Controlling branching structure formation of the salivary gland by the degree of chitosan deacetylation

The salivary gland is characterized by ramified epithelial branches, a specific tissue structure responsible for saliva production and regulation. To regenerate the salivary gland function, it is important to establish the tissue structure. Chitosan is a deacetylated derivative of chitin with wide biomedical applications. Because of its deacetylated nature, chitosan has different properties when prepared with different degrees of deacetylation (DDA). However, the impact of chitosan DDA on the effect of regulating tissue structure formation remains unexplored. In this study, the embryonic murine submandibular gland (SMG) was used as a model to investigate the role of chitosan DDA in regulating tissue structure formation of the salivary gland. When chitin substrates with different DDA were used, the branching numbers of cultured SMG explants changed. Similar effects were observed in the culture with chitosan prepared using different degrees of acetylation. The mRNA expressions of type I and type III collagen were elevated in SMG explants with enhanced branching morphogenesis, as was the protein level. In addition to the amounts of collagen, type I and type III collagen fibers were spatially present in the epithelial–mesenchymal junction of developing branches in the culture with chitosan of a specific range of DDA. The branch-promoting effect of chitosan DDA was abolished when SMG explants were treated with collagenase, both early in the stage of branch initiation and with the establishment of the branching structure. The branch-promoting effect of chitosan DDA disappeared when antisense oligonucleotides were applied to specifically block type III collagen. This study demonstrates for the first time that DDA of chitosan affects tissue structure formation. The different proportions of side-chain components of chitin derivatives regulate structural formation of cultured SMG, indicating that DDA is an important parameter using chitosan as a biomaterial for tissue structure formation of the salivary glands.