Tom Zimmerman

Prevention of Postsurgery-Induced Abdominal Adhesions by Electrospun Bioabsorbable Nanofibrous Poly(lactide-co-glycolide)-Based Membranes

Objectives:The objective of this study was to evaluate the efficacy of nonwoven bioabsorbable nanofibrous membranes of poly(lactideco-glycolide) for prevention of postsurgery-induced abdominal adhesions. Summary Background Data:Recent reports indicated that current materials used for adhesion prevention have only limited success. Studies on other bioabsorbable materials using a new fabrication technique demonstrated the promising potential of generating an improved and inexpensive product that is suitable for a variety of surgical applications. Methods:All rats underwent a midline celiotomy. The cecum was identified and scored using an abrasive pad until serosal bleeding was noted on the anterior surface. A 1 × 1 cm2 of abdominal wall muscle was excised directly over the cecal wound. The celiotomy was then closed in 2 layers immediately (control) after a barrier was laid in between the cecum and the abdominal wall. All rats underwent a second celiotomy after 28 days to evaluate the extent of abdominal adhesions qualitatively and quantitatively. Results:Cecal adhesions were reduced from 78% in the control group to 50% in the group using biodegradable poly(lactide-co-glycolide) (PLGA) nonwoven nanofibrous membranes (n = 10, P = 0.2) and to 22% in the group using membranes containing PLGA and poly(ethylene glycol)/poly(D,L-lactide) (PEG-PLA) blends (n = 9, P = 0.03). Electrospinning method also enabled us to load an antibiotic drug Cefoxitin sodium (Mefoxin; Merck Inc., West Point, PA) with high efficacy. The electrospun PLGA/PEG-PLA membranes impregnated with 5 wt% cefoxitin sodium, which amounts to approximately 10% of the systemic daily dose typically taken after surgery in humans, completely prevented cecal adhesions (0%) in rats. Conclusions:Electrospun nonwoven bioabsorbable nanofibrous membranes of poly(lactide-co-glycolide) were effective to reduce adhesions at the site of injury using an objective rat model. The membrane acted as a physical barrier but with drug-delivery capability. The combined advantages of composition adjustment, drug-loading capability, and easy placement handling (relatively hydrophobic) make these membranes potentially successful candidates for further clinical evaluations.

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.

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.

Resonant and notch behavior in intracranial pressure dynamics.

OBJECT The intracranial pulse pressure is often increased when neuropathology is present, particularly in cases of increased intracranial pressure (ICP) such as occurs in hydrocephalus. This pulse pressure is assumed to originate from arterial blood pressure oscillations entering the cranium; the fact that there is a coupling between the arterial blood pressure and the ICP is undisputed. In this study, the nature of this coupling and how it changes under conditions of increased ICP are investigated. METHODS In 12 normal dogs, intracarotid and parenchymal pulse pressure were measured and their coupling was characterized using amplitude and phase transfer function analysis. Mean intracranial ICP was manipulated via infusions of isotonic saline into the spinal subarachnoid space, and changes in transfer function were monitored. RESULTS Under normal conditions, the ICP wave led the arterial wave, and there was a minimum in the pulse pressure amplitude near the frequency of the heart rate. Under conditions of decreased intracranial compliance, the ICP wave began to lag behind the arterial wave and increased significantly in amplitude. Most interestingly, in many animals the pulse pressure exhibited a minimum in amplitude at a mean pressure that coincided with the transition from a leading to lagging ICP wave. CONCLUSIONS This transfer function behavior is characteristic of a resonant notch system. This may represent a component of the intracranial Windkessel mechanism, which protects the microvasculature from arterial pulsatility. The impairment of this resonant notch system may play a role in the altered pulse pressure in conditions such as hydrocephalus and traumatic brain swelling. New models of intracranial dynamics are needed for understanding the frequency-sensitive behavior elucidated in these studies and could open a path for development of new therapies that are geared toward addressing the pulsation dysfunction in pathological conditions, such as hydrocephalus and traumatic brain injury, affecting ICP and flow dynamics.

A simple orchidometric method for the preliminary assessment of maturity status in male cynomolgus monkeys (Macaca fascicularis) used for nonclinical safety studies.

INTRODUCTION The identification and use of mature male non-human primates in nonclinical toxicology studies could be important for evaluating candidate drugs for which the profile of toxicity may differ depending on sexual maturity. This investigation sought to establish operational criteria to complement the current standard of histological evaluation for defining sexual maturity in male cynomolgus monkeys (Macaca fascicularis) used for toxicology studies, and to identify a practical non-invasive measure to select mature males for study. METHOD Retrospectively, the relationships between body weight, testicular weight and testis histology were established in control males (n=126) used in previous toxicology studies. Prospectively, testicular volumes were measured in-life by orchidometry using comparative scrotal palpation (n=23 males used for study), then compared to testicular weights measured at necropsy. RESULTS Consistent with previous literature, a weak relationship was observed between body weight and testicular weight. There was, however, a very good relationship between testicular weight and histological maturation level, which was based upon microscopic examination of testes, epididymides and prostates. Orchidometric measurement of testicular volume was found to be a reasonable predictor of testicular weight and served to rapidly select sexually mature males for study, and a total testicular volume (left and right combined) of >20 ml correlated with the histological appearance of maturity. CONCLUSION Based upon this preliminary exploratory study, the initial simple measurement of testicular volume by orchidometry may provide a non-invasive alternative approach for assessing the sexual maturity of male cynomolgus monkeys in research colonies or during toxicology studies that will require more thorough validation.

An in-vivo study of the wound-bursting strengths of octyl-cyanoacrylate, butyl-cyanoacrylate, and surgical tape in rats.

BACKGROUND Several non-invasive wound-closure devices are available. Clinical studies of low-tension lacerations suggest similar clinical outcomes with these devices. OBJECTIVE We compared the wound-bursting strengths (WBS) of octyl-cyanoacrylate (Dermabond), butyl-cyanoacrylate (Histoacryl Blue), and adhesive tape (Steri-Strips). METHODS Design-randomized, controlled, blinded experiment. Setting-university-based division of laboratory animal research. Subjects-15 Sprague-Dawley rats weighing 250-350 g. Interventions-standardized 2-cm full-thickness incisions were made in duplicate on both sides of the rats dorsum with a #15 surgical blade and closed with one of the three study wound-closure devices following manufacturer instructions. The order of closure was randomized. Measurements-WBS was measured after wound closure with a validated vacuum-controlled wound chamber device (BT-2000) that measures the pressure required to disrupt the closed wound. Data analysis-between-group comparisons were performed with pair-wise t-tests and chi-squared tests. This study had 80% power to detect a 75-mm Hg between-group difference in WBS (two-tailed alpha = 0.05). RESULTS We evaluated 30 incisions in 15 rats. The mean WBS of octyl-cyanoacrylate (298 +/- 58 mm Hg) was significantly higher than that of butyl-cyanoacrylate (199 +/- 87 mm Hg; difference 98 mm Hg [95% confidence interval (CI) 32-165], p = 0.006) or Steri-Strips (129 +/- 67 mm Hg; difference 169 mm Hg [95% CI 112-227], p < 0.001). The WBS of butyl-cyanoacrylate was stronger than that of Steri-Strips; difference 71 mm Hg (95% CI 4-138), p = 0.035. CONCLUSIONS Octyl-cyanoacrylate tissue adhesive has a higher WBS than butyl-cyanoacrylate, whose WBS is greater than that of surgical tape.

Healing of mid-dermal burns in a diabetic porcine model.

Wound healing is delayed in diabetic patients. We developed a diabetic-porcine burn model and compared the healing of partial-thickness burns in normal and diabetic pigs. We hypothesized that wound healing would be delayed in the diabetic swine. Diabetes mellitus was chemically induced in three domestic pigs (25–50 kg) by intravenous injection of streptozotocin 130 mg/kg over 30 minutes. Glucose levels were maintained between 250 and 500 mg/dl by injecting short-acting or long-acting insulin 1 unit/kg daily as needed. Three weeks later, 14 partial-thickness burns were created on the backs and flanks of each of the anesthetized pigs with a 2.5 × 2.5-cm aluminum bar preheated to 80°C and applied for 20 seconds. A similar number of burns were created on three control nondiabetic pigs. The burns were treated with a topical antibiotic, and 3-mm full-thickness biopsies were taken from all wounds at 7, 10, 14, and 21 days for histomorphologic evaluation using hematoxylin and eosin staining by a board-certified dermatopathologist masked to the type of pig. The main outcome was the percentage of the wound in cross section that was reepithelialized. Comparison of outcomes between normal and diabetic pigs was performed with Student’s t-tests. The diabetic pigs gained less weight, and their skin was considerably thinner than in the control pigs. Although the absolute depth of the burns was similar, the relative depth was greater in the diabetic pigs. The percentage of wound reepithelialization was lower in diabetic than in normal pigs at 7 days (1.8% [95% CI: 0–5.5] vs 65.0% [95% CI: 54.2–75.9]; P < .001) as well as at 10 days (19.2% [95% CI: 6.0–32.4] vs 76.9% [95% CI: 59.8–94.0]; P < .001) and 14 days (43.9% [95% CI: 30.4–57.4] vs 99.9% [95% CI: 92.6–100]; P < .001). All burns were completely reepithelialized at 21 days, and none of the wounds were infected. Reepithelialization of partial-thickness burns is delayed in streptozotocin-induced diabetic pigs when compared with normal pigs. It is unclear whether the delay in healing is due to the thinner skin or the metabolic consequences of diabetes or their combination.

Porcine wound healing in full-thickness skin defects using Integra™ with and without fibrin glue with keratinocytes.

BACKGROUND An artificial dermal matrix such as Integra (Integra Life Sciences Corporation, USA) provides a wound bed template for vascular and fibrocyte ingrowth as well as collagen remodelling. Dermal repair leads to epidermal and basement membrane regeneration. Burn wounds in particular have been shown to benefit from Integra by enhanced wound healing. OBJECTIVE To evaluate the effect of fibrin glue to modify the integration of Integra in large excised cutaneous wounds. It was hypothesized that applying fibrin glue on a wound bed would reduce the time needed for matrix vascularization and incorporation of Integra and take of the cultured keratinocytes. METHODS Four separate full-thickness wounds were created on the dorsum of two swine. Wound beds were randomly assigned to either application of fibrin glue or no application of fibrin glue before application of Integra. Full-thickness biopsies were performed at days 7, 14, 21, 29 and 35. On day 21, keratinocytes were applied either as sheets or aerosolized fibrin glue suspension. RESULTS Histological analysis revealed a wave of inflammatory cells and early granulation tissue ingrowth into the Integra from the fascia below on day 7. Only this initial phase was augmented by application of fibrin glue to the wound bed. By day 14, most and by day 21, all of the Integra thickness was incorporated. Accelerated dermal repair proceeded from the base with new collagen deposition in Integra spaces. There was no evidence of keratinocyte engraftment, although re-epithelialization occurred at wound edges extending onto the incorporated Integra. CONCLUSIONS It appears there is an acceleration of early phase (day 7 to day 21) dermal incorporation with fibrin glue application to the wound bed, perhaps secondary to increased cellular migration. Day 21 appears to be too early to apply cultured keratinocytes either as sheets or aerosolized suspension.

Rosiglitazone, a PPAR-γ Ligand, Reduces Burn Progression in Rats

Burns induce the activation of an inflammatory cascade that generates reactive oxygen radicals and lipid peroxidation leading to burn wound progression and extension. Peroxisome proliferation-activated receptor-gamma is a nuclear hormone receptor that is activated by transcription factors and plays an important role in the regulation of cellular proliferation and inflammation. We hypothesized that treatment of burns with rosiglitazone, a peroxisome proliferation-activated receptor-gamma ligand, would reduce burn wound progression. This is a randomized controlled study of 20 Sprague-Dawley rats. Two burns were created on each animal’s 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 separated by three 5 × 20 mm unburned interspaces (zone of ischemia). Animals were randomized to rosiglitazone 4 mg/kg or vehicle by oral gavage 30 minutes after injury and at 24 and 48 hours after injury. Wounds were observed at 1, 2, 3, and 4 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 interspaces that progressed to necrosis was compared with &khgr;2 tests. Forty comb burns with 120 unburned interspaces were evenly distributed between rosiglitazone and vehicle. The number of interspaces that progressed to full thickness necrosis at 1, 2, 3, 4, and 7 days after injury in the rosiglitazone and vehicle groups were 9/60 (15%) versus 13/60 (21%) (P = .48), 16/60 (27%) versus 15/60 (20%) (P = 1.00), 24/60 (40%) versus 46/60 (77%) (P = .001), 35/60 (58%) versus 53/60 (88%) (P = .001), and 43/60 (72%) versus 54/60 (90%) (P = .02), respectively. Treatment with oral rosiglitazone reduces the percentage of unburned skin interspaces that progress to full necrosis in a rat comb burn model.

Controlled mild hypothermia prolongs survival in a rat model of large scald burns.

OBJECTIVES Early surface cooling of burns reduces pain and depth of injury and improves healing. However, there are concerns that cooling of large burns may result in hypothermia and worsen outcomes. In contrast, controlled mild hypothermia improves outcomes after cardiac arrest and traumatic brain injury. The authors hypothesized that controlled mild hypothermia would prolong survival in a rat model of large scald burns. METHODS Thirty-six Sprague-Dawley rats (250-300 g) were anesthetized with 40 mg/kg intramuscular ketamine and 5 mg/kg xylazine, with supplemental inhalational isoflurane as needed. A single full-thickness scald burn covering 40% of total body surface area was created on each of the rats using a Mason-Walker template placed in boiling water (100 °C) for a period of 10 seconds. The rats were then randomized to hypothermia (n = 18) or no hypothermia (n = 18). Core body temperature was continuously monitored with a rectal temperature probe. In the experimental group, mild hypothermia was induced by applying ice packs over the prone rats until their rectal temperature was reduced by 2 °C for a period of 2 hours. After 2 hours of hypothermia, the rats were rewarmed back to their baseline temperature with a heating pad. The control rats were not cooled. The rats were monitored until death or for a period of 7 days, whichever was greater. The primary outcome was time to death. The difference in survival between the groups was determined using Kaplan-Meier analysis and the log-rank test. RESULTS   Hypothermia was induced in all experimental rats within a mean of 22 minutes (95% confidence interval [CI] = 17 to 27). The numbers of nonhypothermic and hypothermic rats that were dead at each time point were as follows: 2 hours, five versus none; 18 hours, 16 versus five; 24 hours, 18 versus eight; and 48 hours, 18 versus 13 (p = 0.05). There were no additional deaths after 48 hours. The mean time to survival of the hypothermic rats was significantly greater than that of the nonhypothermic rats (p < 0.001). CONCLUSIONS Induction of brief, mild hypothermia prolongs survival and increases the survival rate in nonresuscitated rats with large scald burns.