Anti-inflammatory properties of a liposomal hydrogel with povidone-iodine (Repithel) for wound healing in vitro.

A liposomal hydrogel with 3% povidone-iodine (PVP-ILH, Repithel) has shown clinical benefit in settings where inflammation and/or reactive oxygen species are thought to impede wound healing (e.g., burns, chronic wounds and in smokers). This in vitro study investigated whether PVP-ILH is able to reduce inflammatory events responsible for the impairment of the wound healing process in such patients. Therefore, the following assays were conducted with PVP-ILH (and derived control hydrogels to identify the component responsible for the effect): inhibition of reactive oxygen species production by human polymorphonuclear neutrophils (PMNs) and in a cell-free system, oxygen consumption assay of PMNs (prior to oxidative burst), inhibition of human complement (limiting the generation of complement factors), mast cell degranulation, nitric oxide production by murine macrophages and TNF-alpha production by human monocytes/macrophages. Where toxicity could cause cell inhibition, cell viability was assessed. PVP-ILH and its components interacted in our series of bioassays at various stages in the inflammation cascade. Scavenging of superoxide anions was the most pronounced effect. Furthermore, povidone-iodine inhibited PMN production of reactive oxygen species (inhibition of oxygen consumption) and a mast cell inhibitory (stabilising) activity was observed. Based on these results, the clinically observed, beneficial wound healing effects of PVP-ILH may also be attributed to an impediment of inflammatory activity, mainly by iodine's free radical scavenging. Controlling oxidative stress in the wound may be of great importance, especially since further reactions as, e.g., the formation of peroxynitrite from NO and ROS are prevented.

Evaluation of acellular dermis for closure of abdominal wall defects in a rat model.

BACKGROUND: Abdominal wall repair can be performed with synthetic or biological materials. Biological materials may reduce the risk of infections and fibrosis. The aim of this study was to evaluate two acellular human dermis products. MATERIALS AND METHODS: A rat model was used to compare the two materials. One was prepared using low concentrations of NaOH; the other material was SureDerm, which is commercially available. Full thickness defects were prepared in the abdominal wall and closed with the materials. Rats were sacrificed at 1 or 4 months after operation and the numbers of adhesions to the bowels were scored. Samples were taken for histological analysis and to measure the breaking strength. RESULTS: In both groups a good functional integration of the implants with the abdominal wall was observed. There was no adhesion formation with the bowels in the group with the NaOH prototype. In the SureDerm group, 4 out of 7 rats showed only small adhesions at 4 months after operation. Breaking strength of the healed tissue was significantly higher in the NaOH prototype group at 4 months after operation (p < 0.0026). CONCLUSIONS: The results indicate that both human acellular dermis products may be used in clinical trials for closure of abdominal wall defects.

An in vitro examination of the antioxidant and anti-inflammatory properties of buckwheat honey.

OBJECTIVE: Hydroxyl radical and hypochlorite anion formed at the wound site from superoxide anion produced by activated polymorphonuclear neutrophils (PMNs) are considered important factors in impaired wound healing. Superoxide anion may also react with nitric oxide produced by macrophages to form peroxynitrite, a third strong oxidant that damages surrounding tissue. In order to select honey for use in wound-healing products, different samples were compared for their capacity to reduce levels of reactive oxygen species (ROS) in vitro. METHOD: Honey samples were tested in assays for inhibition of ROS production by activated human PMNs, antioxidant activity (scavenging of superoxide anion in a cell-free system) and inhibition of human complement (reducing levels of ROS by limiting formation of complement factors that attract and stimulate PMNs). For buckwheat honey (NewYork, US), moisture and free acid content were determined by refractive index measurement and potentiometric titration respectively. Honey constituents other than sugars were investigated by thin layer chromatography, using natural product reagent to detect phenolic compounds. Constituents with antioxidant properties were detected by spraying the chromatogram with DPPH. RESULTS: Although most honey samples were shown to be active, significant differences were observed, with the highly active honey exceeding the activities of samples with minor effects by factors of 4 to 30. Most pronounced activities were found for American buckwheat honey from the state of NewYork. Phenolic constituents of buckwheat honey were shown to have antioxidant activity. CONCLUSION: As buckwheat honey was most effective in reducing ROS levels, it was selected for use in wound-healing products. The major antioxidant properties in buckwheat honey derive from its phenolic constituents, which are present in relatively large amounts. Its phenolic compounds may also exert antibacterial activity, whereas its low pH and high free acid content may assist wound healing.

Development of a dermal matrix from glycerol preserved allogeneic skin.

Dermal substitutes can be used to improve the wound healing of deep burns when placed underneath expanded, thin autologous skin grafts. Such dermal matrix material can be derived from xenogeneic or human tissue. Antigenic structures, such as cells and hairs must be removed to avoid adverse inflammatory response after implantation. In this study, a cost-effective method using low concentrations of NaOH for the de-cellularization of human donor skin preserved in 85% glycerol is described. The donor skin was incubated into NaOH for different time periods; 2, 4, 6 or 8 weeks. These dermal matrix prototypes were analyzed using standard histology techniques. Functional tests were performed in a rat subcutaneous implant model and in a porcine transplantation model; the prototypes were placed in full thickness excision wounds covered with autologous skin grafts.An incubation period of 6 weeks was most optimal, longer periods caused damage to the collagen fibers. Elastin fibers were well preserved. All prototypes showed intact biocompatibility in the rat model by the presence of ingrowing blood vessels and fibroblasts at 4 weeks after implantation. An inflammatory response was observed in the prototypes that were treated for only 2 or 4 weeks with NaOH. The prototypes treated with 6 or 8 weeks NaOH were capable to reduce wound contraction in the porcine model. In neo-dermis of these wounds, elastin fibers derived from the prototype could be observed at 8 weeks after operation, surrounded by more random orientated collagen fibers. Thus, using this effective low cost method, a dermal matrix can be obtained from human donor skin. Further clinical studies will be performed to test this material for dermal substitution in deep (burn) wounds.

Immunology of skin transplantation.

Untreated viable allogeneic skin is highly immunogenic. Epidermal Langerhans migrate after transplantation out of the donor skin into the lymph node of the recipient where they can activate T cells capable to mediate rejection. Allogeneic skin is used as a temporary coverage of burn wounds, often in combination with autologous skin grafts. Several methods to pretreat the allogeneic skin have been used to delay the rejection process. Processing of allogeneic skin in 85% glycerol results in a non-viable skin with a well-preserved structure. Experiments in a full thickness porcine wound model showed that rejection of glycerol treated allogeneic skin grafts was up to six days delayed. Viable, untreated allogeneic skin grafts were rejected predominantly by CD8 positive T cells whereas in the glycerol treated grafts the influx of host cells was lower and the majority of the cells were macrophages. The outgrowth of the autologous skin grafts underneath glycerol treated allogeneic skin was three days earlier completed when compared to grafts in combination with untreated allogeneic skin. Thus, by processing the allogeneic skin into 85% glycerol, the direct route to induce graft rejection is blocked since the Langerhans cells are non-viable. The glycerol-preserved skin grafts are finally rejected via an indirect route mediated by macrophages; this process is less disturbing for the outgrowth of autologous cells.

Migration of rat skin dendritic cells.

This study examines the in vivo migration of rat skin dendritic cells (including Langerhans cells) after skin transplantation. As donor animals, PVG-RT7b rats were used. The leukocytes of these rats bear an epitope of the leukocyte common antigen that can be recognized by use of the antibody His 41. The cells of allogeneic (ACI) recipient strains do not label with this antibody. Four days after transplantation of PVG-RT7b skin on allogeneic recipients, His 41+ cells showing a dendritic morphology were present in the T cell area of the draining lymph nodes. During culture of rat skin explants, dendritic cells migrated spontaneously into the medium. These in vitro migrated cells showed a high capacity to stimulate allogeneic T cells. When these cells, obtained from PVG-RT7b skin, were injected into the hind footpads of allogeneic recipients, they migrated to the same compartments of the draining lymph node. These data indicate that the cells that migrate from a transplanted allogeneic skin grafts are the same cells that migrate in vitro from explants. Most probably, they initiate graft rejection in the draining lymph nodes of the recipient.

Migration of dendritic cells to the draining lymph node after allogeneic or congeneic rat skin transplantation.

BACKGROUND: After transplantation, donor dendritic cells (DC) migrating to the draining lymph node of the recipient are thought to play an important role in the initiation of graft rejection. In this study, we compared the in vivo migration of DC after allogeneic skin transplantation with that after congeneic skin transplantation. METHODS: A rat model was used with the PVG-RT7b rats as donor animals. These rats have leukocytes bearing an epitope of the leukocyte common antigen that can be recognized by the monoclonal antibody His 41. The cells of the allogeneic (ACI) and congeneic (PVG) recipient animals do not express this marker. RESULTS: In both recipient rat strains, graft-derived His 41+ DC could be detected in the T cell areas of the draining lymph nodes after skin transplantation. However, the number of migrated His 41+ cells present was lower in the allogeneic recipients. Similar results were obtained when skin DC isolated from the PVG-RT7b rats were injected subcutaneously into the hind footpads of allogeneic and congeneic recipients. Although the numbers of migrated His 41+ DC present were lower, the lymph nodes of the allogeneic recipients were much more enlarged and the grafts were rejected which did not occur in the congeneic recipients. CONCLUSIONS: The presence of donor-derived DC in the graft draining lymph nodes underlines the importance of the direct route of allo-activation. The lower numbers of migrated His 41+ DC in lymph nodes of allogeneic recipients may be the result of killing of the cells after presentation of the allo-antigens to the recipient T cells.

Cross-linking of dermal sheep collagen with tannic acid.

The purpose of this study was to investigate cross-linking of (damaged) collagen by tannic acid, with a view to reconsider its use as a possible therapeutical agent in the treatment of burn wounds. Because of contradictory reports in the literature, and increased purity of tannic acid, this method has again become valuable for re-evaluation. A laboratory study using dermal sheep collagen was conducted to analyse the influence of several metal ions on collagen cross-linking with tannic acid. The tannic acid concentration vs degree of cross-linking, tannic acid uptake and release, influence of the addition of metal ions, and the rate of degradation of treated collagen were established. We have shown that tannic acid mediated collagen cross-linking in a concentration-dependent manner. Cross-linking was influenced by the presence of metal ions: Fe3+ and Ag+ were shown to exert a stimulatory effect on the degree of cross-linking by a 2% tannic acid solution, whereas Zn2+ had an inhibitory effect Ce3+ Ca2+ and Na+ did not influence the degree of cross-linking. The degree of cross-linking was proportional to the uptake of tannic acid, which variod between 6 and 35 wt%. Reversibility of cross-linking was established. Tannic acid-treated dermal sheep collagen showed a slow degradation rate relative to differently cross-linked collagen materials when subjected to collagenase or pancreatic proteolytic enzymes. The results of this study suggest that tannic acid could have a function in vivo in burn treatment by binding burn toxins and inhibiting degradation of the (remaining) dermal matrix, and allows combination with metal ions as antimicrobials. Optimal cross-linking was obtained using a 2 wt% tannic acid solution; combination with Ce3+ as a potential antimicrobial agent is possible without diminishing cross-linking.

Porcine wound models for skin substitution and burn treatment.

Skin regeneration is an important field of tissue engineering. Especially in larger burns and chronic wounds, present treatments are insufficient in preventing scar formation and promoting healing. Initial screening of potentially interesting products for skin substitution is usually done by in vitro tests. Before entering the clinic, however, in vivo studies in immunocompetent animals are necessary to prove efficacy and provide information on safety aspects. We have obtained extensive experience using the domestic pig as test animal for studies on skin replacement materials, including tissue engineered skin substitutes, and burn wound treatment. Two models are described: an excisional wound model for testing of dermal and epidermal substitutes and a burn wound model for contact and scald burns, which allows testing of modern wound dressings in comparison to the present gold standards in burn treatment. The results of these experiments show that in vivo testing was able to reveal (dis)advantages of the treatments which were not detected during in vitro studies.

Isolation of human skin dendritic cells by in vitro migration.

In the human skin, various types of antigen-presenting cells (APC) are present. In the epidermis, they are identified ultrastructurally as Langerhans cells (LC) by the presence of Birbeck granules. LC are considered to belong to the family of dendritic cells (DC) that are important for the initiation of immune responses (1). In the dermis, macrophages and DC are present (2,3). The expression of CD1a molecules can be used to identify DC in the skin (4,5), because macrophages do not express this marker. In vivo, these skin DC are supposed to take up antigens penetrating in the skin. Thereafter, they migrate via the afferent lymphatics into the draining lymph nodes, where a T-cell response can be initiated (6,7). During migration, the DC mature into potent APC. Besides an increase in MHC class II expression, adhesion (8,9) and B7 co-stimulatory molecules (10) are up-regulated. Most research on skin DC has been carried out with cells isolated from enzyme digested skin (8-10). In this chapter, we describe a method to obtain DC from human skin without enzymes, by making use of their migratory capacities. The cells migrate "spontaneously" out of the skin during culture. Characterization of the cells shows that mature DC are obtained with a marker expression not influenced by enzymes.

History of the Euro Skin Bank: the innovation of preservation technologies.

A skin bank facility is indispensable for burn care. In Holland, a skin bank was established in October 1976 on a national basis for economical reasons. Since 1981, overcoming early rejection by innovation of existing, and development of new, preservation technologies became the main scientific objective. The immunological benefits of glycerolization of skin became clear in 1983. In clinical practice, the use of glycerolized allograft in sandwich grafting changed this technique into a reliable procedure. Early rejection can still be a problem, especially in the non-immunocompromised patient. Future research projects are focused on immunomodulation of the skin by chemical, physical and immunological methods. Even the risk of HIV transmission is not now a drawback for the use of glycerolized skin.

Widely expanded postage stamp skin grafts using a modified Meek technique in combination with an allograft overlay.

A method for obtaining widely expanded postage stamp autografts, first described in 1963 by C.P. Meek, has been evaluated in our burns unit. The technique, in which an expansion ratio of 1:9 is achieved with the aid of special prefolded gauzes, was combined with an overlay of glycerol-preserved allografts applied 1 week after grafting. This delay did not appear to affect graft take. In a series of 16 consecutive skin graft procedures performed on 10 patients with extensive burns, the mean epithelialization rate was 90 per cent (range 70-100 per cent) within 5 weeks. Cosmetic results were comparable to those achieved with widely expanded mesh grafts. The Meek technique utilizes small pieces of autograft, and has proved to be a practical alternative to mesh grafts when donor sites are limited.

Long-term results of treatment of scalds in children with glycerol-preserved allografts.

A retrospective study followed by a clinical examination was carried out 2-5 years postburn to analyse the long-term results of the treatment of scalds in children (n = 45) with glycerol-preserved allografts. In 21 patients (47 per cent), the wounds healed spontaneously. In 24 patients, remaining defects were closed by a split skin autograft in the third week postburn. Healing without scar formation was seen in 24 patients (53 per cent), 9 patients (21 per cent) showed moderate, and 12 patients (26 per cent) severe scar formation. Transplanted areas showed hyperpigmentation, and donor sites depigmentation. The cosmetic results require further improvement, and classification of present and new methods of treatment. Clinical research is limited by the number of observations and the great variation in wounds. A well-standardized experimental animal model is, therefore, essential.

Histopathological evaluation of scalds and contact burns in the pig model.

A method was developed to inflict standardized scald burns in the New Yorkshire pig. The tissue damage was compared to a standardized contact burn model. On each flank, six localizations were marked in a symmetrical way. Eight scald burns covering 45 cm2 were inflicted by exposing the prepared skin to 500 ml tap water in an applicator at 80 degrees C for 10, 20, 30 and 40 s. Four contact burns were inflicted by applying a brass block of 45 cm2 area, weight 450 gram, with a temperature of 170 degrees C for 10 and 20 s on the skin without exerting pressure. The right flank was used for macroscopic observation, the left flank for histopathological evaluation. Full thickness biopsies were taken at postburn days (PBD) 0, 1, 2, 3 and 21. In the contact burns, a clear demarcation zone of viable and necrotic dermal tissue and vascular damage was observed. The tissue damage in the scalds showed an intermingled pattern of intact and damaged collagen. The more severe scald burns showed a structurally intact superficial vascular plexus, in combination with destruction of the deep vascular plexus. Current and new methods of scald treatment can be classified in this standardized animal model.

Virucidal effect of glycerol as used in donor skin preservation.

Glycerol has been used for a long time as a viral preservation medium in tissue samples at a 50 per cent concentration, however after a limited time span viruses could no longer be detected. This fact combined with the dehydrating action of glycerol, raised the suspicion that glycerol in a higher concentration could be virucidal. To test this hypothesis, experiments were done at various concentrations of glycerol at three different temperatures (4, 20 and 37 degrees C), using the following viruses: herpes simplex virus, a virus with an envelope, comparable to human immunodeficiency virus; and poliovirus as an example of small, hard to inactivate viruses without an envelope. Glycerol will dehydrate the skin, the extracted water being replaced by glycerol, preserving the original structure. The remaining water is optimally distributed throughout the tissue. However, the possibility exists that glycerol influences the enzymatic processes of nucleic acid breakdown. Plasmid DNA pBR322 was added to HeLa-cells in the presence and absence of glycerol. The outcome of the experiments showed that glycerol has a strong virucidal action. Preservation in 85 per cent glycerol was preferred, because using this concentration the glycerolized allograft skin retained its suppleness and was easy to manipulate during operations.

Morphology of glycerol-preserved human cadaver skin.

Donor allograft skin preserved in 85 per cent glycerol has been used successfully as a temporary coverage for large burn wounds. The glycerol preservation is a method with low costs and has practical advantages such as antibacterial and virucidal effects. This report shows that the glycerol treatment did not affect the fundamental structural integrity of the skin. Intact keratinocytes and Langerhans cells with their characteristic Birbeck granules were still present in the glycerol-treated skin. After treatment with glycerol, the cells in the prepared epidermal cell suspensions were non-viable. MHC class II positive and CD1a positive cells could still be identified in situ and in the suspension.

Immunogenicity of glycerol-preserved human cadaver skin in vitro.

Donor allograft skin preserved in 85% glycerol is used as a temporary coverage for large burn wounds. Glycerol treatment does not affect the structural integrity of the skin; cells are well preserved but dead. However, cells expressing major histocompatibility class II molecules can still be observed. In this study we investigated the mechanism underlying the clinical observation that glycerol-treated alloskin will be destroyed but after a prolonged period. We compared the in vitro immunogenicity of untreated and 85% glycerol-treated human skin cells. Human purified blood T cells did not proliferate when cultured with allogeneic treated skin cells, whereas untreated cells induced a distinct response. A moderate response was measured after adding T cells and viable antigen presenting cells, such as monocytes, to the allogeneic treated skin cells. However, the response on untreated skin cells was much higher. These results favor the suggestion that after transplantation of glycerol preserved skin is performed, an inflammatory process mediated by infiltrating host monocytes occurs rather than a rejection process mediated by T cells.

A histological comparison of acute inflammatory responses with a hydrofibre or tulle gauze dressing.

OBJECTIVE: This study analysed the physical properties of Aquacel hydrofibre dressing in rat partial-thickness wounds, focusing on the acute inflammatory infiltrate of granulocytes and macrophages in the wound and the dressing. METHOD: Partial-thickness wounds (2 x 2 cm) were made on the back of 60 anaesthetised male Wistar rats and covered with Aquacel (n = 30) and tulle gauze (n = 30). The rats were killed on postoperative days one, two, three, four, seven and 10 (10 animals per day and five per dressing). Re-epithelialisation and Polymorphonuclear (PMN), fibronectin and macrophage activity were then analysed. RESULTS: PMN leucocytes (granulocytes) were captured in the dressing and remained active there, resulting in a reduced number in the wounds when compared with tulle gauze. A fibrin layer formed between the dressing and the wound, creating a physical barrier. Macrophages infiltrated the wound bed but could not be detected in the dressing. Little inflammation was observed in the wound bed and the macrophages operated primarily in the repair mode. Active PMNs in the dressing provided an appropriate antimicrobial environment. Tulle materials became embedded in wounds and were associated with a more disturbed pattern of epithelial outgrowth. Aquacel stayed 'on top' of wounds, with only minimal incorporation into the superficial epidermis. CONCLUSION: The observations of the physical properties of different materials and their histological consequences correlate well with published clinical results, particularly in relation to the speed of re-epithelialisation and the level of scarring.

A novel formulation of metal ions and citric acid reduces reactive oxygen species in vitro.

OBJECTIVE: Reactive oxygen species, including superoxide anions, are thought to play an important role in impairing wound healing. Additionally, superoxide anions react with nitric oxide produced by macrophages to form peroxynitrite, another strong oxidant with detrimental effects on surrounding tissue. This in vitro study investigated whether samples of metal ions and citric acid are able to reduce levels of reactive oxygen species. METHOD: Samples of materials were tested in assays for the following: inhibition of reactive oxygen species production by human polymorphonuclear neutrophils (PMNs); antioxidant activity (scavenging of superoxide anions in a cell-free system); inhibition of human complement (limiting the generation of complement factors that attract and stimulate PMNs, thereby reducing levels of reactive oxygen species). RESULTS: Metal ions were shown to inhibit both PMN production of reactive oxygen species and the activation of complement via the classical pathway, whereas citric acid was found to be a scavenger of superoxide anions. CONCLUSION: The beneficial effects of using formulations containing metal ions and citric acid on chronic wounds may be explained in part by a reduction of reactive oxygen species in these wounds.

[The Amsterdam air disaster--management and fate of casualties].

On Sunday, Oct. 4, 1992, an E1 A1 Boeing cargo plane crashed into a 10-story block of flats in the Bijmermeer, on the outskirts of Amsterdam. About 8 people died at the site of the disaster. 37 patients were treated in local hospitals within 90 minutes of the disaster, 16 of whom were admitted. 3 suffered severe, and 6 moderate burns (> 15% and 10-15% of body surface area, respectively). Although there were relatively few injured, a full-scale disaster response was evoked, providing an opportunity to study the effectiveness of current disaster plans. The rapid response of the ambulance service and the hospitals involved indicates that rehearsing disaster preparations is effective. The dispersal of the injured to several nearby hospitals prevented overloading of casualty departments. Secondary transfer of severely burned patients to the burn center was efficient and followed current guidelines. In retrospect, while communication between the various services might be subject to minor criticism, no problems were encountered which interfered with proper execution of the rescue operation. No one who survived the immediate impact died.