; Immunohistochemistry; Keratins; Leg; Lymphatic Metastasis; Male; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; Neoplasms, Multiple Primary; S100 Proteins; Skin Neoplasms

Two cases of trichilemmal carcinoma (TLC) developing in burn scars are reported. In Case 1, a 73-year-old man developed a TLC on his left lower leg five years after a burn. In Case 2, a 43-year-old man developed a cauliflower-like mass on his head 42 years after a burn. Histologically, tumor cells showed a lobular proliferation in continuity with the epidermis. Tumor nests were mostly composed of large atypical cells with clear cytoplasms containing PAS-positive, diastase sensitive materials. Some of the nests showed trichilemmal-type keratinization. These cases were treated only with surgical excision, and there has been no evidence since of local recurrence or metastasis.

Topics: Adult; Aged; Burns; Cicatrix; Cytoplasm; Epidermis; Humans; Keratins; Leg Injuries; Male; Neoplasms, Basal Cell; Scalp; Skin Neoplasms

Fifteen patients, eight with burn or scald wounds and seven with split-thickness donor sites, were treated with cultured epithelial allografts. Skin was obtained from HIV-negative donors undergoing circumcision and sheets of epithelium were cultured using the 3T3 feeder method. Multiple post-operative biopsies were performed at various time intervals and stained with a panel of monoclonal antibodies against cytokeratins, involucrin, transferrin receptor and epidermal growth factor receptor. Fresh cultured epithelial sheets, normal skin, standard treated donor sites and burns treated with autografts were also studied. Cytokeratin-10 expression was not observed at treated sites until 4 weeks post-grafting, when normal suprabasal levels were observed. Cytokeratins 13 and 16, usually observed in highly proliferative states such as psoriasis, were observed in epithelial-treated sites for up to 6 months. Other proliferation markers such as Ki67 and transferrin receptor were only expressed 2-3 weeks post-operatively. Involucrin, a marker of keratinocyte terminal differentiation, was expressed throughout newly formed epidermis until 15 weeks, when the normal pattern of granular expression was observed. These results indicate that although the cultured 'allograft' does not survive, it may modulate the proliferation and differentiation of spontaneously regenerating epithelium.

Topics: Adult; Aged; Biological Dressings; Burns; Cell Differentiation; Cell Division; Cells, Cultured; Child; Child, Preschool; Epidermal Cells; Epidermis; Epithelium; Humans; Immunohistochemistry; Keratins; Middle Aged; Skin Transplantation; Time Factors; Wounds and Injuries

Biopsy specimens from unburned skin were obtained from three severely burned patients and placed into tissue culture. After 2 to 3 weeks, the cultured keratinocytes were released from the Petri dishes and transplanted onto the patient's burn wound, which had been completely excised down to muscle fascia, thereby removing all cutaneous elements. Healing cultured autografts were found to become repopulated with Langerhans cells within 3 to 6 weeks. A neodermis rich in fibronectin rapidly formed between the autografts and muscle fascia. However, using monoclonal antibodies to cytokeratins as markers of differentiation, we found that the autograft keratinocytes expressed an abnormal pattern of differentiation that was similar to the differentiation seen in hyperproliferative states such as psoriasis. In contrast, healed split-thickness graft donor sites and reepithelialized interstices of mesh grafts maintained the basal keratinocyte staining pattern of normal skin with the AE-1 monoclonal antibody.

Topics: Adult; Burns; Cell Count; Cells, Cultured; Child; Epidermal Cells; Epidermis; Female; Fibronectins; Fluorescent Antibody Technique; Humans; Keratinocytes; Keratins; Langerhans Cells; Male; Skin; Skin Transplantation; Staining and Labeling; Wound Healing

An electron microscopical study of grafted autologous cultured human epithelium is presented. Biopsy samples were collected from four patients with full thickness burns at 9 days, 6 weeks and 5-21 months after grafting of the cultured epithelium. By the sixth week after transplantation, grafted cultured epithelial sheets had developed to consist of 10 to 20 layers of cells and the epithelium showed distinct basal, spinous, granular and horny layers, and a patchy basement membrane had formed. Langerhans cells and melanocytes were identifiable. From 5 months onwards flat basal cells became oval, and oval keratohyalin granules in the keratinocytes also assumed a normal irregular shape. Membrane-coating granules in the keratinocytes increased in number. The fine structures of desmosomes also showed a normal mature appearance. Furthermore, complete extension of the basement membrane could be observed. The maturation of cultured human epithelium is complete by 5 months after grafting.

Topics: Adolescent; Adult; Burns; Cells, Cultured; Child; Epidermis; Epithelium; Female; Humans; Keratins; Langerhans Cells; Male; Melanocytes; Microscopy, Electron; Skin Transplantation

Regeneration of skin from cultured keratinocyte autografts used in the treatment of full-thickness burn wounds was studied in 21 pediatric patients from 6 days to 5 years after grafting. Findings were compared both to controls of age- and site-matched normal skin and to controls for epithelial wound-healing, re-epithelialized interstices of meshed split-thickness skin grafts of comparable postgrafting age. Six days after transplantation, a mildly hypertrophic, flat epidermis with all normal strata had regenerated, and the process of de novo dermal-epidermal junction formation had begun. Hemidesmosomes, basal lamina, and anchoring fibrils reformed conjointly in punctate fashion along the attachment face of the grafts. Within 3 to 4 weeks, the dermal-epidermal junction was complete, but full maturation of anchoring fibrils required more than a year. The process was comparable to that observed in meshed graft interstices. Rete ridges regenerated from 6 weeks to 1 year after grafting. The subjacent connective tissue initially healed to form normal scar, but it remodeled dramatically, regenerated elastin, and resembled a true dermis within 4 to 5 years. Meshed-graft interstice controls showed no rete ridge regeneration, subepithelial connective tissue remodeling, or elastin production up to 5 years after grafting. Langerhans cells repopulated grafts within 1 week, and normal population densities were reached within 2 to 6 months. After 1 year, Langerhans cell densities were increased compared with normal skin but were lower than those in age-matched meshed graft controls. Melanocytes were present in cultures at the time of transplantation, but functional epidermal melanin units were not seen in groin- or axilla-derived grafts for 6 to 8 weeks or in sole-derived epidermis until a year or more after transplantation. Normal histologic features were maintained for years after grafting. Transitory pathologic changes including parakeratosis, dyskeratosis, and intraepithelial friction blister formation were infrequently observed. No dysplastic or premalignant changes were seen.

Topics: Adolescent; Burns; Cell Differentiation; Cells, Cultured; Child; Child, Preschool; Epidermis; Epithelium; Humans; Immunohistochemistry; Infant; Keratins; Melanocytes; Microscopy, Electron; Microscopy, Electron, Scanning; Regeneration; Skin; Skin Physiological Phenomena

The aim of the study was to determine the fate of cultured skin allografts in patients with burns. In situ DNA hybridisation with a Y probe (pHY 2.1) was used to detect cells carrying the Y chromosome (the probe being visualised by the alkaline phosphatase-antialkaline phosphatase method) in biopsy specimens taken from cultured allografts derived from donors of the opposite sex to the recipients (20 patients with burns). Specimens were taken within a week, between one and three weeks, between four and six weeks, and more than six weeks after grafting. Only two of the 27 biopsy specimens contained cells that were the same sex as the donor; both were taken within a week after grafting. In the 25 other specimens the epithelial cells were the same sex as the recipient. Cultured skin allografts showed no evidence of survival in patients with burns, which suggests that they are probably not suitable for long term management of burns but may be useful as short term biological dressings.

Topics: Adolescent; Adult; Aged; Bandages; Biological Dressings; Burns; Culture Techniques; DNA Probes; Epidermal Cells; Female; Humans; Keratins; Male; Middle Aged; Skin; Skin Transplantation; Tissue Survival; Y Chromosome

Early tangential excision and primary grafting of full thickness and deep partial thickness burns has become a widely accepted practice, however, little attention has been paid to the epithelial bridging phenomenon, which occurs most frequently at the edge of these wounds. Over 5 years, 923 burn patients were treated and 407 were grafted, of whom 13 developed sufficiently severe epithelial bridging to require deroofing under general anaesthetic. Histological sections show that the bridges are derived from hair follicles. We advocate that patients with epithelial bridges should be observed in the outpatient department for many weeks and surgical intervention should only be used when they fail to resolve spontaneously.

Topics: Adolescent; Burns; Child; Child, Preschool; Epithelium; Female; Humans; Infant; Keratins; Male; Skin; Skin Transplantation; Wound Healing

Three consecutive studies were performed in 58 patients evaluating the effect of occlusion on the healing of partial-thickness wounds. Mirror-image donor sites were covered with the occlusive hydrocolloid dressing (HCD) (DuoDerm) and compared to fine mesh gauze, and the HCD was subsequently compared to a semi-occlusive dressing of polyurethane film, (Op-site). In addition, partial-thickness burn wounds were covered with the HCD and the remaining burn wound was treated with silver sulfadiazine. The donor sites and burn wounds treated with HCD healed significantly faster than those covered with fine mesh gauze or silver sulfadiazine (p less than 0.001) and with less pain. The HCD and polyurethane film were equivalent. There were no clinical infections with the wounds that were occluded. The exudate collected beneath the DuoDerm and Op-site on donor sites was added to the tissue culture system and resulted in a modest increase in keratinocyte proliferation. However, the exudate from burn wounds under HCD resulted in a marked increase in cell proliferation (p less than 0.001).

Topics: Adolescent; Adult; Aged; Burns; Cell Division; Epidermal Cells; Exudates and Transudates; Female; Humans; Keratins; Male; Middle Aged; Occlusive Dressings; Pain Measurement; Wound Healing; Wound Infection

Epidermal sheets prepared from cultured keratinocytes were autografted to a burn wound on a 71-year-old woman. Grafted epidermis seemed to be well accepted during the first 2 months; however, a significant portion peeled off after blisters formed. Ultimately, 24% of the grafted epidermis survived and thrived.

Topics: Aged; Burns; Cells, Cultured; Female; Humans; Keratins; Skin; Skin Transplantation

Neonatal Fischer 344 rat keratinocytes were cultured on a collagen sheet to create a 'quasi-skin.' The cells were grown in either control medium or a low-calcium supplemented medium to enhance proliferation. The rate of 3H-thymidine incorporation into DNA was assessed at days 3, 7, 10, and 20 to determine the effectiveness of the medium supplements. The cells grown in supplemented medium did have an elevated rate of incorporation. On days 7 and 14 post culture, the quasi-skin was used to cover full-thickness defects on the dorsum of adult male rats. The covered defects were biopsied 45 and 60 days postoperatively. Light microscopy showed the wounds to be completely re-epithelialized. Barr bodies were present at the wound site, confirming the presence of quasi-skin keratinocytes up to 60 days postoperatively.

Topics: Animals; Bandages; Biological Dressings; Biopsy; Burns; Cell Division; Cell Survival; Cells, Cultured; Collagen; Culture Media; Epidermal Cells; Epidermis; Keratins; Male; Rats; Rats, Inbred F344; Time Factors; Wound Healing

Two patients had unusual squamous cell carcinoma (SCC) arising in a burn scar. The SCCs rapidly recurred and metastasized after radical operation, and the patients died of disseminated metastases. Histopathologically, the SCC was poorly differentiated and consisted of acantholytic round cells that diffusely proliferated into the deep dermis. However, small, solid nests composed of squamoid cells were focally observed. Immunohistochemical studies revealed that the acantholytic round neoplastic cells expressed not only keratin but also vimentin, and the coexpression was substantiated with double immunostaining. Vimentin-positive SCC composed of acantholytic round neoplastic cells may be a highly malignant subset of cutaneous SCC.

Topics: Burns; Carcinoma, Squamous Cell; Cicatrix; Female; Humans; Keratins; Male; Middle Aged; Skin Neoplasms; Vimentin

Twenty adult individuals with chronic leg ulcers caused by venous insufficiency, and 5 patients with full-thickness burns were treated. Twenty of the patients (15 with leg ulcers and 5 with burns) were grafted with separated autologous keratinocytes. In these cases the cells were fixed to the wound bed by a fibrin net. Five other patients (with leg ulcers) were treated with fibrin without keratinocytes. In 16 of the 20 patients grafted with keratinocytes in a fibrin net, the defect healed completely in 14 to 21 days. On the other hand, the fibrin net without keratinocytes failed to significantly accelerate the process of reepithelialization. Our experience suggests that a rapid healing of full-thickness skin defects can be achieved through keratinocyte grafting.

Topics: Burns; Epidermal Cells; Humans; Keratins; Leg Ulcer; Transplantation, Autologous

Repair of full-thickness burns requires replacement of both the dermal and the epidermal components of the skin. Use of tissue culture methods allows very large expansions of surface area to be covered by cultured normal human epidermal keratinocytes (HK). Porous and resorbable materials, such as collagen and chondroitin-6-sulfate membranes, may be expected to adhere to wounds and promote fibrovascular ingrowth better than grafts of cultured epidermal keratinocytes alone. This article demonstrates the in vitro formation of biologic attachments between HK and a collagen and chondroitin-6-sulfate dermal skin replacement. Dermal membranes are prepared as generic acellular sheets and stored in the dry state for extended periods. Subconfluent HK cultures in logarithmic phase growth can attach quickly to dermal membranes in vitro, form a confluent epithelial sheet on the surface of each membrane, and exhibit mitotic cells for at least 1 week in vitro. Transmission electron microscopy demonstrates the formation of hemidesmosomes, extracellular matrix, and banded collagen at the interface of the epidermal cells and the dermal membrane. By comparison, HK cultures as confluent sheets released enzymatically with Dispase do not attach to the dermal membranes in vitro, under the conditions tested, although complete coverage of the membrane by the cell sheets is obtained. Growth assays show that subconfluent HK cells retain sufficient growth potential to maintain logarithmic phase growth, but that HK cells disaggregated from confluent sheets become growth arrested in comparison. The composite material has discrete dermal and epidermal compartments, has total thickness comparable to split-thickness skin graft, and can be applied to full-thickness skin defects in a single procedure.

Topics: Burns; Chondroitin; Chondroitin Sulfates; Collagen; Culture Techniques; Desmosomes; Epidermal Cells; Humans; Keratins; Mitosis; Skin Transplantation

Cultured keratinocytes (CK) have been used to resurface large burn wounds with some success. These CK are grown in the presence of fetal bovine serum (FBS) and mouse fibroblasts (MF). Serum from ten patients who received CK grafts as part of burn wound coverage was studied by ELISA technique for antibody to these xenogeneic antigens. All patients had some amount of antibody to FBS but no detectable antibody to MF. The amount of antibody to FBS varied between patients and with respect to time after graft application. Generally, the levels of antibody to FBS were moderate by 30 days, declined, and then rose slowly by 5 to 6 months. The majority of antibody to FBS was against bovine serum albumin (BSA), demonstrated by Western blot technique. The presence of such antibody to FBS might produce clinical problems of graft rejection, anaphylaxis, and serum sickness in patients receiving CK grown in FBS supplemental medium. Further investigation will need to determine the likelihood and potential severity of such clinical problems.

Topics: Adult; Animals; Antibody Formation; Blood; Burns; Cattle; Cells, Cultured; Child; Child, Preschool; Culture Media; Enzyme-Linked Immunosorbent Assay; Epidermal Cells; Epidermis; Fibroblasts; Humans; Keratins; Mice

Human epidermal keratinocytes now can be grown reliably and reproducibly in vitro to form multilayered epithelium. These sheets of cultured keratinocytes have been used successfully to autograft patients with severe burns, leg ulcers and following excision of extensive congenital naevi. Whilst the technique carries the obvious advantage of huge expansion of the initial skin biopsy, thus removing the need for painful and slow healing donor sites, problems have been encountered. The take rate has been lower than with conventional split skin grafts. The take rate can be increased by the provision of a dermis. This may be achieved by providing an allodermis or by the use of a highly meshed autologous split skin graft. The wound is then covered with autologous cultured keratinocyte grafts. Manufactured dermis has been under investigation for some years and animal work suggests this may be an alternative approach. There is a delay of 2 to 3 weeks for culture of the autologous sheets of keratinocytes. This has led to the use of allogeneic grafts in a number of patients. The long term survival of these grafts has been attributed to the loss of antigen presenting cells during tissue culture. However some grafts have been rejected. Studies currently in progress may help resolve these anomalies. Whilst a number of problems remain to be solved the technique of cultured keratinocyte grafting takes wound care into an exciting new era. Skin banks may now become more than a surgeon's dream.

Topics: Bandages; Biological Dressings; Burns; Cells, Cultured; Epidermal Cells; Graft Survival; Humans; Keratins; Leg Ulcer; Methods; Transplantation, Autologous

Topics: Burns; Epidermal Cells; Epidermis; Humans; Keratins; Skin Ulcer; Surgery, Plastic

Stratification of human epidermal cells into multilayered sheets composed of basal and suprabasal layers (resembling the stratum germinativum and stratum spinosum of the epidermis) was studied in a dermal component-free culture system. Although no stratum corneum developed in vitro, this culture system provided a method to study early events in human keratinocyte differentiation. Multiparameter flow cytometric analysis of acridine orange-stained epidermal cells from these cultures revealed three distinct subpopulations differing in cell size, RNA content, and cell cycle kinetics. The first subpopulation was composed of small basal keratinocytes with low RNA content and a long generation time. The second subpopulation consisted of larger keratinocytes, having higher RNA content and a significantly shorter generation time. Finally, the third subpopulation contained the largest cells, which did not divide, and represent the more terminally differentiated keratinocytes. This in vitro approach provides discriminating cytochemical parameters by which the maturity of the epidermal cell sheets can be assessed prior to grafting onto human burn patients.

Topics: Burns; Cell Adhesion; Cell Cycle; Cell Differentiation; Cell Division; Cells, Cultured; DNA; Epidermal Cells; Epidermis; Epithelium; Humans; Keratins; Kinetics; RNA

An adult with burns over 55% of body surface area (80% of which were third degree) was treated with cadaver skin allografts. The allografts were later abraded to remove allogeneic epidermis and resurfaced with autogenous keratinocyte cultures. Complete reconstitution of skin, consisting of epidermal autograft and dermal allograft, was achieved.

Topics: Burns; Cadaver; Cells, Cultured; Epidermal Cells; Epidermis; Humans; Keratins; Male; Middle Aged; Skin Transplantation; Transplantation, Autologous; Transplantation, Homologous

Human keratinocytes can be cultured in vitro and used for autografting of the injured epidermis. In this article, we describe our culture methods for propagating autologous keratinocytes for grafting, and we present an overview of the questions raised by the use of such methods. Our culture method involves a two-phase technique. Phase 1 is performed in serum-free medium and yields large quantities of a homogeneous, highly proliferative, basal cell-like population. Phase 2 involves the induced formation of cohesive, stratified sheets of differentiated keratinocytes resembling normal epidermis that can be used for grafting. This two-phase technique was used for preparation of autografts for full-thickness wounds of a massively burned patient. Important questions about the functional potential of cultured keratinocytes and the long-term behavior of cultured epidermal autografts in vivo remain to be answered.

Topics: Adult; Burns; Cell Differentiation; Cells, Cultured; Culture Media; Epidermal Cells; Epidermis; Humans; Keratins; Male; Methods

Epithelial sheets can be cultivated from isolated epidermal cells; in this way, it is possible to increase the cell number considerably. H. Green and co-workers were the first to make use of such epithelia for the autologous covering of burn wounds. We modified this method and report on our experiences with this technique in a patient with small skin defects.

Topics: Adult; Burns; Epithelium; Female; Humans; Immunoenzyme Techniques; Keratins; Melanoma; Skin Neoplasms; Skin Transplantation; Vimentin; Wound Healing

Topics: Biopsy; Burns; Collagen; Cysts; Elastic Tissue; Epithelium; Granulation Tissue; Humans; Hyperplasia; Keratins; Sebaceous Glands; Skin; Skin Transplantation; Staining and Labeling; Sweat Glands; Transplantation, Autologous; Wound Healing