Study On Reconstruction Of Basement Membrane In Composite Chitosan Tissue-engineered Skin

The skin basement membrane (BM) is a key structure with 0.5 to 1.0μm thickness that establishes the boundary between epidermis and dermis and has many important biological functions. Its most obvious function is to tightly link the epidermis to the dermis. Another obvious function of the BM is to determine the polarity of the epidermis and to provide a barrier to epidermal migration. Under normal circumstances, BM prevents the direct contact of epidermal cells with the dermis. Moreover, the BM has the ability of permeation and barrier, and plays the important exclusive role in supporting tissue integrity.BMs of human skin are heterogeneous aggregates composed of type IV and VII collagens, several kinds of laminins (such as laminins 5, 6 and 10), nidogen and perlecan. Type IV collagen and laminin are two major components of BM. Type IV collagen forms a network that confers the distinct mechanical stability known to BM. Laminin binds to collagen IV and forms a second network by interacting with nidogen. Ultrastructurally, the BM may be morphologically divided into three layers: the lamina lucida, the lamina densa and the lamina fibroreticularis. The lamina lucida is a region between the lamina densa and the basal keratinocytes which forms electrondense plaques, called hemidesmosomes. Anchoring filaments composed primarily of LN-5 accumulate in the lamina lucida opposite to the hemidesmosomes in skin. The lamina densa is a sheet-like structure which is primarily composed of type IV collagen, laminins and perlecan. The lamina fibroreticularis underlies the lamina densa and contains fine fibrils, such as anchoring fibrils, which are composed primarily of type VII collagen at the dermal-epidermal junction.It is a common defect of the tissue-engineered skin substitute at present that epidermis separates from dermis easily for lacking of BM reconstruction between the dermis and epidermis. Keratinocyte-fibroblast interaction in organotypic skin culture can promote synthesis of these BM components such as collagen IV, collagen VII, nidogen and laminins, forming network to keep the stability of the dermal-epidermal junction.The morphological sequence of the lamina densa formation in skin equivalents is as follows: (1) the appearance of hemidesmosomal structures along the inner leaflet of the subepidermal cell membrane of basal keratinocytes; (2) the focal appearance of the lamina densa with anchoring filaments in apposition to the hemidesmosomes; (3) the lateral elongation of the lamina densa.LN-5 is the most efficient substrate for the attachment of keratinocytes. LN-5 has been located to the anchoring filaments that traverse the lamina lucida. In vivo skin, LN-5 was identified as one of the major components of these anchoring filaments, which bridge the basal keratinocytes and the lamina densa at the dermal-epidermal junction. LN-5 was exogenously added into the culture system to promote the assembly of the BM at the dermal-epidermal junctions. It was found that exogenously added LN-5 in skin substitute culture medium can accelerate the assembly of lamina densa in a concentration-dependent manner.In this experiment, keratinocyte-fibroblast interaction was influenced by regulation of calcium concentration in culture medium to promote the BM formation on the composite chitosan tissue-engineered skin model. LN-5 of different level was exogenously added in culture medium at different phase to observe the assembly of BM. Modelling in vivo mechanics environment, vertical stress was applied in the culture system of tissue-engineered skin to observe the biological behavior change of the seed cells and the BM formation.To survey the BM construction, staining with hematoxylin & eosin and PAS were adopted,while collagens IV and VII and LN-5 detected immunohistochemically at the dermal-epidermal junction. The sections were also examined with an electron microscope. Soluble LN-5 in conditioned culture medium was analyzed by western blot to investigate the role of LN-5 in BM construction in tissue-engineered skin. A kind of flow loop system of parallel plate flow chamber to employ gravitate of pouring was established to value the ability of tissue-engineered skin with reconstructing BM in resist to fluid shear stress. The skin was placed on full-thickness excised wound in New Zealand rabbit. The healing of wound was observed.The main results are as follows:(1) The BM was reconstructed successfully in vitro by regulation of calcium concentration in culture medium and addition of LN exogenously. The better circumstance for BM formation is high level calcium medium and added LN into system after 7 days culture, depending on the concentration of laminin supplemented. These results suggest that LN-5 accelerate formation of the lamina densa along the dermal-epidermal junction of the skin equivalents;(2)In model of stress on tissue-engineered skin, vertical stress applied for 15 to 30 minutes every day was helpful to restruct BM in vitro;(3)The major BM components such as collagens IV and VII and LN-5 were detected immunohistochemically at the dermal-epidermal junction in the skin;(4)Hemidesmosomes formation at the dermal-epidermal junction at different level were observed by electron microscopy. Hemidesmosomes in tissue-engineered skin with vertical stress were significant in quantity;(5)Soluble LN-5 was examined in conditioned medium. The vertical stress can promote keratinocyte to secret LN-5. Low level of calcium in medium was helpful for keratinocyte to proliferate and synthesis LN-5. Supplement with exogenous LN can obviously level the concentration of soluble LN-5 in medium;(6)The tissue-engineered skin with reconstruction of BM have better tolerance to shear stress in some degree, especially in skin under stress;(7)The composite chitosan tissue-engineered skin is good for recovery of full-thickness excised wound in rabbit.In conclusion, this experiment had established a model of reconstruction of BM in vitro by regulation of calcium concentration and supplement with exogenous LN in culture medium. To examine the histological analysis of BM, hematoxylin & eosin staining, PAS staining, immunohistochemistry and transmission electron microscopy were applied. The crucial role of LN in the reconstruction of BM in tissue-engineered skin had been explored, and the importance of LN in the formation of BM network was confirmed. By mimesis dynamics environment in vivo, the effect of vertical stress to BM formation was examined. It was revealed that the vertical stress applied was benefitial to the reconstruction of BM and there was significant increase in the hemidesmosome. By parallel-plate flow chamber, dynamic assessment of tissue engineering skin with BM reconstruction was obtained.It was concluded that the best organotypic culture environment for tissue-engineered skin was medium containing 1.95 mM Ca²⁺ and supplemented with laminin 5 at a concentration of 20ug/ml from day 7 culture. The keratinocytes formed a best stratified squamous epithelium within 15 days with the presence of basal, spinous, granular and corneal cell layers. This study revealed the key role of LN-5 in the reconstruction of BM and the effect of the dynamic factor to BM formation, and established the base for further insight into the mechanism of BM formation in vitro.