All animal procedures were approved by the Harvard Medical Area Standing Committee on Animals (Protocol 693) and the Harvard Committee on Microbiological Safety (COMS AR 92-3). All procedures conformed to the regulations related to animal use and other federal statutes. Four female Yorkshire pigs (2 diabetic, 2 non-diabetic; Parson's Farm, MA) weighing 50–60 kg at arrival were allowed to acclimatize for 1 week prior to initiation of the experiment. Animals were kept in smooth-walled stainless steel cages to minimize wound trauma and disruption of applied wound chambers. During procedures pigs were kept in a panepinto sling (Universal Metals, Milford, MA)
Induction of diabetes
Pigs were fasted for 12 hours before diabetes was induced. On the day of the procedure, the animals received induction anesthesia with Ketamine (Hospira, Lake Forest, IL)/Xylazine (Xyla-Ject, Phoenix, St. Josephs, MO) via intramuscular injection and were weighed. While animals were under general anesthesia with isoflurane (2–3 Vol%; Novaplus, Hospira, IL), a 21-gauge intravenous catheter (Becton Dickinson, NJ) was inserted into an ear vein. Streptozotocin (AXXORA, LLC; San Diego, CA 92121 USA) was prepared at a dose of 150 mg/kg body weight diluted in 9.5 ml/mg sterile saline (0.9% NaCl injection USP, Baxter) and sterilized by filtration. To keep the blood glucose concentration between 250 and 500 mg/dl, pigs received daily injections of 10 IU insulin/10 IU NPH insulin (Humulin, Eli Lilly, IN) subcutaneously. Blood glucose was measured on a daily basis during the experiment.
Wounding and chamber treatment
Fourteen days after induction of diabetes, pigs received anesthesia as described above. Oxygen saturation and heart rate were measured with pulse-oximeter ear sensors (Datex Ohmeda, Columbia, MD); respiratory rate and rectal temperature were also monitored during the procedure. Prior to surgery the porcine dorsum was waxed (Nair, Church & Dwight, Princeton, NJ) and shaved to remove hair, and 14 squares measuring 1.5 × 1.5 cm were outlined using a template and skin marker. The outlines of the wounds were retraced using a tattoo gun and black ink (Special Electric Tattoo Marker, Huck Spaulding Enterprises, Inc., Voorheesville, NY). The paraspinal area was washed with alcohol and thoroughly disinfected using 10% povidone iodine paint and then washed with 70% isopropanol after 20 minutes of incubation. Fourteen full-thickness excisional wounds (1.5 × 1.5 × 0.5 cm) were created using a No. 11 blade. Hemostasis was achieved by tamponade with sterile gauze. A thin layer of medical adhesive (No. 7730, Hollister, Libertyville, IL) was applied to the skin surrounding the wound, and an adhesive polyurethane chamber (Corium International, MI) was placed over each wound. The edges of the wound chamber were secured with adhesive Leukotape (BSN medical Ltd, Brierfield, BB9 5NJ, England) bandage. After the animal recovered from anesthesia, buprenorphine (0.005 mg/kg BW) was given intramuscularly every 12 hours for 48 hours. From each chamber, wound fluid was collected and measured every day and the glucose concentration determined (Ascensia Elite, Bayer Healthcare, NY). Wound chambers were reinjected daily with 1 ml 0.9% sterile saline.
For bacterial inoculation a methicillin-sensitive strain of S. aureus (American Type Culture Collection no.25923) was used. A single colony of S. aureus was removed from the stock, inoculated into Brain Heart Infusion (BHI) culture media (Becton&Dickinson, Franklin Lakes, NJ USA 07417), and incubated for 18 hours at 37°C. The culture was centrifuged, the supernatant was discarded, and the bacterial pellet was resuspended in sterile phosphate buffered saline (PBS) and adjusted to a final concentration of 2 × 108 colony-forming units (CFU)/ml. To immerse the enclosed wound surface, 1 ml of the bacterial suspension was injected into each chamber. Control wounds were injected with 1 ml of sterile PBS (carrier control). After an inoculation period of 48 hours, solutions were removed on a daily basis and replaced by 1 ml of sterile 0.9% saline solution.
On the final day of the experiment (day 12) pigs were euthanized (Euthasol; Virbac AH, Fort Worth, TX) and 2 mm cross-sectional wound biopsies were taken from the remaining wounds. Samples were fixed in 4% buffered paraformaldehyde and routinely processed for hematoxylin-eosin and tissue gram staining for reepithelialization and localization of bacteria in the wound tissue.
Assessment of re-epithelialization
Re-epithelialization was calculated by scanning the slides (Epson Perfection 3600, Epson, CA) and measuring the epithelial tongues from the computerized image with Paintshop Pro 7.0 software (Jasc Software, MN) using the formula (Sum of epithelial tongues)/(Distance between tattoo marks) × 100.
Wound contraction was determined by digitized planimetry of the tattooed margins. The area of the wounds at specific days was measured using Scion image software (Scion, MD), and the percentage of contraction was calculated by the formula (area at biopsy day)/(area on wounding day) × 100.
Quantification of bacteria
Biopsy specimens (3 mm punch-biopsy) for bacterial quantification in tissue were obtained on days 4, 8, (n = 3 wounds for S. aureus-inoculated wounds) and day 12 (n = 4 wounds for all groups with 4 punch biopsies per wound). All biopsied wounds were excluded from further study. The numbers of CFU/ml were counted in 100-μl aliquots of wound fluid. At days 4, 8, and 12 after wounding, the overlaying fibrinous wound clot was carefully removed and 4 punch tissue biopsies specimens were taken per wound (one per quadrant). Each tissue sample was subjected to surface decontamination by rinsing with 95% ethanol for 2 seconds, two times ignition for 5 seconds, followed by three serial washes with sterile PBS. Samples were then individually weighed and homogenized in 1 ml of sterile 0.9% saline solution using a sterilized tissue homogenizer (Pro Scientific Inc, Oxford, CT USA). Serial dilution was performed, and samples were plated onto trypticase soy agar containing 5% sheep blood agar (PML microbiologicals, Warwick, RI USA) for total colony counts, mannitol salt agar (PML microbiologicals, Warwick, RI USA) for selective S. aureus detection, and Cetrimide selective medium (PML microbiologicals, Warwick, RI USA) for P. aeruginosa. Further confirming tests included Gram-positive cocci:catalase test and bacitracin disk test. All specimens were incubated aerobically at 37°C for 24 hours. An additional specimen obtained from the final wash solution for each biopsy sample was also plated to detect potential surface contamination. Findings on these plates precluded any additional analysis of a biopsy sample. All colony counts were expressed as log10 colony-forming units (CFU) per gram of tissue and ml wound fluid. Bacterial counts >1 × 105 were considered to denote bacterial infection[28, 29].
This study included a total of 56 wounds in 2 diabetic pigs and 2 non-diabetic pigs divided into 4 experimental groups:
A: Diabetic wounds treated with a sterile 0.9% saline solution (n = 14) B: Diabetic wounds inoculated 2 × 108 CFU S. aureus (n = 14) C: Non-diabetic wounds treated with sterile 0.9% saline solution (n = 14) D: Non-diabetic wounds inoculated with 2 × 108 CFU S. aureus (n = 14). Values are presented as means ± SE. Groups were compared using the independent t-test, and statistical calculations were performed with GraphPad Instat software (GraphPad Software, CA). A p-value < 0.05 was considered statistically significant.