The Effects of TGF-b3 Modulation on Scar Tissue Formation in the Pig

Nathan D. Schwade, PhD, James J. Fowler, MD, Joseph Leach, MD, Department of Otolaryngology - Head and Neck Surgery, University of Texas Southwestern Medical Center, Dallas, Texas

Disclosures

Wounds. 2000;12(2) 

In This Article

Materials and Methods

The pig was chosen as a model since its skin architecture closely resembles that of a human.[16] Two young Yorkshire Domestic female pigs, weighing 16-18kg each, were used for this study. One pig was designated to be sacrificed on post-operative day 7 and the second on postoperative day 14. This allowed the opportunity to evaluate the wounds for changes in both breaking strength and histology over a period of time as the scar matured. Each animal was treated in accordance with the University of Texas Southwestern Medical Center Institutional Animal Care and Research Advisory Committee (IACRAC) guidelines.

The TGF-b3 isomer and TGF-b3 antibody dosages were prepared from stock bottles containing TGF-b3 isomer (R&D Systems Minneapolis, MN) and neutralizing antibody to TGF-b3 (R&D Systems Minneapolis, MN). The TGF-b3 antibody was suspended in phosphate-buffered saline (PBS) and diluted to yield 0.2cc solutions containing 0.1µg, 1.0µg, and 10µg of TGF-b3 antibody respectively. The stock solution of TGF-b3 isomer was diluted in 4mm HCL and bovine serum albumin (1mg/ml) to 0.2cc solutions containing 1ng, 10ng, and 100ng respectively. The varying solutions were then grouped as such: group 1 (10µg anti-TGF-b3); group 2 (1µg anti-TGF-b3); group 3 (0.1µg anti-TGF-b3); group 4 (100ng TGF-b3 isomer); group 5 (10ng TGF-b3 isomer); and group 6 (1ng TGF-b3 isomer). Group 7 represented the vehicle control (PBS).

The pigs were anesthetized with five percent isoflurane via facemask until an adequate level of anesthesia was reached. At that point the isoflurane was reduced to two percent throughout the remainder of the procedure. Supplemental oxygen was given at a rate of 2L/minute via facemask. The animals were placed on the operating table. The dorsum of each animal was then prepped and draped in a sterile fashion.

After cleaning the dorsum of each pig, 42 1.5cm incision sites were drawn using a surgical marker. At each site, a #15 surgical blade was used to incise the skin and subcutaneous tissue down to the underlying paraspinous musculature. At this point, the isomer or antibody was injected into the opposing aspects of the dermis created by the incision. Attempts were made to prohibit escape of the solution into the open wound. After administration of the TGF-b3 isomer and antibody, Benzoin adhesive was applied between each incision site, and a sterile occlusive dressing was placed over the dorsum. This served to localize the transudate from the wound and minimized any spread to adjacent wound sites. No sutures were used on the incisions.

On postoperative days 1 and 2, the pigs were re-anesthetized and injected with identical concentrations of either TGF-b3 antibody or isomer. Sterile occlusive dressings were again applied and the animals were then returned to their pens where they were provided with appropriate food and water. The animals were observed daily for any signs of infection. On postoperative day 7 the first animal was painlessly sacrificed according to IACRAC protocol. The entire section of skin covering the dorsum of the animal was harvested as one piece. Five-mm plugs at the distal end of each wound were removed and placed in formalin for histologic examination. The remainder of the section was sent fresh for tensiometry. Breaking strength measurements were performed using a tensiometer (Instron Corp Series IX Automated Materials Testing System 1.34). Specimens measured 8mm x 10mm with a grip distance of 20mm. Machine parameters were as follows: sample rate 10 points/sec, crosshead speed 10mm/min, humidity 50 percent, and temperature 73[ring]F.

Samples of treated and control wounds were sent for pathologic examination. Each was evaluated in a blinded fashion by the pathologists. Wound samples were evaluated after hematoxylin/eosin staining for abnormalities in the epidermis, dermis, and subdermis. The epidermis was evaluated for crust formation, focal acanthosis, and spongiosis. The dermis was evaluated for degree of fibroplasia, presence of reactive vascular endothelium, mast cell infiltrate, lymphocytoplasmic infiltrate, giant cell infiltrate and histiocytic infiltrate.

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