Tumor Necrosis
Factor
and Wound Healing
DAVID P. MOONEY, M.D., MICHAEL O'REILLY, M.S., and RICHARD L. GAMELLI, M.D., F.A.C.S.
Tumor necrosis factor alpha (TNF), 1 to 500 ng in saline (PBS) or collagen, was applied to the wounds of normal and Adriamycinimpaired mice and wound disruption strength (WDS) and histology were examined. Also wounded mice were administered TNF 25 to 75 ,ug/kg IP daily and WDS was determined. Wound histology was examined 6 months after wounding and local TNF application. Local TNF 5 to 500 ng in PBS did not significantly affect WDS. Local TNF 5 to 50 ng in collagen increased WDS 33% to 65% in Adriamycin-impaired animals (p = 0.05 to p < 0.02). Local TNF 50 to 500 ng in collagen increased WDS 23% to 49% in normal animals (p = 0.08 to p < 0.01). Adriamycin-impaired animals demonstrated improved wound histology with local TNF in collagen. Systemic TNF did not significantly affect WDS. Local TNF in collagen did not induce histologic pathology at 6 months. TNF may modulate macrophage function and local TNF in collagen can improve WDS in normal and Adriamycin-impaired animals.
T n UMOR NECROSIS FACTOR alpha (TNF), a product of activated macrophages, was initially described by Carswell et al.' for its ability to induce the necrosis of implantable soft-tissue sarcomas. Since that description TNF has been reported to be a mediator of such diverse processes as cancer cachexia,2 endotoxic shock,3 and hematopoiesis.4 More recently experimental observations suggest that TNF may modulate wound healing; TNF stimulates replication of human diploid fibroblasts,5 chemotaxis by bovine endothelial cells, and angiogenesis in the rat cornea and chick chorioallantoic membrane.6 Based on these observations, we examined the effect on wound disruption strength (WDS) of the topical application of recombinant human TNF alpha to both normal and Adriamycin-impaired animals. WDS was also analyzed in normal animals that were administered TNF systemically. Address correspondence and request of reprints to Richard L. Gamelli, M.D., Department of Surgery, Given Building, University of Vermont, Burlington, Vermont 05401. Accepted for publication: May 12, 1989.
From the Department of Surgery, University of Vermont, Burlington, Vermont
Materials and Methods Experimental Design In a series ofexperiments, groups of 12 mice each were wounded then randomized by body weight to receive locally, in a blinded fashion, PBS, TNF in PBS, collagen, or TNF in collagen. Animals were maintained for 11 days, and were weighed on days 5 and 11 as a measure ofclinical health and gross nutritional status. Eleven days after wounding the animals were killed and WDS determined. A second series of experiments were performed in which groups of 12 animals were wounded then received TNF IP for 11 days, at which time they were killed and WDS, splenic weight, white blood cell count, and differential cell count were determined. In a final experiment, groups of 6 animals were randomized, wounded, and received locally nothing, collagen, or TNF in collagen, were maintained for 6 months before being killed, and histologic analysis of their wounds was made. Mice Male B6DF1 mice (18 to 20 g; Jackson Labs, Bar Harbour, ME) were acclimatized for at least 1 week in a central animal care facility with a 12-hour light/dark cycle and controlled temperature and humidity. Animals randomized by weight before wounding were housed 12 per cage and given food and water ad libitum. TNF
Recombinant TNF, with an activity of 1.89 X I07 units and endotoxin level of less than 2.5 EU/mg (LAL), the gift of H. Michael Shepard of Genentech, Inc., S. San Francisco, California, was diluted to the appropriate con-
124
VoL. 211 -*No. 2
TUMOR NECROSIS FACTOR AND WOUND HEALING
125
centrations in phosphate-buffered saline (PBS) or collagen before use.
animals per group are being maintained for 1 year before wound analysis.
Collagen Modified type I bovine collagen (Zyderm II), gift of the Collagen Corp., Palo Alto, California, was diluted to the appropriate concentration in PBS.
Systemic TNF After wounding as above, four groups of eight animals each underwent daily IP injection of 25, 50, or 75 ,g/kg of TNF in PBS or an equal volume of PBS for 11 days, were killed with ether, and their pelts were harvested. WDS was determined as above along with tail vein white blood cell count, differential cell count, and splenic weight.
Adriamycin Adriamycin (Adria Labs, Columbus, OH) was diluted to 2 mg/mL in saline immediately before use and 15 mg/ kg or an equal volume of saline was administered to each animal by single intraperitoneal (IP) injection synchronous with wounding. Wounding Technique Under ether anesthesia groups of 12 animals each, which received either Adriamycin or an equal volume of saline IP, underwent a 3-cm aseptic surgical incision through their shaved dorsal skin and panniculus carnosus. After wounding and before wound closure with clips, animals were randomized to receive the local application to the cut wound surface of PBS, TNF in PBS, collagen, or TNF in collagen. The skin clips were removed and animals were weighed five days after wounding. The animals were killed on day 11 by ether anesthesia, were weighed, and their pelts were rapidly harvested. Dumbbell-shaped skin strips were cut perpendicularly across the wound and WDS was determined using a tensiometer connected to an X-Y plotter and load transducer via Wheatstone bridges. The maximal load required for disruption was recorded via a digital readout and load deformation curves were obtained in duplicate fashion for each animal.
Statistical Analyses Data was entered into an IBM PC XT (IBM, Boca Raton, FL) and statistical analyses were performed using Duncan's multiple-range method with BMDP statistical software on the University of Vermont Academic Computing Center VAX 8600 system. Results The Effect of Local Administration of TNF on Wound Healing in Normal Animals The local administration of the collagen vehicle alone did not result in any significant difference in WDS when compared to animals that received PBS only when analyzed 11 days after wounding (data not shown). The local administration of 10, 50, 100, or 500 ng of TNF in PBS to the wounds of normal animals did not result in any significant difference in WDS or body weight when compared to animals that received PBS only when analyzed 11 days after wounding (data not shown). The administration of 10, 50, 100, or 500 ng of TNF in the collagen vehicle locally to the wounds of normal
Histology One skin strip per animal was fixed in 10% buffered formaldehyde solution (Anderson Labs, Fort Worth, TX) for at least 24 hours; then six skin strips per group were sectioned and stained with trichrome and hematoxylin and eosin (H&E) stains before examination in a blinded fashion by light microscopy.
The Effect of Topically Delivered TNF on Wound Strength in Normal Animals
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Factor
and Wound Healing
DAVID P. MOONEY, M.D., MICHAEL O'REILLY, M.S., and RICHARD L. GAMELLI, M.D., F.A.C.S.
Tumor necrosis factor alpha (TNF), 1 to 500 ng in saline (PBS) or collagen, was applied to the wounds of normal and Adriamycinimpaired mice and wound disruption strength (WDS) and histology were examined. Also wounded mice were administered TNF 25 to 75 ,ug/kg IP daily and WDS was determined. Wound histology was examined 6 months after wounding and local TNF application. Local TNF 5 to 500 ng in PBS did not significantly affect WDS. Local TNF 5 to 50 ng in collagen increased WDS 33% to 65% in Adriamycin-impaired animals (p = 0.05 to p < 0.02). Local TNF 50 to 500 ng in collagen increased WDS 23% to 49% in normal animals (p = 0.08 to p < 0.01). Adriamycin-impaired animals demonstrated improved wound histology with local TNF in collagen. Systemic TNF did not significantly affect WDS. Local TNF in collagen did not induce histologic pathology at 6 months. TNF may modulate macrophage function and local TNF in collagen can improve WDS in normal and Adriamycin-impaired animals.
T n UMOR NECROSIS FACTOR alpha (TNF), a product of activated macrophages, was initially described by Carswell et al.' for its ability to induce the necrosis of implantable soft-tissue sarcomas. Since that description TNF has been reported to be a mediator of such diverse processes as cancer cachexia,2 endotoxic shock,3 and hematopoiesis.4 More recently experimental observations suggest that TNF may modulate wound healing; TNF stimulates replication of human diploid fibroblasts,5 chemotaxis by bovine endothelial cells, and angiogenesis in the rat cornea and chick chorioallantoic membrane.6 Based on these observations, we examined the effect on wound disruption strength (WDS) of the topical application of recombinant human TNF alpha to both normal and Adriamycin-impaired animals. WDS was also analyzed in normal animals that were administered TNF systemically. Address correspondence and request of reprints to Richard L. Gamelli, M.D., Department of Surgery, Given Building, University of Vermont, Burlington, Vermont 05401. Accepted for publication: May 12, 1989.
From the Department of Surgery, University of Vermont, Burlington, Vermont
Materials and Methods Experimental Design In a series ofexperiments, groups of 12 mice each were wounded then randomized by body weight to receive locally, in a blinded fashion, PBS, TNF in PBS, collagen, or TNF in collagen. Animals were maintained for 11 days, and were weighed on days 5 and 11 as a measure ofclinical health and gross nutritional status. Eleven days after wounding the animals were killed and WDS determined. A second series of experiments were performed in which groups of 12 animals were wounded then received TNF IP for 11 days, at which time they were killed and WDS, splenic weight, white blood cell count, and differential cell count were determined. In a final experiment, groups of 6 animals were randomized, wounded, and received locally nothing, collagen, or TNF in collagen, were maintained for 6 months before being killed, and histologic analysis of their wounds was made. Mice Male B6DF1 mice (18 to 20 g; Jackson Labs, Bar Harbour, ME) were acclimatized for at least 1 week in a central animal care facility with a 12-hour light/dark cycle and controlled temperature and humidity. Animals randomized by weight before wounding were housed 12 per cage and given food and water ad libitum. TNF
Recombinant TNF, with an activity of 1.89 X I07 units and endotoxin level of less than 2.5 EU/mg (LAL), the gift of H. Michael Shepard of Genentech, Inc., S. San Francisco, California, was diluted to the appropriate con-
124
VoL. 211 -*No. 2
TUMOR NECROSIS FACTOR AND WOUND HEALING
125
centrations in phosphate-buffered saline (PBS) or collagen before use.
animals per group are being maintained for 1 year before wound analysis.
Collagen Modified type I bovine collagen (Zyderm II), gift of the Collagen Corp., Palo Alto, California, was diluted to the appropriate concentration in PBS.
Systemic TNF After wounding as above, four groups of eight animals each underwent daily IP injection of 25, 50, or 75 ,g/kg of TNF in PBS or an equal volume of PBS for 11 days, were killed with ether, and their pelts were harvested. WDS was determined as above along with tail vein white blood cell count, differential cell count, and splenic weight.
Adriamycin Adriamycin (Adria Labs, Columbus, OH) was diluted to 2 mg/mL in saline immediately before use and 15 mg/ kg or an equal volume of saline was administered to each animal by single intraperitoneal (IP) injection synchronous with wounding. Wounding Technique Under ether anesthesia groups of 12 animals each, which received either Adriamycin or an equal volume of saline IP, underwent a 3-cm aseptic surgical incision through their shaved dorsal skin and panniculus carnosus. After wounding and before wound closure with clips, animals were randomized to receive the local application to the cut wound surface of PBS, TNF in PBS, collagen, or TNF in collagen. The skin clips were removed and animals were weighed five days after wounding. The animals were killed on day 11 by ether anesthesia, were weighed, and their pelts were rapidly harvested. Dumbbell-shaped skin strips were cut perpendicularly across the wound and WDS was determined using a tensiometer connected to an X-Y plotter and load transducer via Wheatstone bridges. The maximal load required for disruption was recorded via a digital readout and load deformation curves were obtained in duplicate fashion for each animal.
Statistical Analyses Data was entered into an IBM PC XT (IBM, Boca Raton, FL) and statistical analyses were performed using Duncan's multiple-range method with BMDP statistical software on the University of Vermont Academic Computing Center VAX 8600 system. Results The Effect of Local Administration of TNF on Wound Healing in Normal Animals The local administration of the collagen vehicle alone did not result in any significant difference in WDS when compared to animals that received PBS only when analyzed 11 days after wounding (data not shown). The local administration of 10, 50, 100, or 500 ng of TNF in PBS to the wounds of normal animals did not result in any significant difference in WDS or body weight when compared to animals that received PBS only when analyzed 11 days after wounding (data not shown). The administration of 10, 50, 100, or 500 ng of TNF in the collagen vehicle locally to the wounds of normal
Histology One skin strip per animal was fixed in 10% buffered formaldehyde solution (Anderson Labs, Fort Worth, TX) for at least 24 hours; then six skin strips per group were sectioned and stained with trichrome and hematoxylin and eosin (H&E) stains before examination in a blinded fashion by light microscopy.
The Effect of Topically Delivered TNF on Wound Strength in Normal Animals
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