Mucosal tissue model was constructed in vitro by using fibroblasts and epithelial cells from rabbit oral mucosa, and collagen gel. First, the method to construct the mucosal tissue model was discussed. Secondary, the process of epithelial differentiation in this model was observed histopathologically. Finally, the model was evaluated for clinical use as mucosal graft material.
The following results were obtained:
1. The mucosal tissue model was constructed by culturing epithelial cells on the surface of submucosal tissue model. The submucosal tissue model had been formed with fibroblasts and hydrated collagen gel.
2. In culturing the fibroblasts in hydrated collagen gel, collagen gel contracted, and reduced its size from an early stage to 7th day of culture. The contraction rate of the gel depended upon the collagen concentration and the number of fibroblasts. The lower the collagen concentration in the gel and the more the number of fibroblasts, the higher rate of the contraction of the lattice was observed.
3. The fibroblasts in the submucosal tissue model extended cellular processes into the surrounding collagen gel and became bipolar shaped. Growth-rate of fibroblasts in the submucosal tissue model was markedly suppressed compared to that on the plastic culture dish. These results indicate that fibroblasts demonstrate more in vivo-like behavior when cultured in the collagen gel three dimensionally than on the plastic culture dish two dimensionally.
4. On the 7th-day of culture, mono-layer of epithelial cells was observed on the submucosal tissue model.
On the 14th-day of culture, 3-4 epithelial cell layers were seen. The basal cells, at intervals, extended its cytoplasmic protrusions into the submucosal tissue model.
On the 21th-day of culture, complete epithelial differentiation and the formation of rete-peg were observed.
On 28th-day of culture, the multi-layered epithelial cells resembled normal epithelium. The change of basal cell shape, from spherical or cuboidal through elliptical to extremely flattened, paralleled the tendency of cell maturation.
5. Concerning the relationship between the collagen concentration in the gel and the differentiation of epithelial layer, mono-layer of epithelial cells was observed on the substrata containing more than 0.2% of collagen by weight, multi-layered structure of epithelium was observed more than 0.4%, multi-layered structure and differentiation of epithelium was observed more than 0.6%, and the rete-peg formation was observed more than 1.5%. Therefore, it is obvious that the collagen concentration of submucosal tisse is deeply related to the epithelial differentiation.
6. In conclusion, this study provides an in vitro model of mucosa useful for cell biological study of mucosa. Furthermore, we suggest this model is applicable as a mucosal graft in the future. In this case, it seems to be proper that the concentration of collagen in submucosal tissue model is more than 1.5%, and the culture period of epithelium is at least more than 7 days.
