Laminin-1 Peptides Conjugated to Fibrin Hydrogels Promote Salivary Gland Regeneration in Irradiated Mouse Submandibular Glands

Abstract

Previous studies demonstrated that salivary gland morphogenesis and differentiation are enhanced by modification of fibrin hydrogels chemically conjugated to Laminin-1 peptides. Specifically, Laminin-1 peptides (A99: CGGALRGDN-amide and YIGSR: CGGADPGYIGSRGAA-amide) chemically conjugated to fibrin promoted formation of newly organized salivary epithelium both in vitro (e.g., using organoids) and in vivo (e.g., in a wounded mouse model). While these studies were successful, the model's usefulness for inducing regenerative patterns after radiation therapy remains unknown. Therefore, the goal of the current study was to determine whether transdermal injection with the Laminin-1 peptides A99 and YIGSR chemically conjugated to fibrin hydrogels promotes tissue regeneration in irradiated salivary glands. Results indicate that A99 and YIGSR chemically conjugated to fibrin hydrogels promote formation of functional salivary tissue when transdermally injected to irradiated salivary glands. In contrast, when left untreated, irradiated salivary glands display a loss in structure and functionality. Together, these studies indicate that fibrin hydrogel-based implantable scaffolds containing Laminin-1 peptides promote secretory function of irradiated salivary glands.

Keywords: biomaterial; hydrogel; irradiated salivary glands; regeneration; saliva; tissue engineering.

FIGURE 1

Radiation treatment and local L_1P-FH delivery used in this study. (A) Mice received a single 15 Gy radiation dose with a customized lead shield having a 1 cm slit aligned to the mouse’s neck. (B) Radiation treatment caused saliva flow rates to be significantly reduced. The symbol (•) indicates non-irradiated group, while the symbol (■) indicates irradiated group. (C) DyLight 680 conjugated L_1P-FH were successfully delivered to the mouse submandibular glands when applied via transdermal injection. White arrows indicate the site of L_1p-FH injection.

FIGURE 2

Treatment with L_1p-FH preserves epithelial integrity when applied after radiation treatment. Hematoxylin and eosin (A,C,E,G,I,K) as well Masson’s trichrome (B,D,F,H,J,L) staining of mouse submandibular glands from group 1 [non-irradiated, (A–D)], group 2 [irradiated without L_1p-FH injection, (E–H)] and group 3 [irradiated with L_1p-FH injection, (I–L)] were performed and tissue morphology was analyzed using a Leica DMI6000B. Scale bars represent 100 µm. Representative image from a total of five mice per group.

FIGURE 3

Treatment with L_1p-FH maintains epithelial polarity and functional marker expression. Salivary structural and functional marker organization was analyzed using confocal microscopy with specific antibodies against ZO-1 [green; (A–E)], E-cadherin [red; (A–E)], TMEM16A [green; (G–K)], Na⁺/K⁺-ATPase [red; (G–K)], and DAPI (blue; everywhere). Scale bars represent 100 µm. Yellow-dotted areas indicate fibroblast-like areas. Representative image from a total of five mice per group. ZO-1 (F) and TMEM-16A (L) positive pixels were analyzed using ImageJ.

FIGURE 4

L_1p-FH promotes macrophage polarization. Macrophage marker expression was analyzed using confocal microscopy with specific antibodies against iNOS (A–F), Arg-1 (G–L), and DAPI (blue; everywhere). Scale bars represent 100 µm. White and red arrows indicate iNOS or Arg-1 positive cells, respectively. Representative image from a total of five mice per group. iNOS (F) and Arg-1 (L) positive cells were analyzed using ImageJ and GraphPad Prism 6. Data represent the means ± SD of n = 5 mice per condition with statistical significance assessed using one-way ANOVA (*p < 0.01) and Dunnett’s post-hoc test for multiple comparisons to group 2 (irradiated with no L_1p-FH injection at day 30).

FIGURE 5

L_1p-FH increases saliva secretion after radiation treatment. Mice were anesthetized and stimulated with pilocarpine and isoproterenol at days 8 and 30 with saliva collected for 5 min. Data represent the means ± SD of n = 5 mice per condition with statistical significance assessed using one-way ANOVA (*p < 0.01) and Dunnett’s post-hoc test for multiple comparisons to group 1 (non-irradiated mice at day 30). The symbol (+) indicates L_1p-FH injection, while the symbol (−) indicates no L_1p-FH injection, and n. s indicates no significant differences from group 1 (non-irradiated mice at day 30).