Alfredo Gragnani

Is There an Ideal Animal Model to Study Hypertrophic Scarring

Wound healing in hypertrophic scarring and keloid animal models presents significant differences when compared with humans. A brief review is presented about hypertrophic scarring in animal models during the last 5 years. Models were described by animals and scientific artifices to cause hypertrophic scarring. They were divided into 1) heterologous hypertrophic scarring or keloid implants in immunodeficient animals (athymic mice and rats); 2) heterologous hypertrophic scarring or keloid implant in immune privileged site (hamster cheek pouch); 3) hypertrophic scarring or keloid induction via chemically mediated injury (guinea pigs); 4) hypertrophic scarring or keloid induction in anatomic specific site (rabbit ear); and the 5) porcine model. The ideal model would allow to research pathophysiology, histology, and molecular events during time and to test prophylactic and therapeutic treatments for humans. Some of these animals were useful to study specific steps of the scarring process and better understand abnormal wound healing, but none of them have a widespread use. Most recently, the female red Duroc pigs were validated as a new model, demonstrating its similarity to human conditions in different ways. Full-thickness human skin grafts in nude mice also represent improvement in the search of an ideal hypertrophic scarring animal model.

Topical tamoxifen therapy in hypertrophic scars or keloids in burns

As acute burn patients have experienced increasing survival rates, the number of patients who need specific care due to aberrant scarring is also increasing. The burned skin often responds with fibrotic tissue proliferation, which can lead to a hypertrophic scar or a keloid. Non-physiologic scars are mostly not acceptable for the burn patient. Intradermal and topical therapy in burns comprise the treatment of the skin injury and its possible texture, elasticity and color alterations with the aid of active substances that result in fibroblastic modulation. An alteration of cytokine levels may mediate these effects, and evidences suggest that keloid scar formation may be mediated, in part, by deranged growth factor activity, including that of transforming growth factor (TGF)-β1. The addition of tamoxifen, a non-steroidal anti-estrogen, usually used in breast cancer, to standard treatment may lead to improved wound healing in keloids by decreasing the expression of TGF-β1, with the consequent inhibitions of both fibroblast proliferation and collagen production. Topical tamoxifen citrate chemical treatment has been shown to improve scarring. However, prospective studies must be undertaken to validate the inclusion of tamoxifen into standard clinical practice.

Experimental model for fibroblast culture

The use of cell culture methods in Plastic Surgery opened a new horizon in the research of cellular mechanisms of proliferation and biosynthesis functions. Several types of cells have been investigated in the cutaneous compartment. Keratinocytes and fibroblasts have been studied aiming the possibility of developing biomaterial for skin substitution. The present study describes the standardization for the development of fibroblast primary culture, its utilization in experiments and its storage.

Control of the skin scarring response

There comes a time when the understanding of the cutaneous healing process becomes essential due to the need for a precocious tissue repair to reduce the physical, social, and psychological morbidity. Advances in the knowledge on the control of interaction among cells, matrix and growth factors will provide more information on the Regenerative Medicine, an emerging area of research in medical bioengineering. However, considering the dynamism and complexity of the cutaneous healing response, it is fundamental to understand the control mechanism exerted by the interaction and synergism of both systems, cutaneous nervous and central nervous, via hypothalamus hypophysis-adrenal axis, a relevant subject, but hardly ever explored. The present study reviews the neuro-immune-endocrine physiology of the skin responsible for its multiple functions and the extreme disturbances of the healing process, like the excess and deficiency of the extracellular matrix deposition.

Gene expression profile of cytokines and receptors of inflammation from cultured keratinocytes of burned patients

INTRODUCTION At all stages of wound healing, growth factors and cytokines play a particularly important role in the interaction with keratinocytes cellular receptors. Keratinocytes have received little attention about their potential to act as a source and target of cytokines. Changes in the cytokine levels after the burning occur prior to the metabolic abnormalities. Thus, it may be possible to develop therapeutic interventions that can mitigate the acute inflammatory response and modulating expression of these cytokines. The objective was to evaluate the expression of 84 genes mediators of the inflammatory response by using PCR array in a primary human epidermal cultured keratinocytes from patients with burns. METHODS Keratinocytes cultured from normal skin around injury from small and large burn patient were treated for DNA synthesis. The samples were analyzed by the PCR Superarray(®) assay and curve analyses were performed for 84 relevant human genes and their involvement in the inflammatory cytokines pathway and receptors. These genes were checked for the up or down regulation. And it was used MetaCore™ for the analysis of networks and Gene Ontology (GO) processes. RESULTS Chemokines of the CXC family were more expressed in the large burn group, except CXCL12. The C, CC and CX3C chemokine family were downregulated, especially in the small burn group. The interleukins IL8 and IL1B were more expressed in large burn than in small burn; except IL13RA1, IL13 and IL5RA that were downregulated, mainly in the small burn group. CONCLUSIONS The cytokine profile showed some important differences between the large and small burn patients, and from this original database, we can create new interventional trials in acute inflammation in burns.

Rat an experimental model for burns: A systematic review

PURPOSE To revise and systematize scientific knowledge of the experimental model for cutaneous burns in rats. METHODS A bibliographical review from 2008 up to January 2011 in PubMed, EMBASE and LILACS was undertaken. Were used the keywords: animal models, burns and rats. 221 studies were identified, and 116 were selected. RESULTS It was found that: 54/86 (62.7%) had third degree burns; 55/73 (75.3%) studied the back; 45/78 (57.6%) used heated water and 27/78 (35.9%) incandescent instruments; 39/78 (50%) studied systemic effects; 22/71 (31%) used ketamine associated with xylazine; 61/64 (95.3%) performed depilation with appropriate equipment; 36/72 (50%) used microscopy; more than 50% did not describe analgesia or antibiotics during the postoperative period; in 42/116 (36.2%) postoperative fluid therapy was performed; and the time interval after the burn, up to the beginning of the results analysis varied from 7s up to four weeks. Legislation issues on burn experiments are discussed. CONCLUSION The hot water was the main method to induce burns those of third degree on the back, with anesthesia using ketamine and xylazine, after depilation. These were evaluated microscopically, without using analgesia or an antibiotic during the postoperative period. The studies were not very reproducible.

Hemicellulose dressing versus rayon dressing in the re-epithelialization of split-thickness skin graft donor sites: a multicenter study

The aim of this study was to compare the effectiveness of a hemicellulose dressing with that of rayon dressing in the healing of split-thickness skin graft donor sites. Twenty-eight patients were selected from five different hospitals and randomized into two groups: hemicellulose dressing group and rayon dressing group. All patients underwent split-thickness skin grafting for various reasons, and the skin graft donor site wounds were covered with hemicellulose dressing (n=14) or rayon dressing (n=14). The donor site was assessed on postoperative days 1, 7, 14, 21, and 28 for hyperemia, pruritus, pain, exudate level, and adherence of the wound dressing. At the 60-day follow-up visit, the donor site was assessed again for pruritus and pain. Touch-pressure, thermal, and pain sensibility tests were performed preoperatively and on postoperative day 60 together with the assessment of color and texture of the re-epithelialized area. In all patients, re-epithelialization was completed between 14 and 21 days after surgery. There were no significant differences between the two groups with regard to pain, hyperemia, pruritus, exudate, and final appearance (color and texture) of the skin graft donor site. The rayon dressing provided significantly better adherence than the hemicellulose dressing, and both dressings showed similar results with regard to the parameters evaluated when used in the treatment of split-thickness skin graft donor sites.

Portland cement with additives in the repair of furcation perforations in dogs

PURPOSE To evaluate the use of Portland cements with additives as furcation perforation repair materials and assess their biocompatibility. METHODS The four maxillary and mandibular premolars of ten male mongrel dogs (1-1.5 years old, weighing 10-15 kg) received endodontic treatment (n=80 teeth). The furcations were perforated with a round diamond bur (1016 HL). The perforations involved the dentin, cementum, periodontal ligament, and alveolar bone. A calcium sulfate barrier was placed into the perforated bone to prevent extrusion of obturation material into the periradicular space. The obturation materials MTA (control), white, Type II, and Type V Portland cements were randomly allocated to the teeth. Treated teeth were restored with composite resin. After 120 days, the animals were sacrificed and samples containing the teeth were collected and prepared for histological analysis. RESULTS There were no significant differences in the amount of newly formed bone between teeth treated with the different obturation materials (p=0.879). CONCLUSION Biomineralization occurred for all obturation materials tested, suggesting that these materials have similar biocompatibility.

Root perforations treatment using mineral trioxide aggregate and Portland cements

PURPOSE: Clinical, radiological and histological evaluation of root perforations treated with mineral trioxide aggregate (MTA) or Portland cements, and calcium sulfate barrier. METHODS: One molar and 11 premolar teeth of a male mongrel dog received endodontic treatment and furcations were perforated with a high-speed round bur and treated with a calcium sulfate barrier. MTA, Portland cement type II (PCII) and type V (PCV), and white Portland cement (WPC) were used as obturation materials. The teeth were restored with composite resin and periapical radiographs were taken. The animal was euthanized 120 days post-surgery for treatment evaluation. RESULTS: Right lower first premolar (MTA), right lower third premolar (PCV), left lower second premolar (MTA), and right lower second premolar (WPC): clinically normal, slightly radio-transparent area on the furcation, little inflammatory infiltrate, and new-bone formation. Left lower third premolar (PCII), right upper first premolar (WPC), right upper third premolar (PCII), and left upper first molar (PCV): clinically normal, radiopaque area on the furcation, and new-bone formation. Right upper second premolar (MTA), left upper second premolar (WPC), left upper third premolar (PCII): presence of furcation lesion, large radiolucent area, and intense inflammatory infiltrate. CONCLUSION: All obturation materials used in this study induced new-bone formation.

Keratinocyte growth factor, tumor necrosis factor-alpha and interleukin-1 beta gene expression in cultured fibroblasts and keratinocytes from burned patients

PURPOSE To evaluate the gene expression of KGF, TNF-alpha and IL-1 beta in skin fibroblasts and keratinocytes cultured from burned patients. METHODS Three patients with large burns and three patients with small burns, as well as two controls, were included. The cell culture was initiated by the enzymatic method. After extraction and purification of mRNA, qPCR was used to assess the gene expression of KGF, TNF-alpha and IL-1 beta. RESULTS The expression of KGF was increased on average 220-fold in large burns and 33.33-fold in small burns in fibroblasts, and 11.2-fold in large burns and 3.45-fold in small burns in keratinocytes compared to healthy patients (p<0.05). Expression of TNF-alpha was not observed. IL-1 beta is down-regulated in fibroblasts of burned patients, and much more repressed in small burns (687-fold, p<0.05). In keratinocytes, the repression of IL-1 beta expression occurs in patients with small burns (28-fold), while patients with large burns express this gene intensively (15-fold). CONCLUSIONS The study showed a quantitative pattern in the expression of KGF gene, which is more expressed according to the size of the burn. TNF-alpha was not expressed. A qualitative pattern in the expression of IL-1 beta gene was demonstrated.