Previously, skin seed cells (such as keratinocytes) cultured in petri dishes for tissue culture, although the monolayer cells can be observed, but without epidermal hierarchical structure and cuticle skin. However, the seed cells were seeded in dermal substitutes, combined with air-liquid culture technology, will receive a complete epidermis, and be able to express keratin, envelope protein and other components[20]. It can be seen, the dermal substitute not only provides the three-dimensional environment for the growth and development of seeded cell culture, the dermal substitute tissue matrix protein components also play a key role on inducing cell differentiation. Salag et al[21] mixed the cultured fibroblasts with collagen gel according to certain percentages, this mixture formed gel within a few minutes in the petri dish. A few days later, the gel became more dense and similar to dermal tissue structures, and then inoculated with keratinocytes on surface, to obtain a composite skin of epithelial-like structure by culture. Later, Coulomb[22] applied this dermal matrix to study epidermal development, but the dermal matrix is obviously insufficient: if the collagen gel would severely contract by 70%-80%; collagen gel is poorly resist to wound collagenase digestion and degradation; collagen gel brittleness is relatively large, with weak compression resistance or traction resistance. In order to overcome the shortcomings of the collagen gel skin substitute, someone tries to use collagen-glycosaminoglycan sponges as scaffolds to incubate fibroblast as dermal substitutes, and then inoculating keratinocytes to form a composite skin substitute[23]. Recently, people use synthetic or natural hybrid stents mix collagen and fibroblasts as a dermal substitute. Artificial materials commonly used should have good histocompatibility, easy degradability, good surface activity and certain adhesion, such as polyglycolic acid, polylactic acid, L-polylactic acid, as well as the polymers of above two kinds of materials, also adding into collagen sponge by the use of good biocompatibility and biodegradable properties of chitosan. Apligraf is a dermal substitute produced by type I bovine collagen adding to neonatal foreskin fibroblasts, and then incubating keratinocytes, some people use it to treat epidermolysis bullosa syndrome and can shorten healing time[24].
Natural dermal substitutes have been conducted early research and application, its production methods and processes is relatively simple, therefore used widely. Such dermal substitute materials are mainly derived from animal and human skin, in order to avoid allogeneic or xenograft rejection following graft into the receptor, all cells in the epidermis and the dermis should be removed and killed, to form acellular dermis[9,15,25]. Some studies have described a dermal substitute derived from the human skin, because human-derived dermal substitute tissue composition is closest to the autologous skin, the human seed cells (keratinocytes or epidermal stem cells) have homology[22]. In recent years, it is commonly used in research and clinical treatment of artificial skin and seed cell development[9,18,26]. However, human acellular dermal substitute is produced in different methods according to previous reports, the names are also different, such as Alloderm used hypertonic saline and sodium lauryl sulfate to remove human cadaver skin epidermis and dermis cell components[27]. There are also reports of acellular dermal matrix can be obtained epidermal separation with the enzymes treated by Triton X-100[18]. Although the literature dealing with different methods, the obtained acellular dermis all retain the previous fiber composition and the basic tissue structure, and parts of the basement membrane components still exist at the acellular dermal surface, so as to provide a guarantee for seed cells in vitro development[28-30]. In this study, histology and immunohistochemistry of vimentin staining results showed the active cells have been missing in DED, the dermis is rich in collagen fibers and elastic fibers, scanning electron microscope and transmission electron microscope demonstrated dermal collagen three-dimensional structure arranged bundles in the dermis, this structure is conducive to infiltrate the culture medium in the DED matrix and to nourish seed cell development. In addition, the dermis has a certain inter-fiber pore size, which is conducive to fibroblasts and capillary ingrowth in post-transplant tissues, thus facilitating wound healing. Previous research data show that the basement membrane and its components play an important role in the epidermal proliferation and differentiation. The existence of dermal substitute basement membrane components, or components of dermal substitutes can produce basement membrane components, is the key of seed cells developing into a full skin structure[31]. In this experiment, the Periodic Acid-Schiff staining and immunohistochemistry have proved that the DED surface and appendages catheter retained rich mucopolysaccharides and type IV collagen, which were consistent with the basement membrane components previously reported in the human-derived acellular dermal matrix surface[18-19]. The DED basement membrane components, in particular the presence of type IV collagen, can provide a micro-environmental conditions for inducing epidermal development and differentiation[15-16,31], it could serve as a biomimetic dermal scaffold for tissue-engineered skin construction[9,16]. In addition, it provides a good tissue model for the study addressing the in vitro tumor invasion.
