Trimers Conjugated to Fibrin Hydrogels Promote Salivary Gland Function

Abstract

New strategies for tissue engineering have great potential for restoring and revitalizing impaired tissues and organs, including the use of smart hydrogels that can be modified to enhance organization and functionality of the salivary glands. For instance, monomers of laminin-111 peptides chemically conjugated to fibrin hydrogel (L_1pM-FH) promote cell cluster formation in vitro and salivary gland regeneration in vivo when compared with fibrin hydrogel (FH) alone; however, L_1pM-FH produce only weak expression of acinar differentiation markers in vivo (e.g., aquaporin-5 and transmembrane protein 16). Since previous studies demonstrated that a greater impact can be achieved when trimeric forms were used as compared with monomeric or dimeric forms, we investigated the extent to which trimers of laminin-111 chemically conjugated to FH (L_1pT-FH) can increase the expression of acinar differentiation markers and elevate saliva secretion. In vitro studies using Par-C10 acinar cells demonstrated that when compared with L_1pM-FH, L_1pT-FH induced similar levels of acinar-like cell clustering, polarization, lumen formation, and calcium signaling. To assess the performance of the trimeric complex in vivo, we compared the ability of L_1pM-FH and L_1pT-FH to increase acinar differentiation markers and restore saliva flow rate in a salivary gland wound model of C57BL/6 mice. Our results show that L_1pT-FH applied to wounded mice significantly improved the expression of the acinar differentiation markers and saliva secretion when compared with the monomeric form. Together, these positive effects of L_1pT-FH warrant its future testing in additional models of hyposalivation with the ultimate goal of applying this technology in humans.

Keywords: biocompatibility; bioengineering; biomaterial(s); cell-matrix interactions; regeneration; saliva.

Figure 1.

L_1pM-FH and L_1pT-FH composition, modeling, and rheology. (A) Mass spectrometry analysis was performed as described in the Materials and Methods section, with peaks confirming the presence of (A₁) RGD monomer, (A₂) RGD trimer, (A₃) YIGSR monomer, and (A₄) YIGSR trimer. (B_1-4) Molecular modeling shows the following peptide features: backbone in solid lines, side chains in red and blue, and salt bridges in dotted lines. (C) Rheologic studies show the (C₁) strain dependence of L_1pM-FH and L_1pT-FH at fixed angular frequency (1 rad/s), assessed to determine the linear viscoelastic region for the materials, as well as (C₂) the oscillation time experiments for L_1pM-FH and L_1pT-FH based on 50% strain, 1-rad/s angular frequency, 10–mN × m torque limit at 37 °C. Data are expressed as the mean ± SE from 3 experiments. L_1pM-FH, monomers of laminin-111 peptides chemically conjugated to fibrin hydrogel; L_1pT-FH, trimers of laminin-111 peptides chemically conjugated to fibrin hydrogel.

Figure 2.

Par-C10 cell features in vitro. (A) Cell cluster size and (B) lumen diameter were measured for 7 consecutive days with a Carl Zeiss 700 confocal microscope as described in the Materials and Methods section. (C) [Ca²⁺]_i mobilization in response to carbachol (100 µM) was measured at day 3 with Leica Application Suite X software. (D) Expression of rabbit anti-ZO-1 (green) followed by anti-rabbit Alexa Fluor 488 IgG secondary antibody, F-actin expression detected by phalloidin (red), and nuclei detected by TO-PRO 3 iodide (blue) were analyzed with a Carl Zeiss 700 confocal microscope, where scale bars represent 25 µm. Data are expressed as the mean ± SD from 3 experiments. *P ≤ 0.05, significant difference from Par-C10 cells cultured in L_1pM-FH. n.s., no significant difference. L_1pM-FH, monomers of laminin-111 peptides chemically conjugated to fibrin hydrogel; L_1pT-FH, trimers of laminin-111 peptides chemically conjugated to fibrin hydrogel.

Figure 3.

Expression of aquaporin-5 (AQP-5) and cytokeratin 7 (CK7) in vivo. Submandibular gland frozen sections (3 µm) were incubated with rabbit anti-AQP-5 (green) and mouse anti-CK7 (red), followed by anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 568 IgG secondary antibody, and counterstained with TO-PRO-3 iodide (blue). Next, 10× and 40× images of each group were obtained to show the unwounded/wounded area interface and detailed salivary gland structures, respectively. All images were analyzed with a Carl Zeiss 700 LSM confocal microscope, where scale bars represent 50 µm. Yellow dotted lines indicate the wound edge; green arrows, AQP-5; and red arrows, CK7. Finally, the percentage of positively stained areas for each protein in the wounded area was calculated at postsurgery day 8 with ImageJ and analyzed by 1-way analysis of variance (P ≤ 0.05) and Dunnett’s post hoc test for multiple comparisons, where the asterisk (*) indicates significant differences from the L_1pT-FH group. Data represent the means ± SD of n = 6 mice per condition. L_1pM-FH, monomers of laminin-111 peptides chemically conjugated to fibrin hydrogel; L_1pT-FH, trimers of laminin-111 peptides chemically conjugated to fibrin hydrogel.

Figure 4.

Expression of TMEM16A and Na⁺/K⁺-ATPase in vivo. Submandibular gland frozen sections (3 µm) were incubated with rabbit anti-TMEM16A (green) and mouse anti-Na⁺/K⁺-ATPase (red), followed by anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 568 IgG secondary antibody, and counterstained with TO-PRO-3 iodide (blue). Next, 10× and 40× images of each group were obtained to show the wounded/unwounded area interface and detailed salivary gland structures, respectively. Yellow dotted lines indicate the interface between the wound and unwounded area, where green arrows indicate TMEM16A while red arrows indicate Na⁺/K⁺-ATPase. All images were analyzed with a Carl Zeiss 700 LSM confocal microscope, where scale bars represent 50 µm. Finally, positive areas of each protein in the wounded area were calculated at postsurgery day 8 with ImageJ and analyzed by 1-way analysis of variance (P ≤ 0.05) and Dunnett’s post hoc test for multiple comparisons, where the asterisk (*) indicates significant differences from the L_1pT-FH group. Data represent the means ± SD of n = 6 mice per condition. L_1pM-FH, monomers of laminin-111 peptides chemically conjugated to fibrin hydrogel; L_1pT-FH, trimers of laminin-111 peptides chemically conjugated to fibrin hydrogel.

Figure 5.

Saliva secretion in vivo. Mice receiving the indicated treatments were anesthetized, and salivary flow was stimulated with pilocarpine and isoproterenol at postsurgery day 20 as described in the Materials and Methods section. Then, saliva was collected for 5 min. Data represent the mean ± SD from 10 mice per condition, and statistical significance was assessed by 1-way analysis of variance (P ≤ 0.05) and Dunnett’s post hoc test for multiple comparisons, where the asterisk (*) indicates significant differences from the L_1pT-FH group. L_1pM-FH, monomers of laminin-111 peptides chemically conjugated to fibrin hydrogel; L_1pT-FH, trimers of laminin-111 peptides chemically conjugated to fibrin hydrogel.