OBJECTIVETo study the infiltration of macrophages and their phenotype in the healing process of full-thickness wound in rat.

METHODSThirty healthy SD rats were divided into control group (n = 6) and injury group (n = 24) according to the random number table. Two round full-thickness skin defects (11 mm diameter) were created on both sides of dorsal spine of rats in injury group with surgical scissors and homemade trephine. After injury, wound area was measured immediately. The wounds were disinfected with iodophor every day. Rats in control group received anesthesia and hair removal only. On post injury day (PID) 1, 3, 7, and 13, respectively, 6 rats of injury group were sacrificed after the measurement of wound area (wound healing rate was calculated). Wound samples were obtained by excision down to healthy fascia along wound edge. Histological study was done with HE staining. The expression of CD68 (the surface marker of macrophage) in the wound tissue was observed with immunohistochemical staining. The double positive expressions of induced nitric oxide synthase (iNOS) plus CD68 (type I macrophage) and arginase 1 (Arg-1) plus CD68 (type II macrophage) were observed with immunofluorescence staining. The levels of interferon-γ (IFN-γ), TNF-α, IL-4, IL-13, IL-10, and IL-12 in wound tissue were assayed by double-antibody sandwich ELISA, and the ratio of IL-10/IL-12 was calculated. Full-thickness skin tissues (11 mm diameter) in rats of control group were excised at the same site as rats in injury group, and the histological observation and cytokines assay were performed as well. Data were processed with one-way analysis of variance or LSD- t test.

RESULTSWound area of rats in injury group was gradually reduced after injury, and the overall difference of the wound healing rate on each PID was statistically significant (F = 358.55, P < 0.01). No abnormal appearance of skin tissue was observed in rats of control group. In injury group, inflammatory cell infiltration was obvious in wound tissue on PID 1 and 3; vascular structure and fresh collagen were observed in wound tissue on PID 7 and 13. Numbers of CD68 positive cells in skin tissue of rats in control group and wound tissue of rats in injury group on PID 1, 3, 7, and 13 were respectively (2.7 ± 1.5), (31.8 ± 3.5), (40.8 ± 4.7), (20.8 ± 2.8), (3.2 ± 2.4) per 200 times visual field (F = 180.55, P < 0.01). Compared with that in control group, the number of CD68 positive cells of rats in injury group was increased on PID 1, 3, and 7 (with t values respectively 18.81, 18.79, 14.05, P values below 0.01). No double positive expression of iNOS plus CD68 or Arg-1 plus CD68 was observed in normal tissue of rats in control group. In injury group, proportions of iNOS plus CD68 double positive cells on PID 1, 3, 7, and 13 were respectively (12.2 ± 2.8)%, (16.5 ± 2.9)%, (4.2 ± 2.3)%, (0.7 ± 0.8)% (F = 72.50, P < 0.01); proportions of Arg-1 plus CD68 double positive cells on PID 1, 3, 7, and 13 were respectively 0, (8.2 ± 1.9)%, (21.5 ± 3.4)%, (4.7 ± 2.0)% (F = 120.93, P < 0.01). In injury group, proportion of iNOS plus CD68 double positive cells on PID 3 was significantly higher than that on other PID (with t values respectively 2.65, 8.17, 12.95, P values below 0.05); proportion of Arg-1 plus CD68 double positive cells on PID 7 was higher than that on other PID (with t values respectively 15.27, 8.25, 10.38, P values below 0.01). Compared with that of Arg-1 plus CD68 double positive cells, proportion of iNOS plus CD68 double positive cells was higher on PID 1 and 3 (with t values respectively 10.71 and 5.88, P values below 0.01) and lower on PID 7 and 13 (with t values respectively 10.24 and 4.60, P values below 0.01). The overall differences of IFN-γ, TNF-α, IL-4, IL-13, and IL-10/IL-12 ratio in skin tissue of rats in control group and wound tissue of rats in injury group on every PID were statistically significant (with F values from 14.08 to 631.03, P values below 0.01). Compared with those in control group, levels of IFN-γ, TNF-α, IL-4, and IL-13 in wound tissue of rats in injury group were significantly higher on every PID (with t values from 4.58 to 9.17, P values below 0.05), while IL-10/IL-12 ratio was significantly higher on PID 1, 3, and 7 (with t values respectively 27.70, 30.51, 9.49, P values below 0.05) . In injury group, IFN-γ level on PID 1 [(61 ± 5) pg/mL] and IL-10/IL-12 ratio on PID 3 (1.647 ± 0.098) were significantly higher than those of control group and those on other PID in injury group [with IFN-γ level respectively (32 ± 4), (54 ± 6), (46 ± 7), (47 ± 4) pg/mL and IL-10/IL-12 ratio respectively 0.328 ± 0.045, 0.960 ± 0.034, 0.530 ± 0.028, 0.289 ± 0.040, with t values respectively from 3.19 to 8.20 and from 16.59 to 31.84, P values below 0.05].

CONCLUSIONSMacrophage infiltration increases in the healing process of full-thickness wound in rat with different phenotypes, among which type I macrophage appears in the inflammatory stage, and type II macrophage predominates in the proliferative stage.

Animals ; Antigens, CD ; genetics ; metabolism ; Antigens, Differentiation, Myelomonocytic ; genetics ; metabolism ; Collagen ; Enzyme-Linked Immunosorbent Assay ; Interferon-gamma ; Interleukin-10 ; Interleukin-12 ; Interleukin-13 ; Interleukin-4 ; Macrophages ; Male ; Phenotype ; Rats ; Skin ; injuries ; Tumor Necrosis Factor-alpha ; blood ; Wound Healing ; genetics

OBJECTIVETo observe the fibrosis of skin after damage to the fat dome structure in skin of pig.

METHODSTotally 4 pieces of skin grafts of intermediate thickness in the size of 5 cm × 5 cm were obtained from both sides beside the spine of back in each of the 4 female red Duroc pigs with pedicle on one side with Humby knife performed by burn specialists, who were rich in clinical experience. These skin grafts were assigned as thin dermis group (TD). Pedicled tissue grafts in the size of 5 cm × 5 cm with the thickness of 1.5 mm were obtained within the wounds resulted from former incision with the same method mentioned above, and these tissue grafts were set as fat dome group (FD). The above-mentioned two groups of skin grafts were sutured back in situ immediately after completion of the former procedures. On post surgery day (PSD) 7, 14, and 21, 5 wounds were respectively selected according to the random number table for gross observation of the surgical areas. Tissue samples were obtained from corresponding surgical area deep to the deep fascia after gross observation at above-mentioned time points. Some of the tissue samples were used for observation of distribution of collagen fibers in the regions of operation of both groups of skin grafts with HE staining, and the breadth of fibrosis was measured; some of the tissue samples were used for observation of distribution of type I or III collagen fibers in the regions of incision of both two groups of skin grafts with Sirius red staining. Data were processed with two independent sample t test.

RESULTSA little scab on the edge of wounds was observed on PSD 7; all the wounds were healed on PSD 14; a few hairs were observed growing in the surgical area on PSD 21. HE staining showed that traces of incision were observed in the superficial layer of dermis and at the junction between dermis and fat dome at each time point; profuse hyperplasia of collagen fibers with parallel and orderly arrangement were observed in the region of incision of skin grafts in groups TD and FD at each time point. The breadth of fibrosis of the region of incision of skin grafts was respectively (251 ± 31), (240 ± 3 7), and (342 ± 69) µm in group TD, (239 ± 36), (286 ± 61), and (332 ± 28) µm in group FD on PSD 7, 14, 21, without significantly statistical difference (with t values respectively 0.750, -1.971, and 0.375, P values above 0.05). Sirius red staining showed that large amount of type III collagen fibers and small amount of type I collagen fibers arranging parallelly were present in the region of incision of skin grafts in groups TD and FD at each time point.

CONCLUSIONSUnder the circumstances of relatively intact restoration of dermal tissue, no excessive fibrosis was observed after simple incisional injury of fat dome in skin of pig.

Animals ; Burns ; surgery ; Dermis ; surgery ; transplantation ; Female ; Fibrosis ; complications ; Graft Survival ; Male ; Skin ; Skin Transplantation ; methods ; Skin, Artificial ; Swine ; Wound Healing