Studies on Construction and Functional Evaluation of 3D-Skin Equivalents with the Dermis Constructed by in vitro Cell Manipulation

概要

Skin, consisting of the epidermis and dermis, is a barrier to protect human body from various stimuli. The stratum corneum an d epidermal tight junctions (TJs) in the stratum granulosum are responsible for the skin barrier function. Dysfunction of these barrier causes serious damage to the homeostasis. Therefore, when skin is lost due to wounds or burns, transplantation with skin equivalents is necessary. In pharmaceutical and cosmetic development, it is also essential to evaluate the skin irritation of compounds using skin equivalents. However, skin equivalents have limitations in application to those fields because they have an immature epidermis and weak barrier compared to native human skin. Some reports clearly suggest that dermal models with fibroblasts contribute to epidermal maturation. On the other hand, it is unclear that dermis contribute to improve epidermal barrier function. In this thesis, I tried to reveal the effect of dermal models on epidermal barrier in order to construct skin equivalents with a higher barrier function in vitro.

For investigation of the effect, I adopted a unique skin equivalent consisting of epidermis and dermis constructed by layer-by-layer (LbL) method (LbL-3D Skin). This is because its dermis consisting of dermal fibroblasts only was easy to construct and control structure. Whereas, its epidermis formed an abnormal structure with no barrier function like dermatitis. Hence, LbL-3D Skin could not be used to evaluate the contribution of the dermis to the epidermal barrier. At first, I tried to establish a reproducible method to construct LbL-3D Skin with a barrier function. In native human skin, the position of proliferating keratinocytes (KCs) is extremely restricted in basal layer of the epidermis. Whereas, proliferating KCs extended above area of basal layer in LbL-3D Skin. In novel construction method, proliferating KCs were restricted in basal layer by controlling the number of seeded KCs on the dermal model. This improvement allowed for the reproducible construction of LbL-3D Skin which has a human-like epidermis with barrier function.

Next, I focused on epidermal TJs because this structure is an essential barrier and regulator for other barrier functions such as the stratum corneum. Although TJs should play an essential role in skin equivalents, the effect of dermal models on epidermal TJ formation and function was unknown. First, I clarified TJs of LbL-3D Skin in terms of expression of TJ-related proteins and dense cell-cell adhesion structure. These results showed that LbL-3D Skin has epidermal TJs consisting of TJ-related proteins such as claudin-1 and ZO-1 closely to human skin. Second, I compared TJs of the epidermal model and LbL-3D Skin to investigate the effect of the dermal model on the epidermal TJs. In the epidermal model, TJs did not function as a permeability barrier, despite the expression of TJ-related proteins. LbL-3D Skin with thick dermis has functional TJs as a barrier for a long time. These results showed that dermal models contribute to the formation and maintenance of functional TJs as in native human skin.

This thesis shows that the dermal model regulates epidermal barrier function through the formation and maintenance of functional TJs in skin equivalents. It is also the first to reveal the involvement of the dermis in the formation and mainten ance of functional epidermal TJs. Thus, these results suggested the necessity of dermal models in the construction of skin equivalents with a higher barrier function. This study is an important piece of research on skin equivalent that contributes greatly to a wide range of applied research such as in regenerative medicine and pharmaceutical and cosmetic discovery as well as basic research on the elucidation of skin barrier function.