The effect of amnion-derived cellular cytokine solution on the epithelialization of partial-thickness donor site wounds in normal and streptozotocin-induced diabetic swine.
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The purpose of this study was to determine whether amnion-derived cellular cytokine solution (ACCS) could improve the quality of epithelialization and accelerate closure of dermatome-created partial-thickness wounds in normal and streptozotocin-induced diabetic pigs.Dermatome-created partial-thickness wounds were sealed with wound chambers in healthy and diabetic pigs and were injected with ACCS. Wound fluid was exchanged daily for total protein concentration, and biopsies were taken on days 6, 8, 10, and 12. Epithelialization, thickness of epidermis, number of epidermal cell layers, and rete ridges were evaluated.The macroscopic appearance of the wounds and speed of healing was similar in all groups at each time point. All wounds were healed by day 6. The epidermis was thicker in the ACCS-treated diabetic wounds than in the controls (140.6 microm vs 82.7 microm on day 12 in diabetic pigs). There were more cell layers (13 vs 7.7) in ACCS-treated diabetic pigs on day 12. The number of rete ridges per 2.5 mm was greater on day 12 in the ACCS-treated diabetic wounds (13 vs 8). There was also a significant increase in the number of rete ridges in ACCS-treated nondiabetic pigs but no difference in epidermal thickness or number of cell layers.In diabetic pigs, we found a significantly thicker epidermis and more cell layers and rete ridges in the ACCS-treated wounds. Healthy pigs showed more rete ridges but no difference in thickness of epidermis or number of cell layers on day 12.Keywords:
Epidermis (zoology)
Dermatome
Amnion
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Wound repair is a complex integration of dynamic processes mediated by humeral messages controlling the levels of cytokines, growth factors, and matrix metalloproteinases in the wound space. Isolated growth factors and growth factor combinations have been used to accelerate wound healing with limited success. A cellular cytokine solution can be collected by harvesting the proteins released from amnion-derived multipotent progenitor cells. The purpose of this study was to compare levels of cytokines/growth factors in amnion-derived cellular cytokine solution with physiological levels reported in the medical literature.Amnion-derived multipotent progenitor cells were grown to confluency, and the proteins secreted were characterized by qualitative and quantitative analysis. These results were compared with physiologic levels reported in the medical literature.The results demonstrated that amnion-derived cellular cytokine solution contained physiologic levels of cytokines relevant to wound healing, including platelet-derived growth factor, vascular endothelial growth factor, angiogenin, transforming growth factor beta 2, tissue inhibitor of metalloproteinase-1, and tissue inhibitor of metalloproteinase-2. The ranges (mean +/- standard deviation) were as follows: platelet-derived growth factor, 86 +/- 33 pg/mL; vascular endothelial growth factor, 5.7 +/- 1.5 ng/mL; angiogenin, 1.0 +/- 0.33 ng/mL; transforming growth factor beta 2, 500 +/- 330pg/mL; tissue inhibitor of metalloproteinase-1, 530 +/- 140 ng/mL; and tissue inhibitor of metalloproteinase-2230 +/- 40 ng/mL. These levels are comparable with the physiologic levels reported in the literature.The physiologic levels of cytokines important to healing found in amnion-derived cellular cytokine solution suggest that amnion-derived cellular cytokine solution may be of benefit in healing certain acute and chronic wounds.
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SUMMARY Preformed collagen gel was topically applied to cutaneous wounds of the equine dorsal fetlock (thoracic limb) and metatarsal regions to evaluate the effect on exuberant granulation tissue production and wound healing. In 6 horses and 3 ponies (< 140 cm high at the withers and < 365 kg), 36 standardized cutaneous limb wounds were surgically induced (4 wounds/animal); 18 wounds were treated topically with collagen gel, and 18 wounds were not treated (controls). Collagen gel was initially applied to the wound at 0, 2, or 7 days after wound formation (groups 1, 2, and 3, respectively). Four measurements were regularly made: amount of wound contraction and the size of the granulation bed, epithelial covering, and total wound. Sequential skin and wound biopsies were evaluated histologically to assess wound healing. Using a computer, data were analyzed for differences in the 4 measurements between treated and control wounds, between fetlock wounds and metatarsal wounds, and among groups 1, 2, and 3. Analyses were performed on days 15 and 45 of wound healing and on the final day of healing. A significant difference ( P > 0.05) in the production of exuberant granulation tissue, rate of epithelialization, or degree of wound contraction was not detected between the collagen-treated and control wounds. Total healing time and final scar size were similar. Wound healing patterns were significantly different ( P < 0.05) in the fetlock wounds and metatarsal wounds. All wounds enlarged up to day 15 with fetlock wounds enlarging significantly more than did the metatarsal wounds. Fetlock wounds had a faster rate of contraction than did the metatarsal wounds at day 45; but, due to their smaller size, metatarsal wounds healed significantly faster than did fetlock wounds. The resulting scar size of fetlock and metatarsal wounds was similar. Metatarsal wounds required more resection of exuberant granulation tissue than did fetlock wounds. Propensity to develop exuberant granulation tissue and the rate of healing were directly correlated to body size of the horse. Larger horses required more frequent resection of the granulating wound bed and healed more slowly, despite the relatively smaller wound size as compared with their larger limb size. Ponies (< 140 cm high at the withers and < 365 kg of body weight) healed faster than horses; wounds of the ponies also healed without exuberant granulation tissue formation. Therefore, ponies are not adequate models of wound healing for horses.
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Healing of skin wound is a multi-factorial and complex process. Proper treatment of diabetic wounds is still a major clinical challenge. Although diabetes mellitus can occur in ruminants, healing of wounds in diabetic ruminants has not yet been investigated. The aim of this study was to evaluate healing of ovine excisional diabetic wound model. Eight 4-month-old Iranian Makoui wethers were equally divided to diabetic and nondiabetic groups. Alloxan monohydrate (60 mg kg(-1), IV) was used for diabetes induction. In each wether, an excisional wound was created on the dorsum of the animal. Photographs were taken in distinct times for planimetric evaluation. Wound samples were taken on day 21 post-wounding for histopathologic evaluations of epidermal thickness, number of fibroblasts and number of new blood vessels. The planimetric study showed slightly delay in wound closure of diabetic animals, however, it was not significantly different from nondiabetic wounds (p ≥ 0.05). Furthermore, epidermal thickness, number of fibroblasts and number of blood vessels were significantly lower in diabetic group (p < 0.05). We concluded that healing of excisional diabetic wounds in sheep may be compromised, as seen in other species. However, contraction rate of these wounds may not be delayed due to metabolic features of ruminants and these animals might go under surgeries without any serious concern. However, healing quality of these wounds may be lower than normal wounds.
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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.
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Objective— To evaluate the effects of porcine small intestinal submucosa (PSIS) on the healing of full‐thickness wounds in dogs, specifically the appearance of granulation tissue, percent epithelialization and contraction, histologic variables of inflammation and repair, and aerobic culture results. Study Design— Prospective, controlled, experimental study. Animals— Purpose‐bred, female dogs (n=10). Methods— Wounds were created bilaterally on the trunk; 1 side as a control and 1 treated with PSIS. First appearance of granulation tissue was recorded. Total wound area, open wound area, and epithelialized area were measured at 21 time points—wound contraction and percent epithelialization were calculated. Aerobic cultures were taken at 4 time points and wound biopsies at 8. Histologic features were graded into an Acute Inflammation Score and Repair Score. Results— There was no difference in first appearance of granulation tissue between PSIS‐treated and control wounds. Wound contraction was significantly faster in control wounds as was percent epithelialization after day 21. Histologic Acute Inflammation Scores were significantly higher in PSIS‐treated wounds compared with control wounds on days 2 and 6. There were no differences in Histologic Repair Scores between PSIS‐treated and control wounds or in aerobic culture results. Conclusion— Wounds treated with PSIS contract more slowly, epithelialize less, and have more pronounced acute inflammation after implantation than control wounds. Clinical Relevance— Acute, full‐thickness wounds in dogs do not benefit from treatment with PSIS.
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It has been evidenced that the fat may have a potential to secret some growth factors or be a source of stem cells. So, we explore the effects of fat on healing of porcine skin wounds so as to provide a new method for clinical skin wound repair after injury. Forty‐eight full‐thickness skin wounds were produced on both sides of the back in 6 male minipigs (8 wounds in each animal). Then these wounds were randomly divided into 4 groups, which were saline control group, fat autografting group, basic fibroblast growth factor (bFGF) treatment group and epidermal growth factor (EGF) treatment group. At day 3, 7, 14 and 21 after wounding, the area and the volume of wounds were measured and the histological examination was performed to evaluate the velocity and quality of wounds healing in different groups. At day 3 and 7, the amount of granulation tissues and vessel density in fat treatment group were significantly more than that in other groups. Wound areas and volume in fat treatment wounds were markedly decreased in compared with those in other groups (P < 0.01). Regenerated epidermis in fat treatment group was thicker than that in other groups. These results confirmed that the wound healing velocity and quality in wounds treated with fat autografting were enhanced. It indicated that fat has a potential to accelerate velocity and improve the quality of wound healing after skin injury.
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The authors investigated whether the application of platelet-derived growth factor (PDGF) to donor site wounds would speed healing in a porcine model. In a red duroc pig model, three wounds that were 3 inches × 3 inches were created with a dermatome (0.06-inch depth) on one side of two different animals. These wounds were digitally and laser Doppler (LDI) imaged and biopsied immediately pre- and postwound creation and every 2 days for 2 weeks. A set of identical wounds were subsequently created on the opposite side of the same animals and treated with topical PDGF (becaplermin gel 0.01%, 4 g/wound) immediately on wounding. PDGF-treated wounds were imaged and biopsied as above. Digital images of wounds were assessed for epithelialization by clinicians using an ordinal scale. Perfusion units (PU) were evaluated by LDI. Wound healing was evaluated by hematoxylin and eosin histological visualization of an epithelium and intact basement membrane. First evidence of partial epithelialization was seen in control and PDGF-treated wounds within 7.7 ± 1.4 and 6.4 ± 1.1 days postwounding, respectively (P=.03). Completely epithelialized biopsies were seen in control and PDGF-treated wounds at 11.7 ± 2.6 and 9.6 ± 1.5 days, respectively (P=.02). Clinician evaluation of digital images showed that on day 9, control wounds were, on average, 48.3 ± 18.5% epithelialized vs 57.2 ± 20.2% epithelialized for PDGF-treated wounds. At day 16, control wounds showed an average of 72.9 ± 14.6% epithelialization and PDGF-treated wounds showed an average of 90 ± 11.8%epithelialization. Overall, PDGF-treated wounds had statistically significantly higher scores across all timepoints (P=.02). Average perfusion units as measured by LDI were similar for control and PDGF-treated wounds at time of excision (225 ± 81and 257 ± 100, respectively). On day 2 postwounding, average PU for control wounds were 803 and were 764 for PDGF-treated wounds. Control wounds maintained higher PU values compared with PDGF-treated wounds at all time points and returned to excision PU values by day 12.2 ± 1.1 postwounding. PDGF-treated wounds reached the same values by day 9.7 ± 2.3 (P=.03). The authors conclude that topical PDGF speeds time to epithelialization of partial-thickness wounds in a porcine model as evidenced by histology, clinical appearance, and time to return to prewounding vascularity.
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To evaluate the effect of a proprietary Manuka honey essential oil hydrogel on the healing of acute, full-thickness wounds in dogs.Prospective, controlled, randomized, experimental study.Purpose-bred, adult, female beagles (n = 10).Two 2 × 2 cm surgical wounds were created bilaterally on the trunk of each dog; each side was randomized to receive HoneyCure® (HOC) or standard-of-care (CON) dressings. Cranial wounds were for histopathological analysis and the caudal wounds for culture and planimetry. Total and open wound areas were measured with digital image planimetry at 15 time points. From these data, percent contraction and percent epithelialization were calculated. Tissue biopsies were obtained at 7 time points and histologic features scored. Cultures were obtained at 2 time points.Epithelialization was 11.7, 10.4, and 10.1 percentage points higher in HOC wounds compared to CON wounds at days 16, 18, and 21 respectively. Wound contraction and histological scores did not differ between groups. Cultures were positive in 7/40 (17.5%) wounds, with Staphylococcus pseudintermedius and Staphylococcus epidermidis isolated evenly. There was no difference of infection rate between the two groups; all infections were superficial and did not require treatment.This study did not provide evidence to support the application of HoneyCure® in small, acute wounds in healthy dogs. However, application may be beneficial in the early proliferative stage of wound healing and in wounds that would benefit from early, robust epithelialization.
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