Experimental Study On The Repair Of Skin Defect In Rat With Tissue-Engineered Skin

Objective:In this experimental study, we transplanted TES in the full-thickness skin defect of rat, and observed the healing process after transplanting. The objective is to investigating the feasibility and validity of repairing full-thickness skin defect with TES, and providing the experimental foundation for the ultimate clinical application of TES.Method:1 Experimental subjects: 64 healthy adult female SD rats of clean grade were employed and divided into 4 groups randomly, 16 cases in each group. Group A: TES graft group, Group B: full-thickness skin auto-graft group, Group C: Collagen membrane (CM) graft group, Group D: blank control group.2 The establishment of full-thickness skin defect model in SD rat: We designed one piece of wound on rat's back and cut off the whole layer of skin from fat layer along the drawn line. When bleeding stopped thoroughly and the wound was covered with wet physiological saline gauze. Then one wound was prepared in each rat, round in shape and 2.5cm in diameter. 3 Carrying out the transplantation: The graft of each group was covered and interrupted sutured on the wound, avoiding gas vacuole between them. Single-layer oleic gauze, physiological saline gauze and aseptic dry gauze were covered on the graft in turn .The whole wound dressing was about 2cm in thickness. After operation the rats were fed routinely with one in each cage, and the wound dressing was changed weekly.4 General observations and the indexes: (1) At 4 weeks after operation, the survival rate of group A and B (%) were calculated and analyzed statistically. (2) At 15 and 30 days after operation, the percentages of healing wound in each group (%) were calculated and analyzed statistically. (3) Analyzing statistically the healing time in each group (day).5 Histological observations: Biopsy was done by cornea ring drill at 10, 20, 30, 40 days after transplantation separately and the samples were observed under microscope. The results were compared with each other in 4 groups at each time-point.6 Statistical analysis: We used SAS V6.12 software for analysis. Fisher's exact probability test was applied for enumeration data statistics. Analysis of variance (ANOVA) or T test was applied for measurement date statistics ( x±s).Result:1 General observations: Group A: The grafts survived well except 3 failed cases. The surface of recipient area was smooth without any hair. The scar was relatively small, and the color was similar to normal skin. TES adhered on the wound tightly, and till 40 days after transplantation, we didn't find any evidence of acute and chronic immunological rejection. Group B: The grafts survive well except 1 failed case. The adherence of auto-graft was quite good, and the auto-graft emerged into its surrounding normal skin gradually. About 30 days after operation the wound closed to complete healing, and then the difference between auto-graft and normal skin was not obvious. Group C: There were 4 cases caught infection. The grafts were degraded gradually or fell off from the wound. And the wound healed up with a scar. Group D: One rat died during the experiment. Other wounds healed up with a colored scar.2 Graft survival rate: At 4 weeks after transplantation, the survival rate of group A and B were the following: 81.25% and 93.75%, there was not significant difference between the 2 groups (P>0.05).3 The percentage of healing wound: At 15 days after transplantation, the percentage of healing wound in 4 groups were following: 75.54±6.85%, 81.97±6.32%, 41.90±4.94%, 42.99±6.63%. At 30 days after transplantation, the percentage of healing wound in 4 groups were following: 94.23±8.86%, 99.08±9.06%, 80.07±6.55%, 78.04±8.21%. There were significant differences among group A and B, C, D at each time-point. The percentage of group A was lower than that of group B (P<0.05), and significant higher than that of group C (P<0.01) and D (P<0.01).4 Healing time of the wound: The wound healing time in 4 groups were the following: 33.8±4.6d, 27.1±3.6d, 45.5±5.9d, 48.1±6.0d. There were significant differences among group A and B, C, D. The healing time of group A was longer than that of group B (P<0.05), and significant shorter than that of group C (P<0.01) and D (P<0.01).5 Histological observations: Group A: There was a good confluence border between TES and recipient area. 4 layers of cells could be seen in epidermis, but keratinization was not obvious. Dermis was clear with quantities of collagen scaffold and FB. As time went by, the epidermis and dermis became thicker. And the number and diameter of newborn capillaries increased as well. Group B: The auto-graft almost integrated into the recipient area, and its structure was just about normal skin. Group C and D: A great deal of connective tissue proliferations could be seen in both CM group and bland control group.Conclusion:1 TES has similar structure to normal skin, and can be fold, cut or sutured. It is feasibility to apply TES in repairing the full-thickness skin defect.2 There was no evidence of acute and chronic immunological rejection in TES group. The graft survival rate after transplanting had no significantly difference between TES group and auto-graft group. So we can consider that TES has the capability to closing the full-thick skin loss wound.3 The therapy effect of TES group was much better than that of blank control group, and the wound healing time was significantly shorter. These results indicated that TES can promote the growth and differentiation of tissues and accelerate the wound cure. TES was structurally and functionally closed to natural skin. While compared with natural skin TES has some disadvantages. So the constructing method of TES must be improved continuously.4 The failed case in the experiment was perhaps related to the limitation of TES itself. The structure and function of TES have some shortages, which are disadvantaged to the surviving of TES. The main shortcomings are easy to be infected, low survival rate, without skin appendage, no perfect solution to immune rejection problem. So the constructing method of TES should be improved continuously. At the same time, according to the characteristics of TES, selecting suitable operation method and post-operation management will make up the disadvantages of TES.