Sex-dependent Regeneration Patterns in Mouse Submandibular Glands

Abstract

Our previous studies indicated that YIGSR-A99 peptides chemically conjugated to fibrin hydrogel (FH) and applied to wounded submandibular gland (SMG) in vivo, formed new organized salivary tissue, whereas wounded SMG treated with FH alone or in the absence of a scaffold showed disorganized collagen formation and poor tissue healing. While these studies indicated that damaged SMG grow and differentiate when treated with FH containing L1 peptide, they were performed only in female mice. However, there is a well-established sexual dimorphism present in mouse SMG (e.g., males develop well-differentiated granular convoluted tubules, but these structures are poorly developed in females) and little is known about how these sex differences influence wound healing events. Therefore, the goal of this study was to conduct comparative analyses of regeneration patterns in male and female mice using L_1p-FH in a wounded SMG mouse model. Particularly, we focused on sex-dependent wound healing events such as macrophage polarization, vascularization, tissue organization, and collagen deposition, and how these events affect salivary gland functioning.

Keywords: gender differences; materials science; regeneration; saliva; salivary physiology.

Figure 1.

Scaffolds were successfully implanted and displayed similar biostability in vivo in both sexes. Hydrogel stability was assessed in male and female submandibular gland at post-surgery days 1, 3, 8, and 20. Data represent the means ± SD of n=4 specimens per condition.

Figure 2.

L_1p-FH promotes macrophage polarization in female mice only. Immunostaining for macrophage markers (iNOS, red; Arg-1, green) in untreated and L_1p-FH groups in males and females at post-surgery day 1 and post-surgery day 3 (A-D). Yellow dotted lines indicate wounded area. Representative of n=3 specimens. Scale bars = 200 µm. Inset white boxes shown to identify areas of interest, with white arrows indicating iNOS⁺ cells (B) and Arg-1⁺ cells (D). Scale bars = 50 µm. Specimens were analyzed using a confocal Zeiss LSM 700 microscope at 10× magnifications. The number of iNOS⁺ and Arg-1⁺ cells were analyzed in untreated (purple) and L_1p-FH (blue) groups using ImageJ and the M1 to M2 ratio was calculated (E-H). Data represent the means ± SD of n=3 mice per condition and statistical significance was assessed by one-way ANOVA (*p<0.05) and Tukey’s honestly significant differences post-hoc test. Abbreviations: FH, fibrin hydrogel; iNOS, inducible NO synthase.

Figure 3.

L_1p-FH promotes blood vessel formation in females only. Immunostaining for blood vessels markers (VCAM-1, green; ICAM1, red) in untreated and L_1p-FH groups in males and females at post-surgery day 8 (A, B) and post-surgery day 20 (C, D). Yellow dotted lines indicate wounded area. Representative of n=3 specimens. Scale bars = 200 µm. Inset white boxes shown to identify areas of interest, with white arrows indicating blood vessels at post-surgery day 8 (B) and post-surgery day 20 (D). Scale bars = 50 µm. Specimens were analyzed using a confocal Zeiss LSM 700 microscope at 10× magnifications. Representative of n=3 specimens. Abbreviation: FH, fibrin hydrogel.

Figure 4.

L_1p-FH induces wound healing in female mice only. Histology sections of whole SMG in untreated, L_1p-FH, and sham groups in males (A) and females (B) at post-surgery day 20. Whole gland images show extent of wounded area (blue dotted lines). Scale bar = 1 mm. Inset black boxes shown to identify structures of interest, specifically acini (yellow arrows, H&E), ducts (green arrows, H&E), blood vessels (black arrows, H&E), and areas of dense collagen deposition (red arrows, Masson trichrome). Specimens were analyzed using a Leica microscope at 20× magnifications. Experiments are representative of n=3 specimens. Scale bars = 200 µm. Abbreviations: FH, fibrin hydrogel; H&E, Hematoxylin and Eosin; SMG, submandibular gland.

Figure 5.

L_1p-FH promote saliva secretion in females only. Mice were anesthetized and stimulated with pilocarpine (50 mg/kg) and isoproterenol (0.5 mg/kg) in untreated (purple), L_1p-FH (blue), and sham groups (red) at post-surgery day 20. Then, whole saliva was collected for 5 min in male (A) and female (B) mice. Data represent the means ± SD of n=10 mice per condition and statistical significance was assessed by one-way ANOVA (*P < 0.05) and Tukey’s Honestly Significant Differences post-hoc test. Abbreviation: FH, fibrin hydrogel.