Primary Study Of The Wound Repair By Tissue Engineering Skin Made Of Microskin Combined With Artificial Dermal Scaffold

Objective:To study the repairing effect of the wounds on the back of SD rats using tissue engineering skin (TES) made of microskin combined with artificial dermal scaffold.Materials and methods:The SD rats were divided into 4 groups:blank control group(Group A), artificial dermis grafting group(Group B), microskin grafting group(Group C) and TES grafting group(Group D). The wounds were examined at the 1st,3rd and 5th postoperative weeks in order to detect the difference of healing quality among them. Following are the results:1. Overview of wound healing:a macro study of wound healing, the contraction rate, the healing rate and analysis of HE staining.2. The repair of the epidermis:epidermis repairing quality by the full thickness of the epidermis was evaluated by scanning electronic microscopy (SEM). Further examination on the distribution of epidermal stem cells (ESCs) and follicle stem cells (FSCs) were achieved by CK15 and CD29 immunohistochemical staining.3. The repair of the dermis:dermis repair quality was comprehensively evaluated by the analysis of Masson staining results and the full-thickness of the dermis observed by SEM. Further evaluation was based on the results acquired in testing the RT-PCR determination of mRNA expression levels of type-I collagen and type-Ⅲcollagen.Finally, the measurement data obtained from the above tests was analyzed using the statistical software SPSS 13.0.Results:Among 4 groups, the SD rats repaired with TES grafting demonstrated the best results, and achieved the experimental recovery at the 5th week after surgery.At the 1st postoperative week, the contraction rates did not show differences between each others. At the 3rd postoperative week, wound contraction was evident in all the 4 groups. The contraction rate of Group D was the lowest (29.0±2.4%). At the 5th postoperative week, the contraction rate continued to rise in all the 4 groups. Wound contraction rate decreased slightly in Group A. The overall contraction rates of the other 3 groups were less than Group A. Contraction rates of the other 3 groups showed no significant differences between each other(P>0.05). At the 1st postoperative week, the healing rates of Group A and Group B were significantly lower than Group D, while that of Group C was the best. At the 3rd postoperative week, Group B showed the smallest coverage (53.8±5.5%); the healing rates were over 70% in the other 3 groups. At the 5th postoperative week, the healing rate was over 80% in all 4 groups, and the healing rate of 4 groups showed no significant differences between each other (P>0.05). The full-thickness of epidermis layer in Group A was the least in that of all 4 groups by SEM observation at the 5th postoperative week (P<0.01). The epidermal thickness of the other 3 groups showed no significant differences between each other (P>0.05).The results of CK15 and CD29 immunohistochemical staining were consistent to some extent. ESCs and FSCs located in these following structures:the full-thickness epidermis, basement membrane, the hair follicles, new glands in the wound area. The positive cells even scattered in the full-thickness of dermis layers. The decreased CK15 and CD29 immunohistochemical staining positive unit (PU) values reflected that ESCs and FSCs had been consumed in wound healing.HE staining indicated the infiltration of inflammatory cells and angiogenesis. The hair follicles under the epidermis gathered up to the epidermis layer, and participated in epidermal repair. Most of the artificial dermal scaffold had been replaced by the host collagen.The observation of Masson staining showed dermis thicknesses of Group B and Group D were thicker than that of Group A and Group C at the 1st,3rd and 5th postoperative weeks. Collagen remodeling and rearrangement in the dermis occurred during the progress of wound repair. At the 5th postoperative week, the collagen in Group A still had the lowest density, and arranged loosely. The artificial dermis in Group B and Group D had been replaced by new collagen. The new collagen in Group D gradually mixed together with the collagen that originated from the dermis of microskin. There was a higher density of fibroblasts in Group C and Group D when being compared with Group A and Group B.At the 5th postoperative week, with the help of their artificial dermal scaffold, Group B and Group D had the thicker dermis than Group A and Group C. The full thickness of dermis in Group D was 768.2±24.22μm. Dermis in Group C was the thinnest. A further observation of dermal layer in Group D showed that its fine structure contained the advantages in Group B and Group C. Upper layer was made of thick and disordered collagen with larger gaps among the fibers. Lower layer was made of the parallel, compact collagen bundles with small gaps among the fibers. A gradual transition between two layers helped to form a more mature structure of dermis.The mRNA expression levels of type-Ⅰcollagen and type-Ⅲcollagen in Group C and Group D were detected by RT-PCR at the 1st,3rd and 5th postoperative week. The mRNA expression levels of type-Ⅰcollagen were always at a high level in both groups, but the expression level was more prominent in Group D, especially at the 1st postoperative week. The mRNA expression levels of type-Ⅲcollagen were close between Group C and Group D. The expression levels at the 1st week were the highest during the wound healing.Conclusions:The construction of TES grafting by microskin combined with artificial dermal scaffold is simple and effective. This kind of TES could repair the wounds on the back of SD rats, and received a significant improvement on the healed dermis quality.