The Study Of Skin Histological Changes Under Expansion Stress

Skin and soft tissue expansion has become one of the most common plastic surgery treatments for tissue repair and reconstruction. However, there are disadvantages for the application of tissue expansion such as long duration and changes of skin texture. Therefore, obtain a large and short-term expanded skin tissues without significant skin texture alterations is required clinically and, therefore, becomes the focus of the research.The first part in this thesis describes an experimental method for the establishment of a tissue expansion model in rats. A newly designed expander was applied in the experimental animals and the expansion effects were evaluated on the sections of expanded tissue samples stained with HE and anti-PCNA antibody. Our animal model provides a good research tool for the study of the proliferation mechanism of expanded skin.The second part in this thesis describes the dynamic change of the skin collagen fibers during different expansion period. The Masson staining and Picric-Sirius Red staining were used to reveal the changes of skin collagen fibers in skin tissues under mechanical stress. We seek to explore the mechanisms associated with the characteristics of striae gravidarum-like skin and the high rate of skin retraction after expansion. These studies provide clue for further research on how to reduce the rate of skin retraction and regulate the type of collagen in vitro after expansion.The third part in this thesis describes the studies on the effects of dermal stem cells on the proliferation of the major cell types in expanded skin tissues. The results provide the experimental foundation for further research on how to promote proliferation of dermis during the skin expanding processes.Part Ⅰ:Application of the modified expander in a rat model of skin and soft tissue expansionObjective A newly designed pillow-shaped expander with short injection catheter was used to explore the mechanisms underlying the proliferation of skin cells under mechanical stress and to verify the effects of the expansion on skin tissues. We seek to establish a more stable animal model for further related researches.Methods Sixty SD rats were randomly divided into A or B group (30rats in each group) Rats in group A were implanted with the newly designed expander while the rats of group B were implanted with the ordinary expander manufactured by Shanghai Winner company. The expanders were implanted subcutaneously on the back rats. Samples of normal skin and expanded skin were collected from rats in Group A. The structure of skin was evaluated under microscope on the biopsy tissue sections with HE staining, and the distribution of the cells positive for immunohistochemistry stained PCNA were also evaluated.Results Stability of the model in group A was significantly higher than group B. After the expansion, the epidermis became thickened and the dermis were thinned. Punctate distribution of proliferating cells was observed mainly in the basal layer of the epidermis and hair follicle sheath revealed with immunohistochemistry staining.Conclusion In this study, the newly designed expander was proved to be suitable for researches on skin expansion in a rat model. The resultant effective expansion stress promotes the proliferation of skin cellular components and thickens skin tissues.Part Ⅱ:The dynamic changes of the skin collagen fibers in different expansion period.Objective:To analyze the changes in collagen fibers content per unit area of skin, and the changes in the ratio between type Ⅰ and type Ⅲ collagen fibers. To characterize the histological changes of collagen contents during skin expansion.Methods:The expanded skin tissue (1-5weeks after expansion) and unexpanded normal skin tissue samples were collected and the tissue sections were stained with Masson and Picric-Sirius Red staining. Tissue section images were taken under an optical microscope and a polarizing microscope, respectively, and were analyzed by Image-Pro Plus6.0image analysis software.Results:Compared to unexpanded normal skin tissue, the collagen fiber contents per unit area of expanded skin tissue decreased, and more loosely arrangement of collagen fibers was observed. The proportion of type I collagen fibers decreased while the proportion of type Ⅲ collagen fibers increased.Conclusion:Skin expansion leads to the decreased proportion of type I collagen fibers and increased proportion of type III collagen fibers. The changes of collagen contents may likely associate with the changes in skin texture and extensive retraction of expanded skin tissues.Part Ⅲ:An experimental hypothesis and preliminary exploration about dermal stem cells’role in the proliferation mechanism of expanded skin.Objective:To explore the proliferation and differentiation of dermal stem cells under the expanding stress and to provide some experimental foundation for research on how to promote proliferation of dermis in the expanding process.Methods:The adult stem cells in the skin of newborn SD rats were labeled by Brdu reagents, and skin and soft tissue expansion was initiated when the SD rats were5weeks old. At different time points (1-5weeks), the skin biopsies were harvested from the expanding skin sites and unexpanded normal skin sites. The tissue sections from the biopsies underwent PCNA and Brdu Immunofluorescence staining and the positive signals were co-localized by fluorescence microscopy. The293T cells were included as a quality and technique control for Brdu immunofluorescence staining.Results:The green fluorescent PCNA+cells can be clearly observed and more positive cells was obtained in the expanded skin tissue relative to unexpanded control tissues, as similarly demonstrated by the tissue sections with immunohistochemical staining in the first part. The green fluorescent Brdu+cells, however, was rarely found in the unexpanded skin tissues and was not observed in the expanded skin tissue. The Brdu labeling on293T cells verified that the antibodies and the staining techniques used in this study were effective.Conclusion:Brdu can be used as label reagents for skin stem cells in experimental animals. But it is not suitable for labeling the skin stem cells after the skin was expended possibly due to the difficulty in the detection of the fluorescence in the adult stem cells that lose the cellular quiescence and undergo speedy proliferation under the stimulation of mechanical stress, resulting in the expedited decay of the labeled signals.