Chitosan cooperates with mesenchyme-derived factors in regulating salivary gland epithelial morphogenesis

Abstract

Chitosan is a widely used biocompatible biomaterial in the tissue regeneration, but its utility and application in the tissue morphogenesis of salivary gland remains unclear. The study aimed to explore the effects of chitosan on the epithelial morphogenesis of submandibular gland (SMG). With chitosan, the branching morphogenesis of the whole SMG explant was facilitated, and the morphogenetic-promoting effects of mesenchymal tissue on SMG were further enhanced. Furthermore, chitosan was competent to induce recombined SMG epithelium to form branches in the serum-free condition independently. In the presence of chitosan, the morphogenetic efficacy of mesenchyme-derived growth factors responsible for epithelial morphogenesis including fibroblast growth factors 7, fibroblast growth factor 10 and hepatocyte growth factor increased. The specific epithelial phenotype induced by individual growth factor, which was required for the accomplishment of salivary epithelial morphogenesis, was promoted by chitosan. Moreover, the proliferative and the chemotactic properties of these growth factors towards the SMG epithelia were also reinforced by chitosan. Therefore, in orchestrating and intensifying the essential mesenchyme-derived growth factors, chitosan is versatile in mediating SMG epithelium to form a predetermined phenotype more efficiently and comprehensively. This study suggested that chitosan is a morphogenetic-regulating biomaterial for salivary tissue, which might be useful for the future salivary gland investigation and regeneration.

Figure 1

(A) Effects of chitosan on branching morphogenesis of SMG explants. SMG explants were cultured in negative control SMG medium (Cont), mock control SMG medium (Mock), chitosan-containing SMG medium (Chi-M), and chitosan-containing SMG medium without additives (Chi(-A)), respectively. Photographs were taken at 0 and 24 hours of culture. (B) Quantification was performed at 24 hours and was presented as the fold change of bud. Asterisks (*) denote significant differences (P < 0.001) compared with the mock control. Scale bars = 100 μm.

Figure 2

(A) Effects of chitosan on the morphogenesis of SMG epithelium. Representative pictures of SMG epithelium cultured in mock control (M) and chitosan-containing SMG medium (Chi-M) without exogenous growth factors were shown after 24 hours of culture. Quantification was performed at 24 hours and was presented as the fold change of bud. Scale bar = 100 μm. (B) Effects of chitosan on the branching morphogenesis of SMG explants cocultured with the SMG adjacent mesenchymal tissue. The SMG explants were cultured in mock control (M) and chitosan-containing SMG medium (Chi-M) for 24 hours. Quantification was performed at 24 hours and was presented as the fold change of bud. Asterisk (*) denotes a significant difference (P < 0.01) compared with the mock control. Scale bars = 100 μm.

Figure 3

Recombination assay of SMG epithelium with homotypic mesenchyme. Isolated SMG epithelial explants were recombined with mesenchymal rudiments. The recombinants were cultured in mock control medium (M), chitosan-containing medium (Chi-M), or mock control medium added with serum (Serum+M). The photographs were taken in a time-lapse manner with a 24-hour interval. Scale bars = 100 μm.

Figure 4

SMG epithelial explants cultured with 10 ng/ml FGF7. Paired epithelial explants were cultured in mock control medium (M) and chitosan-containing medium (Chi-M) respectively. With the addition of FGF7, remarkable lobular formation was found in the chitosan-containing group. In the mock control group, no new lobes were formed, but only with an epithelial protruding on the surface (arrow). Quantification was performed by counting the number of buds per explant at 48 hours of culture. Asterisk (*) denotes a significant difference compared with the mock control group (P < 0.01). Scale bar = 100 μm.

Figure 5

Effects of chitosan in FGF10-induced morphogenesis of SMG epithelium. One lobule of paired SMG epithelial explants was cultured for 48 hours in either mock control medium (M) or chitosan-containing medium (Chi-M) containing 200 ng/ml FGF10. With FGF10, the formation and elongation of duct-like structures were found in the chitosan-containing group. In the mock control group, the elongation of duct-like structure was found without new duct formation. The result of quantification was shown in bar graphs by counting the number of duct-like structures per explants at 48 hours of culture. Asterisk (*) denotes a significant difference compared with the mock control group (P < 0.01). Scale bar = 100 μm.

Figure 6

Effects of chitosan in HGF-induced SMG epithelial morphogenesis. One lobule of paired SMG epithelial explant was cultured for 48 hours in either mock control medium (M) or chitosan-containing medium (Chi-M) with 100 ng/ml HGF. With HGF, the increased epithelial area and protrusions were found in the chitosan-containing group. In the mock control group, the area of explants extended to a lesser extent. The relative area was presented as the area ratio by counting explants areas of 48 hours over 0 hour. Asterisk (*) denotes a significant difference compared with the mock control group (P < 0.01). Scale bar = 100 μm.

Figure 7

Chitosan effects on SMG epithelial proliferation induced by FGF7, FGF10, and HGF after 48 hours culture. FGF7 (10 ng/ml), FGF10 (200 ng/ml), and HGF (100 ng/ml) were added respectively in either mock control medium (M) or chitosan-containing medium (Chi-M). Each growth factor stimulated the epithelial proliferation, which is shown by BrdU labeling (green). The green fluorescent pixels were measured and expressed as a ratio relative to nuclei-stained pixels per unit area from all optical sections of the explants. Asterisks (*) denote significant differences (P < 0.01) compared to the explants cultured in the mock control medium of each group. Scale bars = 100 μm.

Figure 8

HGF induced chemotaxis and proliferation of SMG epithelium. One lobule of SMG epithelial explant and a bead were placed at a distance of 500 μm. The asterisks marked the original location of the epithelial explant. No proliferation or migration was found in the beads soaked with mock control medium (M bead) or chitosan-containing medium (Chi-M bead). The bead soaked with mock control medium and HGF (HGF M bead) demonstrated a weak chemotactic capacity toward epithelial explants. In the bead soaked with chitosan-containing medium and HGF (HGF Chi-M bead), the epithelial explant increased in size, migrated, and finally surrounded and engulfed the bead after 48-hour culture. Scale bar = 100 μm.