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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
tion. Conclusion: By implanting in vitro derived tendons into an in vivo environment, we have significantly improved their tensile strength. The tensile strengths of the conditioned constructs were similar to native tendons from two week old rats (10,275 kPa vs. 23,800 kPa, respectively). Collagen content also improved significantly as measured by hydroxyproline concentration. Histologically, the constructs’ structure was more similar to mature collagen, with increased collagen fiber diameter, greater organization of the collagen fibers and a decrease in extracellular matrix. Future research will be aimed at optimizing the tensile strength and collagen content via administering active forces across the developing constructs in vivo.
production. Immunoreactive collagen (normalized to GAPDH) increased significantly (p ⬍ 0.05) after TGF-1 (4.20 ⫾ 0.70 fold), TGF-3 (2.45 ⫾ 0.37 fold for the 2.5 ng/mL dose and 2.91 ⫾ 0.15 fold for the 5 ng/mL dose), LPA (2.10 ⫾ 0.17 fold for the 25 M dose) and ET (1.9 ⫾ 0.34 fold for the 50 nM dose) but not after thrombin. These results suggest that while TGF-3 is thought to reduce scar formation, similar increases in CTGF and collagen expression were observed with TGF-3 as with TGF-1 and LPA. A phosphopeptide analogue of the small heat shock related protein, HSP20, conjugated to a protein transduction domain (PTDpHSP20) inhibits scar formation in a hamster model. The effect of PTD-pHSP20 (50 M) on TGF-1, LPA and ET- induced increases in the CTGF and collagen expression were determined. PTDpHSP20 significantly (p ⬍ 0.05) inhibited TGF-1, LPA and ET-1 induced increases in CTGF (30.20 ⫾ 9.35 %, 36.00 ⫾ 11.00 %, and 27.21 ⫾ 8.10 % respectively) and collagen (43.50 ⫾ 7.50 %, 47.50 ⫾ 10.23 %, and 27.70 ⫾ 10.71 % respectively) expression. These results suggest that phosphopeptide analogues of HSP20 can reduce the expression of CTGF and collagen induced by multiple mediators of scar formation (TGF-1, LPA and ET-1). In addition, TGF-3 appears to have the same effect as TGF-1 on CTGF and collagen expression in human keloid fibroblasts. PTD-pHSP20 treatment may represent a novel strategy for the treatment of keloids and other fibrotic disorders.
P36. ANASTOMOTIC LEAKS: A SCIENTIFIC PERSPECTIVE. M. Zerey, R. D. Peindl, B. Paton, W. Hope, W. Newcomb, T. Schmelzer, J. Cristiano, J. Heath, A. E. Lincourt, K. W. Kercher, K. S. Gersin, B. Heniford; Carolinas Medical Center, Charlotte, NC
P35. CTGF AND COLLAGEN EXPRESSION IN HUMAN KELOID FIBROBLASTS. L. Lopes 1, P. Komalavilas 1, E. Furnish 1, G. Yang 2, M. Longaker 2, C. Brophy 3; 1Arizona State University, Tempe, AZ, 2Stanford, Palo Alto, CA, 3Phoenix Vamc, Phoenix, AZ Paracrine factors that have been implicated in keloid formation include transforming growth factor -1 (TGF-1), thrombin, lysophosphatidic acid (LPA), and endothelin (ET). On the other hand, TGF-3 has been implicated in reducing scar formation. We investigated the effect of these mediators on connective tissue growth factor (CTGF) and collagen expression in human dermal keloid fibroblasts. Human keloid fibroblasts were grown to 80% confluence, serum-starved for 48 h, and subsequently stimulated for 24 hr with TGF-1 (2.5 ng/mL), TGF-3 (2.5 or 5 ng/mL), thrombin (25-150 nM), LPA (10 or 25 M), and ET (25 or 50 nM). Untreated cells were used as controls and all experiments were done in triplicate. Immunoreactive CTGF (normalized to GAPDH) increased significantly (p⬍0.05) after TGF-1 (3.26 ⫾ 0.31 fold), TGF-3 (1.75 ⫾0.44 fold for the 2.5 ng/mL dose and 2.60 ⫾ 0.64 fold for the 5 ng/mL dose), LPA (2.15 fold ⫾ 0.64 for the 25 M dose) and ET (1.80 fold ⫾ 0.26 for the 50 nM dose) but not after thrombin. CTGF induces scar formation by increases in collagen
Introduction: Anastomotic leaks are devastating complications in surgery. There is little standardization when assessing anastomotic leaks and reporting anastomotic integrity. The leak rate may be important when: 1) evaluating mechanical staplers and stapling reinforcement products, and 2) predicting clinical outcomes when leaks occur. We present a novel technique quantifying anastomotic leak pressure and leak rate at a given intraluminal pressure. Methods: In an Institution’s Animal Care and Use Committee approved protocol, we performed gastrojejunal anastomoses using a #25 Proximate ILS circular stapler (Ethicon Endo-Surgery, Cincinnati, OH). Individual gastrojejunal anastomoses were excised en bloc and placed in the following leak measurement system (Figure 1). The system delivers pressurized Methylene Blue (MB) to a Plexiglas TM chamber. The gastric cuff is secured along its cicrcumference and the jejunal limb is fed through a 32mm aperture exposing the gastrojejunal anastomosis to clear, de-ionized water in a secondary chamber. The leak pressure is noted at the first visual appearance of MB at the stapled anastomosis. Thereafter, 1mL aliquots are obtained from the water at one-minute time intervals and the spectrophotometric absorbance at 668nm is measured at the initial leak pressure at that time point (DU 65 spectrophotometer, Beckman Instruments Inc; Fullerton, Calif). In order to better understand the behavior of leaks at rising pressures, the delivery pressure of the MB is increased by 0.2PSI increments once the leak rate has remained constant for a determined period of time at a given pressure. The experiment is terminated once the upper limit of measurable absorbance is reached. In order to determine leak volume in experimental samples, a standard curve was performed, noting optical absorbance at 668nm with increasing concentrations of MB solution. Leak rate at a given pressure was calculated by dividing the change in MB solution volume over a measurement period. SigmaPlot version 9.0 (Systat Software Inc., Richmond, CA) was used to plot the data and determine the best fitting linear or quadratic equation. Results: The best-fitting quadratic equation for the standard curve was y ⫽ 0.3644 ⫹ 14.12x ⫹ 4.317x 2 (r ⫽ 0.9997). An example of the data generated is illustrated in
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
tion. Conclusion: By implanting in vitro derived tendons into an in vivo environment, we have significantly improved their tensile strength. The tensile strengths of the conditioned constructs were similar to native tendons from two week old rats (10,275 kPa vs. 23,800 kPa, respectively). Collagen content also improved significantly as measured by hydroxyproline concentration. Histologically, the constructs’ structure was more similar to mature collagen, with increased collagen fiber diameter, greater organization of the collagen fibers and a decrease in extracellular matrix. Future research will be aimed at optimizing the tensile strength and collagen content via administering active forces across the developing constructs in vivo.
production. Immunoreactive collagen (normalized to GAPDH) increased significantly (p ⬍ 0.05) after TGF-1 (4.20 ⫾ 0.70 fold), TGF-3 (2.45 ⫾ 0.37 fold for the 2.5 ng/mL dose and 2.91 ⫾ 0.15 fold for the 5 ng/mL dose), LPA (2.10 ⫾ 0.17 fold for the 25 M dose) and ET (1.9 ⫾ 0.34 fold for the 50 nM dose) but not after thrombin. These results suggest that while TGF-3 is thought to reduce scar formation, similar increases in CTGF and collagen expression were observed with TGF-3 as with TGF-1 and LPA. A phosphopeptide analogue of the small heat shock related protein, HSP20, conjugated to a protein transduction domain (PTDpHSP20) inhibits scar formation in a hamster model. The effect of PTD-pHSP20 (50 M) on TGF-1, LPA and ET- induced increases in the CTGF and collagen expression were determined. PTDpHSP20 significantly (p ⬍ 0.05) inhibited TGF-1, LPA and ET-1 induced increases in CTGF (30.20 ⫾ 9.35 %, 36.00 ⫾ 11.00 %, and 27.21 ⫾ 8.10 % respectively) and collagen (43.50 ⫾ 7.50 %, 47.50 ⫾ 10.23 %, and 27.70 ⫾ 10.71 % respectively) expression. These results suggest that phosphopeptide analogues of HSP20 can reduce the expression of CTGF and collagen induced by multiple mediators of scar formation (TGF-1, LPA and ET-1). In addition, TGF-3 appears to have the same effect as TGF-1 on CTGF and collagen expression in human keloid fibroblasts. PTD-pHSP20 treatment may represent a novel strategy for the treatment of keloids and other fibrotic disorders.
P36. ANASTOMOTIC LEAKS: A SCIENTIFIC PERSPECTIVE. M. Zerey, R. D. Peindl, B. Paton, W. Hope, W. Newcomb, T. Schmelzer, J. Cristiano, J. Heath, A. E. Lincourt, K. W. Kercher, K. S. Gersin, B. Heniford; Carolinas Medical Center, Charlotte, NC
P35. CTGF AND COLLAGEN EXPRESSION IN HUMAN KELOID FIBROBLASTS. L. Lopes 1, P. Komalavilas 1, E. Furnish 1, G. Yang 2, M. Longaker 2, C. Brophy 3; 1Arizona State University, Tempe, AZ, 2Stanford, Palo Alto, CA, 3Phoenix Vamc, Phoenix, AZ Paracrine factors that have been implicated in keloid formation include transforming growth factor -1 (TGF-1), thrombin, lysophosphatidic acid (LPA), and endothelin (ET). On the other hand, TGF-3 has been implicated in reducing scar formation. We investigated the effect of these mediators on connective tissue growth factor (CTGF) and collagen expression in human dermal keloid fibroblasts. Human keloid fibroblasts were grown to 80% confluence, serum-starved for 48 h, and subsequently stimulated for 24 hr with TGF-1 (2.5 ng/mL), TGF-3 (2.5 or 5 ng/mL), thrombin (25-150 nM), LPA (10 or 25 M), and ET (25 or 50 nM). Untreated cells were used as controls and all experiments were done in triplicate. Immunoreactive CTGF (normalized to GAPDH) increased significantly (p⬍0.05) after TGF-1 (3.26 ⫾ 0.31 fold), TGF-3 (1.75 ⫾0.44 fold for the 2.5 ng/mL dose and 2.60 ⫾ 0.64 fold for the 5 ng/mL dose), LPA (2.15 fold ⫾ 0.64 for the 25 M dose) and ET (1.80 fold ⫾ 0.26 for the 50 nM dose) but not after thrombin. CTGF induces scar formation by increases in collagen
Introduction: Anastomotic leaks are devastating complications in surgery. There is little standardization when assessing anastomotic leaks and reporting anastomotic integrity. The leak rate may be important when: 1) evaluating mechanical staplers and stapling reinforcement products, and 2) predicting clinical outcomes when leaks occur. We present a novel technique quantifying anastomotic leak pressure and leak rate at a given intraluminal pressure. Methods: In an Institution’s Animal Care and Use Committee approved protocol, we performed gastrojejunal anastomoses using a #25 Proximate ILS circular stapler (Ethicon Endo-Surgery, Cincinnati, OH). Individual gastrojejunal anastomoses were excised en bloc and placed in the following leak measurement system (Figure 1). The system delivers pressurized Methylene Blue (MB) to a Plexiglas TM chamber. The gastric cuff is secured along its cicrcumference and the jejunal limb is fed through a 32mm aperture exposing the gastrojejunal anastomosis to clear, de-ionized water in a secondary chamber. The leak pressure is noted at the first visual appearance of MB at the stapled anastomosis. Thereafter, 1mL aliquots are obtained from the water at one-minute time intervals and the spectrophotometric absorbance at 668nm is measured at the initial leak pressure at that time point (DU 65 spectrophotometer, Beckman Instruments Inc; Fullerton, Calif). In order to better understand the behavior of leaks at rising pressures, the delivery pressure of the MB is increased by 0.2PSI increments once the leak rate has remained constant for a determined period of time at a given pressure. The experiment is terminated once the upper limit of measurable absorbance is reached. In order to determine leak volume in experimental samples, a standard curve was performed, noting optical absorbance at 668nm with increasing concentrations of MB solution. Leak rate at a given pressure was calculated by dividing the change in MB solution volume over a measurement period. SigmaPlot version 9.0 (Systat Software Inc., Richmond, CA) was used to plot the data and determine the best fitting linear or quadratic equation. Results: The best-fitting quadratic equation for the standard curve was y ⫽ 0.3644 ⫹ 14.12x ⫹ 4.317x 2 (r ⫽ 0.9997). An example of the data generated is illustrated in