A new dermal equivalent: the use of dermal fibroblast culture alone without exogenous materials.
BACKGROUND
During the past decade, several kinds of skin equivalents have been developed. However, the dermal equivalents have all contained exogenous materials, which can be difficult to obtain and a source of infections.


OBJECTIVE
The aim of this study was to develop a new dermal equivalent by culturing dermal fibroblasts alone without exogenous materials and to evaluate its applicability in vitro and in vivo.


METHODS
The postconfulent cultures of dermal fibroblasts in serum containing medium, that was supplemented with epidermal growth factor, insulin, hydrocortisone, transferrin and triiodothyronine for 3 weeks, produced a fibrous sheet that was visible macroscopically. To construct a skin equivalent, epidermal keratinocytes were cultured on the top of the fibrous sheet at the air-liquid interface. To evaluate its fate in vivo, the fibrous sheet was grafted into a nude mouse.


RESULTS
Histologically, the fibrous sheet showed dermis-like tissue that consisted of an extracellular matrix around dermal fibroblasts, and revealed collagen fibers by Masson-trichrome staining. The components of dermal matrix such as type I collagen, type III collagen, elastin, fibrillin-1 and fibronectin were diffusely expressed. Some collagen fibrils were found by electron microscopy. In the skin equivalent, a multilayered epidermis with a horny layer was formed. Some differentiation markers (keratin 1 and 10, and involucrin) and the components of basement membrane (beta4 integrin chain, type IV and VII collagens) were expressed in a similar fashion to those in normal skin in vivo. Ultrastructurally, basement membrane zone such as hemidesmosomes, lamina lucida and lamina densa was found, although it was still incomplete. When the fibrous sheet was grafted in vivo, it revealed blood vessels that were derived from the nude mouse, and persisted for 4 weeks.


CONCLUSIONS
These findings demonstrated that a new dermal equivalent, closely resembling a dermis in vivo, could be constructed by culturing dermal fibroblasts alone in a special culture medium. In addition, the dermal equivalent may be useful for experimental and clinical purposes, such as the reconstruction of a skin equivalent in vitro and grafting in vivo.
Lee
Dong-Youn
DY
Department of Dermatology, Sungkyunkwan University School of Medicine, Samsung Medical Center, Seoul, South Korea. dylee@smc.samsung.co.kr
Lee
Joo-Heung
JH
Yang
Jun-Mo
JM
Lee
Eil-Soo
ES
Park
Kwan-Hyun
KH
Mun
Goo-Hyun
GH