Steve A. McClain

Wound Healing in Ovariectomized Rats Effects of Chemically Modified Tetracycline (CMT-8) and Estrogen on Matrix Metalloproteinases -8, -13 and Type I Collagen Expression

Cutaneous wound healing is a complex process involving interactions of various cell types. Skin, in addition to certain other organs, is dependent on estrogen; and estrogen-deficiency is associated with impaired wound healing. Wound healing involves the action of collagenolytic matrix metalloproteinases (MMPs). We investigated the expression and localization of collagenolytic MMPs -8 and -13 by collagenase activity assay, Western immunoblot analysis, in situ hybridization and immunohistochemical staining as well as type I collagen by hydroxyproline content analysis and immunohistochemical staining in cutaneous wounds from aged Sham and ovarioectomized (OVX) rats. After wounding, OVX rats were treated with either placebo, chemically modified tetracycline-8 (CMT-8) or estrogen. We found that MMP-8 and MMP-13 mRNA were expressed in wound epithelium of all samples examined as evidenced by in situ hybridization. Type I collagen, which was abundant in all groups examined, was decreased in OVX rats, but was increased by both CMT-8 and estrogen treatments to the level of Sham group. Hydroxyproline analysis revealed similar results. Western blot data showed that all forms of MMP-8 and MMP-13 were clearly reduced in the CMT-8 treated group compared to OVX. Analysis of collagenolytic activity confirmed the decreased collagenolysis in skin wound extracts from CMT-treated rats when compared with skin wound extracts from OVX rats. Our results show for the first time that MMP-8 mRNA and protein are expressed in rat wound epithelium. We further show that CMT-8 and estrogen have a beneficial effect on skin wound healing in OVX rats by increasing the collagen content and reducing the MMP-mediated collagenolysis.

Persistent wound infection delays epidermal maturation and increases scarring in thermal burns

We developed a reliable scale measuring epidermal maturation during wound healing and determined the effects of persistent infection on epidermal maturation and dermal scarring after cutaneous burns. A secondary analysis of data from 80 contaminated burns collected during a randomized experiment comparing four topical burn therapies in a contaminated porcine burn model was performed. Persistent infection was defined histologically as the presence of intradermal neutrophils containing bacteria at 14 days. Epidermal maturation at 14 days was classified into one of five categories from least (0) to most (4) mature using strictly defined criteria. Dermal scarring was classified as none, superficial, or deep. The epidermal classification system was highly reliable (ρ = 0.97). At 14 days, 18% of burns were infected. Most infected wounds (79%) had an immature epidermis (types 0–2) while most noninfected wounds (75%) had a mature epidermis (types 3 or 4); χ2 and χ2 for linearity both p < 0.001. Deep scars were more common in infected (93%) than noninfected wounds (29%), p < 0.001. We conclude that our scale is reliable and that persistence of infection 14 days after thermal injury is associated with delayed epidermal maturation and deep scarring. (WOUND REP REG 2002;10:372–377)

Chemically modified tetracycline (CMT-8) and estrogen promote wound healing in ovariectomized rats: Effects on matrix metalloproteinase-2, membrane type 1 matrix metalloproteinase, and laminin-5 γ2-chain

Estrogen deficiency is associated with impaired cutaneous wound healing. Remodeling of the extracellular matrix in wound healing involves the action of matrix metalloproteinases on basement membrane zone components, especially laminin‐5. We studied the effects of estrogen and a potent matrix metalloproteinase inhibitor, chemically modified non‐antimicrobial tetracycline, CMT‐8, on wound healing in ovariectomized rats. At the tissue level, laminin‐5 γ2‐chain expression was decreased and the migration‐inductive 80 kDa form of laminin‐5 γ2‐chain was absent in ovariectomized rats when compared with sham and CMT‐8‐ or estrogen‐treated ovariectomized animals as detected by Western blotting. The highest levels of gelatinolytic activity (matrix metalloproteinase‐2 and ‐9) were found in sham animals. Levels were reduced in ovariectomized rats and were lowest after treating ovariectomized rats with CMT‐8 or estrogen as analyzed by functional activity assay and zymography. The total amount of membrane type 1‐matrix metalloproteinase was unchanged in all groups. We conclude that CMT‐8 and estrogen can promote wound healing in ovariectomized rats, not only by normalizing wound bed total collagen content and structure, but also by recovering the expression and processing of key molecules in wound healing, i.e., laminin‐5 γ2‐chain. This study shows, for the first time, the role of estrogen and CMT‐8 in laminin‐5 γ2‐chain modulation in vivo.

Rapid and selective enzymatic debridement of porcine comb burns with bromelain-derived Debrase: acute-phase preservation of noninjured tissue and zone of stasis.

Deep burns are associated with the formation of an eschar, which delays healing and increases the risk of infection. Surgical debridement of the eschar is, at present, the fastest means to achieve an eschar-free bed, but the process can not differentiate between the viable tissue and the eschar and follow the minute irregularities of the interface between the two. We evaluated the efficacy and selectivity of a novel enzymatic bromelain-based debriding agent, Debrase® Gel Dressing (Debrase®), in a porcine comb burn model. We hypothesized that Debrase® would result in rapid debridement of the eschar without adverse effects on the surrounding uninjured skin. This is a prospective, controlled, animal experiment. Five domestic pigs (20–25 kg) were used in this study. Sixteen burns were created on each animals dorsum using a brass comb with four rectangular prongs preheated in boiling water and applied for 30 seconds, resulting in four rectangular 10 × 20 mm full-thickness burns and separated by three 5 × 20 mm unburned interspaces representing the zone of stasis. The burned keratin layer (blisters) was removed, and the burns were treated with a single, topical, Debrase® or control vehicle application for 4 hours. The Debrase®/control was then wiped off using a metal forceps handle, and the burns were treated with a topical silver sulfadiazine (SSD). The wounds were observed, and full-thickness biopsies were obtained at 4 and 48 hours for evidence of dermal thickness, vascular thrombosis, and burn depth, both within the comb burns and the unburned interspaces in between them. Chi-square and t tests are used for data analysis. A single 4-hour topical application of Debrase® resulted in rapid and complete eschar dissolution of all the burns in which the keratin layer was removed. The remaining dermis was thinner (1.1 ± 0.7 mm) than in the control burns (2.1 ± 0.3 mm; difference 0.9 mm [95% confidence interval: 0.3–1.4]) and was viable with no injury to the normal surrounding skin or to the unburned interspaces between the burns, which represents the zone of stasis. In control burns, the entire thickness of the dermis was necrotic. At 48 hours, Debrase®-treated areas were found partially desiccated under SSD treatment. The unburned interspaces demonstrated partial-thickness necrosis in two third and full-thickness necrosis in one third of wounds. In contrast, full-thickness necrosis was noted in all control interspaces (P = .05). In a porcine comb burn model, a single, 4-hour topical application of Debrase® resulted in rapid removal of the necrotic layer of the dermis with preservation of unburned tissues. At 48 hours, SSD treatment resulted in superficial tissue damage and partial preservation of the unburned interspaces.

Spatiotemporal progression of cell death in the zone of ischemia surrounding burns

Burns are dynamic injuries, characterized by progressive death of surrounding tissue over time. Although central to an understanding of burn injury progression, the spatiotemporal degrees and rates of cellular necrosis and apoptosis in the zone of ischemia surrounding burns are not well characterized. Using a validated porcine hot comb model, we probed periburn tissue at 1, 4, and 24 hours after injury for high‐mobility group box 1 as a marker of necrosis and activated cleaved caspase‐3 as a marker of apoptosis, followed by spatiotemporal morphometric analysis. We found that necrosis was the most prominent mechanism of cell death in burn injury progression, with significant progression between 1 and 4 hours postburn. Apoptosis appeared not to play a role in early burn injury progression but was observed in cells at the interface of necrotic and viable tissue at 24 hours postburn. Our findings imply that intervention within the first 4 hours following injury is likely necessary to limit burn injury progression. Additionally, based on high‐mobility group box 1 staining patterns, we define distinct early, intermediate, and late pathological signs of cell necrosis that may facilitate delineation of causal mechanistic relationships of burn injury progression in vivo.

A novel TGF-beta antagonist speeds reepithelialization and reduces scarring of partial thickness porcine burns.

Scar formation after thermal injury is common and results in significant aesthetic and functional impairment. Transforming growth factor beta (TGF-β) plays a significant role in scar formation. We tested the hypothesis that a novel TGF-β peptantagonist would reduce scar formation and wound contraction in partial thickness burns by using a randomized controlled experiment. The subjects include two domestic pigs (20–25 kg). Forty burns were created on the animal’s dorsum using an aluminum bar preheated to 80°C and applied for 20 seconds resulting in a partial thickness thermal burn extending half way down the dermis. Burns were treated every other day for 1 week, then twice weekly for 3 weeks with a topical TGF-β antagonist or its vehicle. Full thickness biopsies were obtained from all burns at 7, 10, and 14 days after injury. The wounds were completely excised after 28 days for histological assessment. Wound sections were stained with H&&E and evaluated by a dermatopathologist masked to treatment assignment for reepithelialization and depth of scar formation. We also determined the number of wounds at 28 days that healed with contracted, hour-glass shaped scars. Data were compared with &khgr;2 and t-tests. Twenty burns were treated with TGF-β antagonist and 20 with control vehicle. TGF-β antagonist increased the percentage of completely reepithelialized wounds at 14 days (90 vs 45%, P = .002) and reduced the percentage of contracted wounds (35 vs 65%, P = .02) and full thickness scars (10 vs 60%, P = .002) at 28 days. Treatment of partial thickness porcine burns with the TGF-β antagonist speeds reepithelialization and reduces scar formation and wound contraction in partial thickness porcine burns.

Curcumin reduces injury progression in a rat comb burn model.

The oriental spice curcumin has anti-inflammatory and antioxidant effects. When given orally before injury, curcumin reduces burn progression in a rat comb burn model. The authors hypothesized that intravenous administration of curcumin after injury would reduce burn progression and that its effects are mediated through iron chelation. Two comb burns were created on the dorsum of Sprague-Dawley rats (weight, 300 g) using a brass comb with four rectangular prongs preheated in boiling water and applied for 30 seconds resulting in four rectangular 10 × 20 mm full-thickness burns separated by three 5 × 20 mm unburned interspaces (zone of ischemia). Animals were randomized to receive one of four doses of crude curcumin or one of six doses of purified curcumin intravenously 1 and 24 hours after injury. Another set of animals were randomized to deferoxamine or control vehicle. Wounds were observed at 7 days after injury for visual evidence of necrosis in the unburned interspaces. Full-thickness biopsies from the interspaces were evaluated with Hematoxylin and Eosin staining 7 days after injury for evidence of necrosis. The percentage of unburned interspaces undergoing necrosis at 1 week by purified curcumin doses was 0 &mgr;g/kg, 74%; 0.3 &mgr;g/kg, 58%; 1 &mgr;g/kg, 53%; 3 &mgr;g/kg, 37%; 10 &mgr;g/kg, 63%; 30 &mgr;g/kg, 53%; and 100 &mgr;g/kg, 26%. The differences among the groups were significant (P = .03). When compared with controls, the 1 and 3 &mgr;g/kg curcumin treatment groups had significantly less progression of interspaces to necrosis (P = .04 and .002) as did the 30 and 100 &mgr;g/kg treatment groups (P = .03 and <.001). Deferoxamine did not reduce burn progression. When administered intravenously 1 and 24 hours after injury, both crude and purified curcumin reduce the percentage of unburned interspaces that undergo necrosis in a rat hot comb burn model. The effects of purified curcumin appear to be bimodal, suggesting more than one mechanism of action. The effects of curcumin do not appear to be mediated by iron chelation.

Validation of a porcine comb burn model

OBJECTIVE A brass comb burn model that creates 3 full-thickness burns separated by 3 interspaces of unburned skin representing the zone of ischemia has been described in rats. We evaluated this model in pigs. METHODS Design--observational. Subjects--6 pigs (20-25 kg). Interventions--4 burns created on each animal on the dorsum using a brass comb with 4 rectangular prongs preheated in boiling water and applied for 30 seconds resulting in 4 rectangular 10 x 20-mm full-thickness burns separated by three 5 x 20-mm unburned interspaces. Outcomes--wounds observed at 1, 2, 3, and 7 days for evidence of necrosis in unburned interspaces. Full-thickness biopsies from interspaces were evaluated with hematoxylin-eosin staining 7 days after injury for evidence of necrosis. Data analysis--the percentages of interspaces progressing to necrosis were analyzed with descriptive statistics. RESULTS Twenty-four comb burns with 72 unburned interspaces were created evenly distributed between the animals. The percentages of interspaces that progressed to full-thickness necrosis at 1, 2, 3, and 7 days after injury were 88.9% (64/72; 95% confidence interval [CI], 79.6%-94.3%), 88.9% (64/72; 95% CI, 79.6%-94.3%), 88.9% (64/72; 95% CI, 79.6%-94.3%), and 97.7% (70/72; 95% CI, 90.4%-99.2%), respectively. There was perfect agreement between gross inspection and histomorphology. CONCLUSIONS The comb burn model in swine results in the progression of most unburned ischemic interspaces to full-thickness necrosis within 1 to 7 days.

Validation of a vertical progression porcine burn model.

A major potential goal of burn therapy is to limit progression of partial- to full-thickness burns. To better test therapies, the authors developed and validated a vertical progression porcine burn model in which partial-thickness burns treated with an occlusive dressing convert to full-thickness burns that heal with scarring and wound contraction. Forty contact burns were created on the backs and flanks of two young swine using a 150 g aluminum bar preheated to 70°C, 80°C, or 90°C for 20 or 30 seconds. The necrotic epidermis was removed and the burns were covered with a polyurethane occlusive dressing. Burns were photographed at 1, 24, and 48 hours as well as at 7, 14, 21, and 28 days postinjury. Full-thickness biopsies were obtained at 1, 4, 24, and 48 hours as well as at 7 and 28 days. The primary outcomes were presence of deep contracted scars and wound area 28 days after injury. Secondary outcomes were depth of injury, reepithelialization, and depth of scars. Data were compared across burn conditions using analysis of variance and &khgr;2 tests. Eight replicate burns were created with the aluminum bar using the following temperature/contact-time combinations: 70/20, 70/30, 80/20, 80/30, and 90/20. The percentage of burns healing with contracted scars were 70/20, 0%; 70/30, 25%; 80/20, 50%; 80/30, 75%; and 90/20, 100% (P = .05). Wound areas at 28 days by injury conditions were 70/20, 8.1 cm2; 70/30, 7.8 cm2; 80/20, 6.6 cm2; 80/30, 4.9 cm2; and 90/20, 4.8 cm2 (P = .007). Depth of injury judged by depth of endothelial damage for the 80/20 and 80/30 burns at 1 hour was 36% and 60% of the dermal thickness, respectively. The depth of injury to the endothelial cells 1 hour after injury was inversely correlated with the degree of scar area (Pearsons correlation r = −.71, P < .001). Exposure of porcine skin to an aluminum bar preheated to 80°C for 20 or 30 seconds results initially in a partial-thickness burn that when treated with an occlusive dressing progresses to a full-thickness injury and heals with significant scarring and wound contracture.

Development of a Porcine Excisional Wound Model

OBJECTIVES To develop a porcine model for cutaneous tangential excisional wounds. METHODS This was a prospective, longitudinal, blinded experiment on four isoflurane-anesthetized swine. Forty standardized excisional wounds (1 square inch each) were created with an electric dermatome set at a depth of 300, 600, 750, or 900 microm. Full-thickness wounds were created with a surgical blade (n = 8 for each depth). Wounds were treated with polyurethane film. Full-thickness punch biopsies were taken after one, two, three, four, five, and 14 days for blinded histopathological evaluation. The main outcome was the percent wound reepithelialization (REP) calculated by dividing the length of neoepidermis in cross-section by the specimens diameter (interobserver correlation = 0.99). Analysis of variance was used to compare percent REP among wound depths. This study had 80% power to detect a 33% difference in REP across groups (two-tailed alpha = 0.05). RESULTS There were significant differences in percent REP across wound depths for days 2-5 (p < 0.001 for all days). The more superficial wounds (300 to 600 microm) were mostly reepithelialized by day 4, whereas wounds ranging in depth from 750 to 900 micro m were mostly healed by day 5. Full-thickness wounds had no evidence of REP even after 14 days. CONCLUSIONS The authors describe an excisional wound model in swine. Rapid reepithelialization occurs within three to five days in wounds <900 microm deep.