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696 Porcine wound model of persistent inflammation in chronic wounds
ABSTRACTS | Wound Healing and Tissue Remodelling 694
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Comparative analysis of cytokines, proteases and growth factors in human wound exudates obtained by vacuum therapy P Do¨rfler1, W Bauer2, T Go¨rge3, TA Luger3, K Rappersberger4, G Stingl2, T Stockinger4, A Stu¨tz1 and B Wolff-Winiski1 1 Akribes Biomedical, Vienna, Austria, 2 Department of Dermatology, Division of Immunology, Allergy and Infectious Diseases, University of Vienna, Vienna, Austria, 3 Department of Dermatology, University of Mu¨nster, Mu¨nster, Germany and 4 Department of Dermatology, Hospital Rudolfstiftung, Vienna, Austria Wound exudates are considered liquid biopsies of wounds and contain the pathogenic drivers of chronicity. Previous studies have shown elevated levels of pro-inflammatory cytokines and proteases in chronic wound exudates. We obtained 78 wound exudates harvested by negative pressure therapy and compared their levels of 16 cytokines and chemokines, 11 growth factors, 8 metalloproteinases, determined by multiplex analysis, and their enzyme activities of myeloperoxidase, elastase and metalloproteinase. The wounds included healing and nonhealing venous, arterial, pressure and diabetic ulcers as well as healing surgical wounds. Hepatocyte growth factor, the cytokines IL-1 alpha, IL-1 beta, TNF-alpha and CXCL10 as well as metalloproteinases 1, 2, 3, 8, 9, and 12 were elevated in wound exudates of non-healing wounds. Enzyme activities were also increased in non-healing wounds. However, due to the large patient-to-patient variability of these data, the numerical values of any of these parameters do not permit the distinction between healing and non-healing wound phenotypes in individual patients. Only the analysis of consecutive samples of the same patient allowed the detection of differences corresponding to the healing process. In conclusion, wound exudate levels of cytokines, proteases and growth factors are insufficient to distinguish between healing and non-healing wounds.
Local inflammatory response after topically applied burn stimulus investigated in excised human skin K Tiffner1, M Funk2, L Kamolz3 and T Birngruber1 1 HEALTH - Institute for Biomedicine and Health Sciences, Joanneum Research Forschungsgesellschaft m.b.H., Graz, Austria, 2 Bioskinco GmbH, Wu¨rzburg, Germany and 3 Division of Plastic, Esthetic and Reconstructive Surgery, Medical University of Graz, Graz, Austria Local inflammatory response after burn injuries has not been extensively studied by now, because adequate sampling technologies have been missing. In this study, we investigated the local inflammatory response after a topically applied burn and scalding stimulus, respectively in fresh excised human skin with dermal Open Flow Microperfusion (dOFM). dOFM is a continuous sampling technique which enables monitoring of local skin processes by sampling of interstitial fluid (ISF) directly from the dermis. In a feasibility study three test areas were applied on one skin flap. One test area was treated with a burn stimulus induced by a heated metal block, the second with a scalding stimulus induced by boiling water and the third test area was left untreated to serve as negative control site. Three OFM probes were inserted into each test area at a depth of about 0.8 mm. The temperature in the dermis was measured with temperature sensors during each stimulus. ISF was continuously sampled with dOFM directly from the dermis from half an hour before to 48 hours after the respective stimulus was applied. The half-hourly and hourly aliquots were used to screen for approximately 100 inflammatory biomarkers. Burn injuries were histologically verified. We observed a difference in cytokine levels between the areas treated with burn and scalding stimulus, respectively and the control site. The effect remained consistent for the monitoring period of 48 hours. The areas treated with burn and scalding stimulus, respectively showed comparable cytokine levels. Further ex vivo experiments will be performed to assess reproducibility of the results. Additionally, the ex vivo results will be compared with in vivo results to verify the usability of this ex vivo model.
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Porcine wound model of persistent inflammation in chronic wounds N Bucher3, J Adamczak1, H Fahrngruber2, P Reisenegger1, A Stu¨tz2, B Wolff-Winiski2, L Kamolz3, F Sinner1 and T Birngruber1 1 HEALTH - Institute for Biomedicine and Health Sciences, Joanneum Research Forschungsges. mbH, Graz, Austria, 2 Akribes Biomedical, Vienna, Austria and 3 Plastic, Aesthetic and Reconstructive Surgery, Department of Surgery, Medical University of Graz, Graz, Austria Although the majority of wounds respond well to conventional treatment, some chronic wounds fail to heal within an appropriate time frame. We made use of the similarity between human and porcine skin and established a model mimicking persistent inflammation in chronic wounds, based on Liehl et al (ESDR 2014). Resiquimod (R-848), a small molecule TLR7/8 agonist was investigated to induce a chronic wound status in porcine skin (n ¼ 4). Full thickness wounds were generated with 6 mm biopsy punches and were treated with different concentrations of R-848 via topical application of a single daily dose of 50 ml for five days consecutively. Two different strategies were tested: Application of i) a constant concentration of 0.05% R-848 and ii) variable concentrations (0.1% - 0.01% R-848). Vehicle dissolving R848, and human serum served as controls. Wound size and inflammation were scored daily for 15 days and were compared with semi-quantitative digital wound analysis. R-848 effectively induced persistent inflammation in wounds while vehicle and human serum did not. The constant concentration of 0.05% R-848 maintained a persistent inflammation as long as the application was continued. The variable concentrations of R-848 achieved comparable wound progression to the constant dosing. Maximum wound severity was reached on day 6 and inflammation started to decline 2 days after treatment had been terminated. No significant difference was observed between the progression of wounds treated with vehicle and human serum, respectively. Conclusively, we established a preclinical model for persistent inflammation maintained by the application of R-848. This model mimics the inflammatory component of chronic human wounds and has the potential to test candidate drugs for efficacy. Subsequent adaptations will investigate if the chronic status can be prolonged.
Effects of Gadodiamide on cell proliferation and collagen production in cultured human dermal fibroblasts S Hayashi, Y Hamasaki, A Hatamochi and K Igawa Dermatology, Dokkyo medical university, Shimotsuga, Japan Nephrogenic systemic fibrosis (NSF) is a disease characterized by fibrosis of the systemic organs in patients with renal failure. Following the findings of recent epidemiological studies and the finding of gadolinium (Gd) in the skin tissue of NSF patients, it is now definitely known that the use of Gd contrast agents can trigger NSF. To date, however, the exact mechanism underlying the induction of fibrosis in various organs by Gd remains unexplained. The present study was undertaken to evaluate the influence of Gd on the proliferation activity and collagen production of cultured fibroblasts. Normal human dermis-derived fibroblasts were incubated in the presence of gadodiamide (GA). The proliferation activity of the cells was assessed on the basis of the cell counts in the fibroblast growth curve and the DNAsynthetic activity of the cells (indicator; level of 3H-thymidine uptake by cells). The collagen production was evaluated by densitometric measurement of the quantity of collagen through electrophoresis and fluorography after incorporation of 3H-proline into the procollagens. Furthermore, the expression levels of the genes for type I and III collagen were measured by real-time reverse transcription polymerase chain reaction (RT-PCR) assay. The cell count tended to be higher when the fibroblasts were incubated in medium containing GA, furthermore, the DNA synthetic activity also rose in a concentration-dependent manenr in the GA-treated group as compared to that in the control group. No significant changes in either the collagen production or the collagen gene expression levels were noted in cultures containing GA. These results suggest that the formation of sclerosing lesions in patients with NSF may be attributable to the effect of GA of enhancing the growth activity of fibroblasts.
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A novel device for skin type-based treatment while cleansing D Kern1, J Namkoong1, M Riggs1, B Cook1, ZD Draelos2 and HE Knaggs1 1 Nu Skin Enterprises, Inc., Provo, UT and 2 Dermatology Consulting Services, High Point, NC The human skin is an important barrier to protect the inside of the body from the outside environment. A heathy skin forms a tight barrier with proper desquamation. When this barrier is damaged, irritants enter the skin causing problems. To maintain the healthy skin, surface deposits are removed through proper hygiene and treatment. We developed a novel device technology that gently stimulate desquamation and exfoliation leading to the improved skin. 3 different treatment surfaces with accompanying 5 skin type-specific treatment cleansers were developed. Skin types were divided as normal-to-combination, oily, dry, sensitive or acne-prone. This technology consisting of a treatment surface, a treatment cleanser and the device were evaluated for the skin benefit and tolerability in a 12- week clinical study. In total, one hundred female subjects were recruited and grouped based on their specific skin types; twenty subjects were in each group. During the study, the device was used twice a day for two minutes each time. Subjects were evaluated by clinical grading and self-assessment at baseline before usage, after the initial usage and weeks 1, 2, 4, 8 and 12. The treatment/ cleansing was well tolerated by subjects from different skin types without excessive exfoliation, skin irritation or any adverse events. This novel technology was shown to cleanse the skin while also gently exfoliating, maintaining and improving healthy skin. Skin benefits were evident throughout the study period. At the conclusion of the study, the clinical dermatologist reported improvements for all skin types in smoothness, softness, texture, clarity, radiance, firmness, hydration, cleansing ability and overall appearance. In addition, subjects noted improvements in these parameters. Overall, this novel technology consisting of unique treatment surfaces, skin type-based treatment cleansers and a mechano-biological targeted device combination is both gentle and provides significant benefit for healthier skin.
S312 Journal of Investigative Dermatology (2017), Volume 137
Evaluation of colorimetry and biomechanical properties in wounds of dermatologic patients with postoperative infection J Liu, S Wolfe, A Macleod and J Powers Duke University, Durham, VA Diagnosis of post-operative infection based on clinical presentation alone can be challenging. The purpose of this study was to objectively evaluate the appearance of infected and noninfected wounds in Caucasian subjects receiving excisions of non-melanoma skin cancers and atypical nevi. We used a colorimeter (Minolta Chromameter) to measure wound color and a biomechanical tissue characterization device (BTC-2000) to determine stiffness. 11 subjects of average age 61.2 10.7 years were recruited for this single-site study. Patients with prior antibiotic use or site infection were excluded. The most common site was the upper extremity (4/11). 3 patients developed infection, all with Staphylococcus aureus. Data were collected pre-operatively and on postoperative day (POD) 14. Colorimetry data are reported in CIELAB color space, which includes light vs dark (L*), magenta vs green (a*), and yellow vs blue (b*). Between POD 0 and 14, average colorimetry changes among all patients were -5.51, 1.27, -2.33, indicating darkening, and gain in magenta and blue hues, respectively. The average colorimetry changes were -3.67, -0.01, and -0.46 in infected patients, and -7.02, 1.89, and -3.96 in uninfected patients. These data suggest that uninfected wounds are actually more likely to be darker and have more magenta and blue hues, but these differences lack statistical significance and are limited by sample size. The average stiffness measurement among all patients was 79.87 33.28 mmHg/mm. Between POD 0 and 14, stiffness increased by an average of 26.73 mmHg/mm. The average stiffness increase among patients with infection was 22.93 mmHg/mm, compared to 28.63 mmHg/mm in uninfected, with no statistically significant difference. Our measurements suggest that wound infection is not necessarily associated with observable changes in tissue color or stiffness. However, 2 of the 3 infected patients reported pus and drainage, and 1 reported significant pain, which may be a more reliable indicator of wound infection than appearance alone.
695
Comparative analysis of cytokines, proteases and growth factors in human wound exudates obtained by vacuum therapy P Do¨rfler1, W Bauer2, T Go¨rge3, TA Luger3, K Rappersberger4, G Stingl2, T Stockinger4, A Stu¨tz1 and B Wolff-Winiski1 1 Akribes Biomedical, Vienna, Austria, 2 Department of Dermatology, Division of Immunology, Allergy and Infectious Diseases, University of Vienna, Vienna, Austria, 3 Department of Dermatology, University of Mu¨nster, Mu¨nster, Germany and 4 Department of Dermatology, Hospital Rudolfstiftung, Vienna, Austria Wound exudates are considered liquid biopsies of wounds and contain the pathogenic drivers of chronicity. Previous studies have shown elevated levels of pro-inflammatory cytokines and proteases in chronic wound exudates. We obtained 78 wound exudates harvested by negative pressure therapy and compared their levels of 16 cytokines and chemokines, 11 growth factors, 8 metalloproteinases, determined by multiplex analysis, and their enzyme activities of myeloperoxidase, elastase and metalloproteinase. The wounds included healing and nonhealing venous, arterial, pressure and diabetic ulcers as well as healing surgical wounds. Hepatocyte growth factor, the cytokines IL-1 alpha, IL-1 beta, TNF-alpha and CXCL10 as well as metalloproteinases 1, 2, 3, 8, 9, and 12 were elevated in wound exudates of non-healing wounds. Enzyme activities were also increased in non-healing wounds. However, due to the large patient-to-patient variability of these data, the numerical values of any of these parameters do not permit the distinction between healing and non-healing wound phenotypes in individual patients. Only the analysis of consecutive samples of the same patient allowed the detection of differences corresponding to the healing process. In conclusion, wound exudate levels of cytokines, proteases and growth factors are insufficient to distinguish between healing and non-healing wounds.
Local inflammatory response after topically applied burn stimulus investigated in excised human skin K Tiffner1, M Funk2, L Kamolz3 and T Birngruber1 1 HEALTH - Institute for Biomedicine and Health Sciences, Joanneum Research Forschungsgesellschaft m.b.H., Graz, Austria, 2 Bioskinco GmbH, Wu¨rzburg, Germany and 3 Division of Plastic, Esthetic and Reconstructive Surgery, Medical University of Graz, Graz, Austria Local inflammatory response after burn injuries has not been extensively studied by now, because adequate sampling technologies have been missing. In this study, we investigated the local inflammatory response after a topically applied burn and scalding stimulus, respectively in fresh excised human skin with dermal Open Flow Microperfusion (dOFM). dOFM is a continuous sampling technique which enables monitoring of local skin processes by sampling of interstitial fluid (ISF) directly from the dermis. In a feasibility study three test areas were applied on one skin flap. One test area was treated with a burn stimulus induced by a heated metal block, the second with a scalding stimulus induced by boiling water and the third test area was left untreated to serve as negative control site. Three OFM probes were inserted into each test area at a depth of about 0.8 mm. The temperature in the dermis was measured with temperature sensors during each stimulus. ISF was continuously sampled with dOFM directly from the dermis from half an hour before to 48 hours after the respective stimulus was applied. The half-hourly and hourly aliquots were used to screen for approximately 100 inflammatory biomarkers. Burn injuries were histologically verified. We observed a difference in cytokine levels between the areas treated with burn and scalding stimulus, respectively and the control site. The effect remained consistent for the monitoring period of 48 hours. The areas treated with burn and scalding stimulus, respectively showed comparable cytokine levels. Further ex vivo experiments will be performed to assess reproducibility of the results. Additionally, the ex vivo results will be compared with in vivo results to verify the usability of this ex vivo model.
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Porcine wound model of persistent inflammation in chronic wounds N Bucher3, J Adamczak1, H Fahrngruber2, P Reisenegger1, A Stu¨tz2, B Wolff-Winiski2, L Kamolz3, F Sinner1 and T Birngruber1 1 HEALTH - Institute for Biomedicine and Health Sciences, Joanneum Research Forschungsges. mbH, Graz, Austria, 2 Akribes Biomedical, Vienna, Austria and 3 Plastic, Aesthetic and Reconstructive Surgery, Department of Surgery, Medical University of Graz, Graz, Austria Although the majority of wounds respond well to conventional treatment, some chronic wounds fail to heal within an appropriate time frame. We made use of the similarity between human and porcine skin and established a model mimicking persistent inflammation in chronic wounds, based on Liehl et al (ESDR 2014). Resiquimod (R-848), a small molecule TLR7/8 agonist was investigated to induce a chronic wound status in porcine skin (n ¼ 4). Full thickness wounds were generated with 6 mm biopsy punches and were treated with different concentrations of R-848 via topical application of a single daily dose of 50 ml for five days consecutively. Two different strategies were tested: Application of i) a constant concentration of 0.05% R-848 and ii) variable concentrations (0.1% - 0.01% R-848). Vehicle dissolving R848, and human serum served as controls. Wound size and inflammation were scored daily for 15 days and were compared with semi-quantitative digital wound analysis. R-848 effectively induced persistent inflammation in wounds while vehicle and human serum did not. The constant concentration of 0.05% R-848 maintained a persistent inflammation as long as the application was continued. The variable concentrations of R-848 achieved comparable wound progression to the constant dosing. Maximum wound severity was reached on day 6 and inflammation started to decline 2 days after treatment had been terminated. No significant difference was observed between the progression of wounds treated with vehicle and human serum, respectively. Conclusively, we established a preclinical model for persistent inflammation maintained by the application of R-848. This model mimics the inflammatory component of chronic human wounds and has the potential to test candidate drugs for efficacy. Subsequent adaptations will investigate if the chronic status can be prolonged.
Effects of Gadodiamide on cell proliferation and collagen production in cultured human dermal fibroblasts S Hayashi, Y Hamasaki, A Hatamochi and K Igawa Dermatology, Dokkyo medical university, Shimotsuga, Japan Nephrogenic systemic fibrosis (NSF) is a disease characterized by fibrosis of the systemic organs in patients with renal failure. Following the findings of recent epidemiological studies and the finding of gadolinium (Gd) in the skin tissue of NSF patients, it is now definitely known that the use of Gd contrast agents can trigger NSF. To date, however, the exact mechanism underlying the induction of fibrosis in various organs by Gd remains unexplained. The present study was undertaken to evaluate the influence of Gd on the proliferation activity and collagen production of cultured fibroblasts. Normal human dermis-derived fibroblasts were incubated in the presence of gadodiamide (GA). The proliferation activity of the cells was assessed on the basis of the cell counts in the fibroblast growth curve and the DNAsynthetic activity of the cells (indicator; level of 3H-thymidine uptake by cells). The collagen production was evaluated by densitometric measurement of the quantity of collagen through electrophoresis and fluorography after incorporation of 3H-proline into the procollagens. Furthermore, the expression levels of the genes for type I and III collagen were measured by real-time reverse transcription polymerase chain reaction (RT-PCR) assay. The cell count tended to be higher when the fibroblasts were incubated in medium containing GA, furthermore, the DNA synthetic activity also rose in a concentration-dependent manenr in the GA-treated group as compared to that in the control group. No significant changes in either the collagen production or the collagen gene expression levels were noted in cultures containing GA. These results suggest that the formation of sclerosing lesions in patients with NSF may be attributable to the effect of GA of enhancing the growth activity of fibroblasts.
698
699
A novel device for skin type-based treatment while cleansing D Kern1, J Namkoong1, M Riggs1, B Cook1, ZD Draelos2 and HE Knaggs1 1 Nu Skin Enterprises, Inc., Provo, UT and 2 Dermatology Consulting Services, High Point, NC The human skin is an important barrier to protect the inside of the body from the outside environment. A heathy skin forms a tight barrier with proper desquamation. When this barrier is damaged, irritants enter the skin causing problems. To maintain the healthy skin, surface deposits are removed through proper hygiene and treatment. We developed a novel device technology that gently stimulate desquamation and exfoliation leading to the improved skin. 3 different treatment surfaces with accompanying 5 skin type-specific treatment cleansers were developed. Skin types were divided as normal-to-combination, oily, dry, sensitive or acne-prone. This technology consisting of a treatment surface, a treatment cleanser and the device were evaluated for the skin benefit and tolerability in a 12- week clinical study. In total, one hundred female subjects were recruited and grouped based on their specific skin types; twenty subjects were in each group. During the study, the device was used twice a day for two minutes each time. Subjects were evaluated by clinical grading and self-assessment at baseline before usage, after the initial usage and weeks 1, 2, 4, 8 and 12. The treatment/ cleansing was well tolerated by subjects from different skin types without excessive exfoliation, skin irritation or any adverse events. This novel technology was shown to cleanse the skin while also gently exfoliating, maintaining and improving healthy skin. Skin benefits were evident throughout the study period. At the conclusion of the study, the clinical dermatologist reported improvements for all skin types in smoothness, softness, texture, clarity, radiance, firmness, hydration, cleansing ability and overall appearance. In addition, subjects noted improvements in these parameters. Overall, this novel technology consisting of unique treatment surfaces, skin type-based treatment cleansers and a mechano-biological targeted device combination is both gentle and provides significant benefit for healthier skin.
S312 Journal of Investigative Dermatology (2017), Volume 137
Evaluation of colorimetry and biomechanical properties in wounds of dermatologic patients with postoperative infection J Liu, S Wolfe, A Macleod and J Powers Duke University, Durham, VA Diagnosis of post-operative infection based on clinical presentation alone can be challenging. The purpose of this study was to objectively evaluate the appearance of infected and noninfected wounds in Caucasian subjects receiving excisions of non-melanoma skin cancers and atypical nevi. We used a colorimeter (Minolta Chromameter) to measure wound color and a biomechanical tissue characterization device (BTC-2000) to determine stiffness. 11 subjects of average age 61.2 10.7 years were recruited for this single-site study. Patients with prior antibiotic use or site infection were excluded. The most common site was the upper extremity (4/11). 3 patients developed infection, all with Staphylococcus aureus. Data were collected pre-operatively and on postoperative day (POD) 14. Colorimetry data are reported in CIELAB color space, which includes light vs dark (L*), magenta vs green (a*), and yellow vs blue (b*). Between POD 0 and 14, average colorimetry changes among all patients were -5.51, 1.27, -2.33, indicating darkening, and gain in magenta and blue hues, respectively. The average colorimetry changes were -3.67, -0.01, and -0.46 in infected patients, and -7.02, 1.89, and -3.96 in uninfected patients. These data suggest that uninfected wounds are actually more likely to be darker and have more magenta and blue hues, but these differences lack statistical significance and are limited by sample size. The average stiffness measurement among all patients was 79.87 33.28 mmHg/mm. Between POD 0 and 14, stiffness increased by an average of 26.73 mmHg/mm. The average stiffness increase among patients with infection was 22.93 mmHg/mm, compared to 28.63 mmHg/mm in uninfected, with no statistically significant difference. Our measurements suggest that wound infection is not necessarily associated with observable changes in tissue color or stiffness. However, 2 of the 3 infected patients reported pus and drainage, and 1 reported significant pain, which may be a more reliable indicator of wound infection than appearance alone.