Polly Carson

Full-thickness wounding of the mouse tail as a model for delayed wound healing: accelerated wound closure in Smad3 knock-out mice

Experimentally induced wounds in animal models are useful in gaining a better understanding of the cellular and molecular processes of wound healing, and in the initial evaluation of the safety and effectiveness of potential therapeutic agents. However, studying delayed healing has proved difficult in animals, whose wounds heal within a few days. In this report, we describe a novel method for establishing mouse wounds that require up to 3 weeks or more for complete closure, and we show the validity of this model in Smad3 null mice, which are known to display accelerated healing. Full‐thickness wounds, measuring 0.3 by 1.0 cm, were made down to fascia on the dorsal aspect of the mouse tail in Smad3 knock‐out mice and control littermates, approximately 1 cm distal to the body of the animal. The wounds were left to heal by secondary intention and were assessed histologically by computerized planimetry for wound closure at various times after wounding. The wounds in wild‐type mice displayed delayed healing, with full closure occurring between 14 and 25 days after wounding. Complete closure of similar wounds in Smad3 null mice healed 30 percent faster (p < 0.01). By immunostaining for ki67, a marker for proliferation, Smad3 null animals also showed increased proliferation of dermal wound cells by day 4 after wounding. Cultured dermal fibroblasts from Smad3 null mice had increased baseline DNA synthesis and, interestingly, an enhanced response to transforming growth factor‐β1. By Western blot analysis, Smad3 null mice fibroblasts showed a compensatory increase in mitogen‐activated protein kinase phosphorylation in response to transforming growth factor‐β1, suggesting that mitogen‐activated protein kinase overcompensation together with loss of Smad3 may be involved in the modulation of faster healing. We conclude that this novel tail‐wounding model may be useful for studying delayed wound closure.

The safety and efficacy of a proteolytic ointment in the treatment of chronic ulcers of the lower extremity.

BACKGROUND Elase is a widely used ointment consisting of a combination of 2 proteolytic enzymes, fibrinolysin and desoxyribonuclease (DNAse). It is said to promote debridement of necrotic and purulent debris from skin ulcers. OBJECTIVE Our purpose was to assess the efficacy and safety of this ointment and its components in the treatment of chronic ulcers of the lower extremity. METHODS This was a double-blind, randomized, prospective study of 84 patients with leg ulcers exhibiting necrotic and purulent debris, who were treated for 21 days with twice-daily applications of the ointment, fibrinolysin, DNAse, or who received the ointment vehicle (placebo). We assessed 6 efficacy features: ulcer size, purulent exudate, necrotic tissue, erythema, pain, and overall condition of the lesion at days 8, 15, and 21 after initiation of treatment. We also assessed the frequency of adverse effects. RESULTS All treatments produced some improvement in the efficacy parameters and overall condition of the ulcers by week 3, but no statistically significant difference was found when compared with placebo. No serious adverse effects were noted. A later retrospective reanalysis of the data found a statistically significant reduction of purulent exudate only at days 3 and 7 of treatment in the group treated with the complete ointment, but not in the other features. CONCLUSION The proteolytic ointment provides no long-term clinical benefit in reducing purulent exudate, pain, erythema, necrotic tissue, or overall condition of chronic leg ulcers when compared with either of its two components or placebo.

Human β-defensin-2 expression is increased in chronic wounds

First identified in psoriatic epidermis and subsequently in other inflammatory cutaneous lesions, human β‐defensin‐2 (hβD‐2) is one of two endogenous antimicrobial peptides related to defensins in plants and animals. Our objective was to determine the expression of hβD‐2 after injury and in chronic wounds. Biopsies of normal ipsilateral thigh skin and wound edges were taken from nine consecutive patients with venous leg ulcers (day 1) and from the same biopsy sites 2 days later (day 3). Sequential samples were also obtained from intact or meshed bilayered bioengineered skin consisting of neonatal human keratinocytes and dermal fibroblasts in a collagen matrix. Specimens were processed and immunostained for hβD‐2 using a polyclonal rabbit antibody. In both human tissues and bioengineered skin, staining for hβD‐2 was confined to the upper epidermal layers, sparing the basal cells. Analysis of 26 tissue samples from patients showed that normal skin had no hβD‐2 expression but that marked up‐regulation occurred after wounding by day 3. Conversely, chronic ulcers showed moderate‐to‐strong immunostaining for hβD‐2 at baseline on day 1, with little or no change in intensity after wounding by day 3. In vitro, bioengineered skin showed increased distribution of cytoplasmic hβD‐2 immunostaining after meshing. We conclude that the expression of hβD‐2 is up‐regulated after injury. Chronic wounds uniformly show a constitutively high baseline expression of hβD‐2, possibly due to ongoing tissue injury and bacterial colonization.

Wound edge biopsy sites in chronic wounds heal rapidly and do not result in delayed overall healing of the wounds.

Wound biopsies are an essential diagnostic component in the management of chronic wounds. First, the possibility of malignancy or infection in the wound often requires sampling of the wound edge and its bed. Secondly, several practice guidelines recommend biopsying wounds that have not responded to treatment after 2–6 weeks. However, there has always been a concern that the biopsy may worsen the wound and delay overall healing. In this report, we investigated the safety and effects of wound biopsies on overall chronic wound healing rates (advance of the wound edge per week toward the center) before and after the biopsy was performed. In a cohort of 14 consecutive patients with chronic wounds of the lower extremity, we found that postbiopsy chronic wound healing rates (0.99±1.18 mm/week; mean±SD) were not decreased and were actually higher than prebiopsy chronic wound healing rates (0.49±0.85 mm/week; mean±SD, p<0.05). In addition, we documented that healing of the biopsy sites up to the original wound edge occurred within 6 weeks in 11 of the 14 subjects. Therefore, we conclude that chronic wounds do not worsen after being biopsied and that wound biopsies are a safe procedure that does not delay overall healing of the chronic wound.

Mupirocin treatment of exudative venous ulcers

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Safety and Efficacy of a Bilayered Skin Construct in Full-Thickness Surgical Wounds

This was a prospective, randomized, controlled clinical trial assessing the safety and efficacy of a living bilayered skin construct (BSC; Organogenesis, Canton, MA) in treating full‐thickness surgical excision wounds. We enrolled 31 patients needing excision of a non‐melanoma skin cancer. The patients consisted of 18 females and 13 males, with an average age of 67 years (range: 44 to 84 years). Patients were randomized to either receive a single application of BSC or to heal by secondary intention. Endpoints to assess efficacy included time to complete wound closure, intensity and duration of post‐operative pain, cosmetic appearance, patient satisfaction, and quality of the healed wound. Endpoints to assess safety included infection at the wound site and rejection of the BSC. Findings indicate that BSC is safe in the post‐operative treatment of acute surgical wounds for removal of non‐melanoma skin cancer. The data also suggest that BSC may facilitate management by decreasing post‐operative pain. It is unclear whether or not BSC decreases healing time of acute wounds or results in a better cosmetic outcome.

Topically applied recombinant tissue plasminogen activator for the treatment of venous ulcers. Preliminary report.

background Increasing evidence suggests that fibrin deposition is an important pathogenic component of venous ulceration and that fibrin removal could accelerate ulcer healing. objective We sought to determine whether topical application of recombinant tissue plasminogen activator (tPA) compounded in 1% hyaluronate acid (HA) can be used safely in venous ulcers and whether it can accelerate healing. methods Twelve patients were randomized in a double‐blind fashion in three sequential groups of four subjects each, so as to receive daily topical application of either placebo (HA alone, one patient) or tPA/HA (three patients) at escalating doses of 0.25, 0.5, and 1.0 mg/ml of tPA for 4 weeks. results No safety problems occurred, and we found a close direct correlation between mean ulcer reepithelialization, fibrin removal, and the dose of topically applied tPA (r = 0.991). conclusion In this first study to examine its usefulness, topically applied tPA appears to be a safe and promising agent for treating venous ulcers.

Differential keratin expression during epiboly in a wound model of bioengineered skin and in human chronic wounds.

Epiboly represents the process by which keratinocytes migrate to envelop a surface. The authors have been investigating a living bilayered skin construct (BSC) that is used in the treatment of lower extremity wounds due to venous insufficiency and diabetes. The construct demonstrates epiboly after injury and incubation in vitro, and this model may be useful for studying epidermal migration and the process of skin maturation. Punch biopsies of the construct in vitro were cultured and immunostained for specific keratins at baseline and at 24 to 72 hours. For comparison, skin biopsy specimens from human chronic venous ulcers and acute healing wounds were similarly processed. The authors found that K1 and K10 were fully expressed in the epidermis of the fully epibolized surface on BSC. K1 was also present in the migrating edge of specimens, whereas K10 was not detectable. K16 and K6 were evident in normal skin and the epibolized area of the construct; K6 expression was very prominent in the migrating edge. Importantly, K17 was distinctly limited to the epibolized surface and the migrating edge, and its expression was very similar to that observed in healing human wounds. In conclusion, differential expression of keratins in this epiboly model closely reflects in vivo studies and supports keratin specificity in the processes of migration and differentiation of new epidermis. Therefore, these findings provide further and important validity for the study of epithelialization and the hope of developing prognostic markers for venous ulcer healing.

Bone Marrow Cell Mobilization by the Systemic Use of Granulocyte Colony-Stimulating Factor (GCSF) Improves Wound Bed Preparation

Innovative approaches are needed to accelerate the healing of human chronic wounds not responding to conventional therapies. An evolving and promising treatment is the use of stem cells. Our group has previously described the use of expanded (in vitro) autologous stem cells aspirated from human bone marrow and applied topically in a fibrin spray to human acute and chronic wounds. More recently, we have sought ways to mobilize stem cells directly from the bone marrow, without in vitro expansion. In this report, we show that systemic injections of granulocyte colony-stimulating factor (GCSF) can mobilize stem cells from bone marrow into the peripheral blood and then to the wound site. Our objectives were to optimize parameters for this method by using mouse models and proof of principle in a human chronic wound situation. Mice were injected for 5 days with 2 different formulations of GCSF and compared to control saline. To monitor stem cell mobilization, flow cytometric measurements of Sca-1 and c-Kit and colony-forming cell assays were performed. Full-thickness tail wounds in mice were created and monitored for healing, and polyvinyl alcohol sponges were implanted dorsally to assess collagen accumulation. To determine bone marrow stem cell homing to the wound site, chimeric mice transplanted with Green Fluorescent Protein bone marrow cells were scanned by live imaging. Additionally, as proof of principle, we tested the systemic GCSF approach in a patient with a nonhealing venous ulcer. Our findings lay the ground work and indicate that the systemic administration of GCSF is effective in mobilizing bone marrow stem cells into the peripheral blood and to the wound site. These findings are associated with an increased accumulation of collagen and promising results in terms of wound bed preparation and healing.

Topical delivery of cultured stem cells to human non-healing wounds: GMP facility development in an academic setting and FDA requirements for an IND and human testing.

With increasing emphasis on translational research, the need for appropriate regulatory oversight and approval has become essential. The requirements of the Food and Drug Administration (FDA) for Investigational New Drug (IND) exemption in studies that are investigator-initiated have become increasingly stringent. Moreover, academic institutions have not had substantial experience in establishing Good Manufacturing Practice (GMP) facilities required for manipulating human cells in vitro and for chemical or biochemical manufacturing. GMP regulations are established by the FDA under the authority of the Federal Food, Drug and Cosmetic Act. In this report, the authors outline the general strategy and some critical steps that an investigator and the institution may find helpful in developing a GMP facility, especially in an academic center. Also, more specifically and as proof of principle, we describe our approach to culturing autologous bone marrow-derived human mesenchymal stem cells (MSCs) and delivering them to non healing wounds. The lessons learned in this often lengthy and challenging process may be helpful to other academic institutions and investigators embarking on manipulating and delivering viable cells for human experimentation.