|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1996 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1995 : ¥1,200,000 (Direct Cost : ¥1,200,000)
This study includes the development of supporting tissues, collagen IV for example, on which epidermal cells can adhere preferencially, and administering laminin 5, preferably located on the epidermal-dermal junction, thereby promoting the lamina densa formation.
We tried to form the skin equivalents by seeding keratinocytes on the dermal equivalents, contracted collagen I gels containing fibroblasts, and continued to culture for additional one week in the culture medium containing laminin 5 at the concentration of 0,1,5, and 20mug/ml. After 2 weeks culture, the epidermal-dermal junction was examined by electronmicroscopy.
The recognizable laminae densae were rarely detected in the absence of laminin 5. By contrast to the control culture, the laminae densae were frequently observed along the epidermal-dermal junction of the skin equivalents cultured in the presence of laminin 5 (1,5,20mug/ml). Morphometrical data of the experiments were confirmed by measuring the total length of the lamina densa found in the unit length of the epidermal-dermal junction. The data indicated that the lamina densa was developed approximately 3 times as long as that of control in the skin equivalents cultured in the presence of laminin 5 at the concentration of 5mug/ml, and 6 times at 20mug/ml. By immuno-electronmicroscopy, laminin 5 was detected at the lamina densa in the skin equivalents.
Keratinocytes sheets were grafted to the panniculus carnosus of the nude mice. The samples were taken and assessed by the electronmicroscopy. At day 3 after grafting, the lamina densa of grafts supplemented with laminin 5 were more continuous than that found in the control transplantation. The supplement of laminin 5 promoted the formation of the lamina densa resulting in the improvement of Keratinocytes coverage.
In conclusion, exogenous laminin 5 being present at a sufficient concentration allowed to form the lamina densa and promote the keratinocytes coverage.