2.2. Mouse Excisional Wound-Healing Model

Animals. All experiments were approved by the animal care committee at the Sackler Faculty of Medicine, Tel Aviv University, Israel and the Israeli Ministry of Health (M-12-071). WT mice and diabetic db/db mice (BKS.Cg-Dock7m +/+ Leprdb/J, #000642) were obtained from Jackson Laboratories (Bar Harbor, ME). Homozygous db/db mice possess a genetic mutation in the leptin receptor and represent a model of type 2 diabetes mellitus characterized by hyperglycemia, obesity, hyperinsulinemia, and impaired wound healing. These mice become obese at approximately three to four weeks of age. Elevations of plasma insulin begin at 10 to 14 days and elevations of blood sugar at 4 to 8 weeks. All of the animals used in this study were 10–12 weeks old. Mice were housed one per cage in a 12-h light/dark cycle and provided standard food and water.

PCR genotyping of mice tail DNA. Tail DNA was obtained from 14 day-old, heterozygous F2 littermates. Genotyping for WT and diabetic mice was carried out by PCR (KAPA_Mouse_Genotyping_Kit_TDS, KAPA biosystems), using primers specific for the wild-type and the mutant alleles and a common primer (WT- AGC CAC TAC AAT CCA CCC CTT G; Mutant- GCA GTG CAC AGG CTC AGG AA; Common- GCT GCA GAA TGG ACG GTT GA). The PCR data were verified by the color of the mice, the blood sugar levels, and the weight.

Diagnosis of diabetes. Diabetic mice (db/db genotype) were defined as those with confirmed hyperglycemia, expressing non-fasting blood glucose levels > 250 mg/dL from tail vein (Contour, Bayer).

Preparation of stem cells for transplantation. Cells for transplantation were collected from cultures at 80–90% confluency. Cells were washed with PBS, harvested with trypsin-EDTA, counted and resuspended at a concentration of 3 × 10⁶ in 200 µL in PBS. To prevent aggregation, cells were prepared immediately before transplantation.

Experimental wound-healing model. A model of excisional skin wound healing described previously by Galiano et al. [21], was used with changes detailed hereinafter. Briefly, WT and db/db mice were anesthetized using inhalation of isoflurane. Hair was shaved from the dorsal side of each animal. The skin surface to be excised and its surrounding area were rinsed with povidone-iodine 10%. A pain-relief subcutaneous injection (Rimadyl) was administered. In order to prevent skin contraction at the wound site, a doughnut-shaped silicone splint with an 8 mm inner diameter was sutured to the skin by 4-0 nylon continuous sutures. A sterile 6 mm diameter biopsy punch tool (Kruse) was used in the center of the silicone splint to make a full-thickness wound extending through the panniculus carnosus. The animals were housed in the institutional animal facility. Animals were divided into 4 groups: WT PBS-treated (control), WT PBS + mOMSCs-treated, db/db PBS-treated (control), and db/db PBS + mOMSCs-treated, each consisting of 6 to10 mice per group.

In the treated groups 200 µL PBS containing 3 × 10⁶ suspended mOMSCs generated from the lamina propria of the oral mucosa of allogeneic Balb/c mice was injected (insulin syringe, 30G) subdermally around and under the wound. In the control groups 200 µL PBS injections were injected at the same sites as in the treated group. Wounds were dressed with Tegaderm sterile dressing (3M Healthcare, St Paul, MN, USA).

Luciferase-labeled mOMSCs were injected into the wound. Cells were monitored on days 1, 7, and 14 days post-wounding using in vivo bioluminescence imaging performed after the luciferin administration.

Following surgery, the mice were placed in individual cages under a lamp, and allowed to fully recover from anesthesia.

Wound morphology analysis. Digital photographs of the wounds (Nikon camera) were taken at the time of surgery and every 3–4 days thereafter until wound closure. Wounds were considered as closed when the wound bed was completely filled in with new tissue. The wound area was measured at each time point using ImageJ software (NIH, Bethesda, MD, USA). The global wound area (%) was calculated according to the residual wound area on a given day (tx) relative to the wound area measured on the day of surgery. A wound was considered to be completely closed when the wound area was equal to zero. The rate of wound closure in the treated db/db was monitored and compared to control animals.

Histomorphometry. We analyzed histological sections of PBS + mOMSCs-treated and PBS-treated diabetic animals (n = 5 per time point) at 0, 7, and 11 days following wounding.

Histomorphometry of the Epithelial Gap. At the time of sacrifice, wounds were excised, and fixed in 10% formalin for 6 days. The samples underwent routine histological processing for paraffin embedding. The tissue was sectioned and stained with hematoxylin and eosin (H&E). All histological sections were scanned using Aperio ImageScope. The epithelial gap (EG) was digitally analyzed and defined as the distance between the advancing edges of epidermal keratinocytes. Three to six serial sections were averaged to determine the EG at each time point. An EG of zero represented a completely re-epithelialized wound.

Granulation tissue staining. The histological images were analyzed for the total area of GT using digital analysis software. The area of GT was calculated by tracing regions of GT and calculating pixel area. The total area of granulation was the sum of these regions.

Statistical Analysis. Results are expressed as mean ± SEM. Unpaired, two tails Student’s t-test and one-way analysis of variance for multiple comparisons with Tukey post hoc test were used to determine statistical significance, which was defined by a P value < 0.05.