Viability and Apoptosis of Wound Fibroblasts After Cryopreservation
Abstract: The optimal serum concentration for cryopreserving dermal fibroblasts has not been properly defined. Using flow cytometry, the authors determined whether human dermal fibroblasts from acute and chronic human wounds display differences in viability and apoptosis depending on the percent of fetal bovine serum (FBS) used for their cryopreservation. Fibroblast cultures were established from venous ulcers and from three-day-old acute wounds made on the thigh of each patient by punch biopsy. Cultures in their three to five in-vitro passage were cryopreserved for four weeks in Dulbecco?s Modified Eagle?s Medium (DMEM) plus dimethyl sulfoxide (DMSO, 10%) and 0-, 10-, 20-, 50-, and 80-percent FBS. After thawing, cells were allowed to attach to tissue culture plastic for four hours. Flow cytometry using propidium iodide (PI) to test for cell viability and Annexin V for early apoptosis was performed on attached cells and those still in the culture supernatant. Attached cells from acute and chronic wounds showed no differences in cell viability (greater than 90%) or apoptosis (less than 5%) at the various FBS concentrations. However, in unattached fibroblasts from acute wounds, increasing FBS concentrations were associated with up to 30 percent more viability and less apoptosis (p=0.0203 and p=0.0317, respectively; two-way analysis of variance). The authors then tested dermal fibroblasts cultured from chronic wounds, because they are known to be senescent and are more difficult to cryopreserve. Using the same methodology, the authors found that 20-percent FBS was best for preserving cell viability (p=0.019) and that higher FBS concentrations had no additional beneficial effect on cell viability and apoptosis. The authors conclude that maximum FBS concentrations, up to 80 percent, may be best for cryopreserving dermal fibroblasts from acute wounds. However, it may not be possible to extrapolate these findings to senescent fibroblasts or fibroblast cultures from chronic wounds or diseased skin.
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Editorial Message
In the article by Weed, et al. The use of in-vitro culture systems is important for understanding cell behavior seen in wound healing. Etöz, et al.
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Wound Bed Preparation: The Science Behind the Removal of Barriers to Healing [PART 2]
Abstract: Wound healing involves a well-orchestrated, complex process leading to repair of injured tissues. However, chronic wounds do not follow the normal pattern of repair. This is due to underlying physiological problems associated with their development, which unless corrected would continue to cause wound deterioration. The key to effective wound care lies in a combination of three approaches: treatment of underlying medical problems, assessment and treatment of local wound bed, and effective management of any patient-centered concerns. An essential component of this recommended approach is restoration of healthy granulation tissue in the wound bed. Wound bed preparation brings a number of existing procedures, including debridement, treatment of infection, and management of exudate levels, together into a systematic approach to help restore the chronic wound bed environment. The aim of wound bed preparation is to remove the barriers to healing and initiate the repair process. This review explores the scientific rationale behind this concept and examines how wound bed preparation offers healthcare professionals an improved paradigm for the treatment of chronic wounds. By implementing wound bed preparation, the formation of healthy granulation tissue will be optimized and the efficiency of biotechnological therapies improved, which would ultimately reduce the time to wound closure.
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Debridement: Rationale and Therapeutic Options
Abstract: Debridement is commonly defined as the process of removing necrotic, devitalized tissue and foreign material from a wound. The presence of necrotic tissue within a wound may impair wound repair processes by stimulating inflammation and delaying granulation and epithelialization. However, the above definition of debridement may not tell the whole story. Debridement may additionally remove senescent cells from the wound bed and nonmigratory cells from the ulcer edge and also remove excessive or abnormal bacteria; all of which may allow for improved availability of growth factors. This supplement will review the rationale for debridement, existing clinical data supporting debridement, and the various debridement options available. The supplement was supported through an unrestricted education grant from Healthpoint Ltd., Ft. Worth, Texas.
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Functions of Chicken IL-8 (cCAF) in Wound Healing
Abstract: The chicken IL-8 (ortholog of human IL-8) was the first inducible chemokine gene to be discovered. cIL-8 was originally named 9E3/CEF4 from the name of the cDNA clone when it was first isolated. Later, the product of the gene was named after its biological functions as the chicken chemotactic and angiogenic factor (cCAF). This chemokine is expressed at very low levels in normal tissues, but expression becomes elevated rapidly upon wounding. The most potent natural activator of cIL-8 expression is thrombin, an enzyme that is abundantly produced at sites of wound and tissue damage. The author has previously shown that this chemokine is chemotactic for leukocytes and fibroblasts, is angiogenic, and stimulates differentiation of fibroblasts into myofibroblasts. Chemotaxis for leukocytes requires the full-length protein and contributes to the inflammatory phase of healing, whereas chemotaxis for fibroblasts provides the wound with additional fibroblasts, cells that play a major role in development of the granulation tissue. Angiogenesis can be accomplished by the C-terminal 28 amino acids alone and contributes to the proper oxygenation and supply of nutrients to the wound. It involves destabilization of the endothelium with subsequent activation of enzymes that are critical for basal lamina degradation and endothelial cell migration. The differentiation-inducing effects of cIL-8 contribute to wound closure and contraction and can be accomplished by the N-terminal 15 amino acids alone. Furthermore, cIL-8 and its N-terminus stimulate expression of tenascin, a very important extracellular matrix molecule in wound healing. Based on these findings, this article provides a model by which cIL-8 and potentially hIL-8 function in accelerating and improving the healing process. These effects on the repair process may have important applications to healing of impaired wounds. A potentially very important characteristic of IL-8 chemokines is that they are inducible and, therefore, can be more safely targeted than const
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Use of Mouse Footpad Model to Test Effectiveness of Wound Dressings
Abstract: Acemannan, a complex carbohydrate consisting of a b-(1,4)-linked polymannose acetate, is a major carbohydrate component of aloe vera leaf pulp. To test the effectiveness of acemannan in a wound environment, we utilized a unique mouse footpad model. Chemically pure acemannan was applied topically to incised mouse tarsal wounds either in solution at three different concentrations or in the form of a gel. The wounds were scored by a double-blind procedure that permitted a controlled comparison between treated and control wounds. Statistical analysis determined wounds treated topically with acemannan resulted in significantly better wound healing as reflected by lower wound scores in treated feet compared to controls. This effect was dependent upon the dose of acemannan applied to the wound and was detectable when acemannan was applied in solution or in a gel. Other carbohydrate solutions were tested but showed no effect on wound healing. Because acemannan was suspected to stimulate macrophage cytokine production, mouse peritoneal macrophages were exposed to different doses of acemannan and other carbohydrates in vitro and resulted in a dose-dependent release of IL-1b, TNFa, and IL-6. We hypothesize the effect of acemannan on wound healing is probably mediated through local cytokine release from wound macrophages. Disclosure: This research was funded in part by Carrington Laboratories, Irving, Texas. Dr. Chinnah was employed by Texas A & M University during the period this research was performed. Dr. Tizard received a grant from Carrington Laboratories.
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PART II: Modulation of Radiation-Induced Delay in the Wound Healing by Ascorbic Acid in Mice Exposed to Different Doses of Hemi-Body Gamma Radiation
Abstract: Hemi-body irradiation in multiple fractionated doses is frequently used alone or in combination with surgery for the treatment of cancer. It produces both acute and late effects on the skin that have profound effects on surgical wounds. Because of the crucial practical importance of radiation exposure associated with skin wounds, it is imperative to investigate the efficacy of cost-effective nutritional factors in the reconstruction of irradiated wounds. Therefore, the effect of ascorbic acid treatment was studied on the healing of excision wounds in mice exposed to 2, 4, 6, or 8 Gy hemi-body gamma radiation. A full-thickness skin wound was created on the dorsum of 8- to 10-week-old Swiss albino mice after hemi-body exposure to 2, 4, 6, or 8 Gy. The progression of wound contraction was monitored periodically by capturing video images of wounds. Collagen, hexosamine, deoxyribonucleic acid (DNA), nitric oxide (NO), and histological profiles of excision wounds were also evaluated and either treated or not treated with ascorbic acid before exposure to 0 or 6 Gy. Irradiation caused a dose-dependent delay in wound contraction and wound healing time, while ascorbic acid pretreatment resulted in a significant elevation in the rate of wound contraction and a decrease in mean wound healing time. Treatment with ascorbic acid before irradiation enhanced the synthesis of collagen, hexosamine, DNA, and NO, while histological assessment revealed an improved collagen deposition and an increase in fibroblast and vascular densities. The present study demonstrates that ascorbic acid pretreatment has a beneficial effect on irradiated wounds and could be part of a strategy to ameliorate radiation-induced delay in wound repair.
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The Application of a Fibroblast Gel Contraction Model to Assess the Cytotoxicity of Topical Antimicrobial Agents
Abstract: Fibroblast culture systems are routinely used to investigate wound contraction under a wide range of experimental conditions. These include the effects of irradiation, inhibition of chronic inflammatory cell mediators, and the biocompatibility of wound management products. In particular, these in-vitro cell systems have been routinely used to assess the cytotoxicity of topical antimicrobial agents and dental materials. L929 cells, derived from an immortalized mouse fibroblast cell line, are internationally recognized cells that are routinely used in in-vitro cytotoxicity assessments. In these studies, it is proposed that equine granulation tissue fibroblasts, cultured from slow healing wounds or from granulating wounds with exuberant granulation tissue removed during normal surgical debridement, may also be used to assess in-vitro cytotoxicity, in particular with respect to topical wound healing products. This model demonstrated that granulation tissue fibroblasts behave similarly to L929 fibroblasts in that they were effective in differentiating the toxicity of a variety of topical iodine-containing formulations. The data presented in this report suggests that currently marketed iodine-containing antiseptic agents show variable toxicity to fibroblasts involved in wound healing. These results suggest that currently available topical iodine antiseptic agents could be detrimental to wound healing if treatment is prolonged.
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Debridement: Rationale and Therapeutic Options
Abstract: Debridement is commonly defined as the process of removing necrotic, devitalized tissue and foreign material from a wound. The presence of necrotic tissue within a wound may impair wound repair processes by stimulating inflammation and delaying granulation and epithelialization. However, the above definition of debridement may not tell the whole story. Debridement may additionally remove senescent cells from the wound bed and nonmigratory cells from the ulcer edge and also remove excessive or abnormal bacteria; all of which may allow for improved availability of growth factors. This supplement will review the rationale for debridement, existing clinical data supporting debridement, and the various debridement options available. The supplement was supported through an unrestricted education grant from Healthpoint Ltd., Ft. Worth, Texas.
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