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Diabetic wounds result in significant morbidity, prolonged hospitalization, and enormous health-care expenses. Pigs have been shown to have wound healing resembling that in humans. The aim of this study was to develop a large-animal model for diabetic wound healing. Diabetes was induced by streptozotocin injection in Yorkshire pigs. Full-thickness wounds were created and dressed with a sealed chamber. Nondiabetic pigs with or without high glucose wound fluid concentration served as controls. Glucose concentration in serum and wound fluid was measured and collected. Wound contraction was monitored, and biopsies were obtained for measurement of reepithelialization. Wound fluid was analyzed for insulin-like growth factor-1 (IGF-1), platelet-derived growth factor, and transforming growth factor. Glucose concentration in wound fluid initially followed serum levels and then decreased to undetectable on day 9. Reepithelialization was significantly delayed in diabetic pigs. In nondiabetic pigs, wounds treated in a local hyperglycemic environment, and thus excluding the effects of systemic hyperglycemia, showed no difference in wound closure compared with controls. This suggests that delayed wound healing in diabetes is not induced by local high-glucose concentration itself. Analysis of growth factor expression showed a marked reduction in IGF-1 in the diabetic wounds. Diabetic pigs have impaired healing that is accompanied by a reduction of IGF-1 in the healing wound and is not due to the local hyperglycemia condition itself.

We have previously reported that endogenous vascular endothelial growth factor (VEGF) concentration in older pig wounds peaked later and at one-fourth the level of young pigs. These data suggested that VEGF might play a major role in the healing of full-thickness wounds in the aged pig. By in vivo gene transfer using the microseeding technique, we treated full-thickness wounds with different doses of VEGF-expressing adenoviral vector (Ad-VEGF) varying from 1 x 10 7 to 2.7 × 10 1 1 particles per wound (ppw). We found that the VEGF expression in wound fluid followed a dose-response pattern. However, when wounds were microseeded with the highest concentration of Ad-VEGF (2.7 x 10 1 1 ppw), diminished healing rates were found. We then determined the minimal functional concentrations of Ad-VEGF. We used five aged Yucatan minipigs, all retired breeders, to analyze the role of overexpression of 1 x 10 8 and 1 x 10 9 ppw of Ad-VEGF (n = 78) in terms of healing of full-thickness wounds, all 2.5 x 2.5 x 1 cm in size (n = 158). The Ad-VEGF solutions were delivered to the wound floor and borders by in vivo microseeding. Control wounds (n = 80) were microseeded with Ad-Lac-Z (n = 25), treated with saline (n = 49) or treated dry (n = 6). All wounds except for the dry-treated ones were covered with a wound chamber and a wet environment was created by injecting 2.5 ml saline into the chamber. Peak VEGF expression (2300-4000 pg/ml) was detected on days 2 or 3 post gene delivery. This level of VEGF expression was not seen in the saline (n = 49) or Ad-null (n = 25) control groups. The VEGF expression in wounds treated with 1 x 10 8 and 3 x 10 8 ppw (n = 39) exhibited a slower onset with a peak concentration of 400-920 pg/ml on days 5-7. Although high levels of VEGF expression were achieved in the local wound environment, we could not show a significant increase in neovascularization as compared to saline-treated wounds. No significant differences were observed in the rate of reepithelialization and wound contraction among groups of full-thickness wounds treated with Ad-VEGF, Ad-null mutant, or saline in the aged "wet wound healing" pig model. These results indicate that increased levels of VEGF in wounds produced by in vivo gene transfer have little effect on the healing of full-thickness wounds in the aged pig model. Moreover, significantly higher levels of VEGF expression by Ad-VEGF could lead to impaired wound healing.

Wound treatment in a flexible transparent chamber attached to the perimeter of the wound and containing a liquid has been extensively tested in preclinical experiments in pigs and found to offer several advantages. It protects the wound; the liquid medium or saline in the chamber provides in vivo tissue culture-like conditions; and antibiotics, analgesics, and various molecules can be delivered to the wound through the chamber. The wound chamber causes no injury to the wound itself or to the surrounding intact skin. Topical delivery of, for instance, antibiotics can provide very high concentrations at the wound site and with a favorable direction of the concentration gradient. A series of 28 wounds in 20 patients were treated with a wound chamber containing saline and antibiotics. Most patients had significant comorbidity and had not responded to conservative or surgical management with débridement and delayed primary closure or skin grafts. Six wounds had foreign bodies present; four of these were joint prostheses. Seven patients were on corticosteroids for rheumatoid arthritis, lupus, or chronic obstructive pulmonary disease, and four patients had diabetes. Most patients were treated with the wound chamber in preparation for a delayed skin graft or flap procedure, but one was treated with a wound chamber until the wound healed. Twenty-five of the wounds (89 percent) healed, and five wounds (18 percent) required additional conservative management after the initial chamber treatment and grafting procedure. Of the three wounds that did not heal, one healed after additional chamber treatment, one had a skin graft that did not take, and one required reamputation at a higher level. Antibiotic delivery was less than one intravenous dose daily, which avoided the potential for systemic absorption to toxic levels. Antibiotics such as vancomycin and gentamicin could be used in concentrations of up to 10,000 times the minimal inhibitory concentration. Forty-eight hours after application, 20 percent or more of the original antibiotic concentration was present in the wound chamber fluid. In conclusion, the wound chamber provides a safe, powerful tool in the treatment of difficult infected wounds.

It has been recognized that the rate of cutaneous wound healing declines with age, yet the molecular processes that affect this decline remain poorly understood. The purpose of this study was to compare reepithelialization and contraction rates, and growth factor profiles in full-thickness wounds in swine of various ages. Multiple full-thickness excisional wounds were created on the dorsum of 24-month-old (n=2), 4-month-old (n=2), and 2-month-old (n=2) Yucatan Minipigs. The extent of reepithelialization was shown to decrease with increasing age in a manner that was statistically significant among the 2-month-old (79%), 4-month-old (48%), and 24-month-old pigs (22%). Enzyme-linked immunosorbent assay results showed that endogenous vascular endothelial growth factor concentrations in the 2- and 4-month-old animals peaked on day 4, reaching levels of 482 pg/ml and 420 pg/ml, respectively. In the 24-month-old pigs the vascular endothelial growth factor concentration peaked later (day 6), and was present at a lower level (229 pg/ml). On day 4 the vascular endothelial growth factor levels in the older pigs reached only 120 pg/ml, representing a four-fold decrease in concentration compared to the younger pigs. A comparison of platelet-derived growth factor-BB concentrations across the age groups showed similar patterns in the 2- and 4-month-old pigs (peaks of 77 and 91 pg/ml on days 2 and 3, respectively), and levels in the 24-month-old were below the sensitivity level (31.5 pg/ml) of the assay. Transforming growth factor-beta1 levels across the age groups did not differ in a manner that was statistically significant, and all age groups peaked on day 9. Wound contraction showed no statistical differences among the age groups from days 3 to 9. On day 11, however, wound contraction in 2-month-old pigs was about 10% faster than in 24-month-old pigs (p < 0.05). These data suggest a possible new algorithm for treating wounds in aged skin, by which exogenous growth factors can be added to the wound microenvironment in doses and at times that match the growth factor profiles observed in wounds made in younger skin.