Laminin-111-derived peptide conjugated fibrin hydrogel restores salivary gland function

Abstract

Hyposalivation reduces the patient quality of life, as saliva is important for maintaining oral health. Current treatments for hyposalivation are limited to medications such as the muscarinic receptor agonists, pilocarpine and cevimeline. However, these therapies only provide temporary relief. Therefore, alternative therapies are essential to restore salivary gland function. An option is to use bioengineered scaffolds to promote functional salivary gland regeneration. Previous studies demonstrated that the laminin-111 protein is critical for intact salivary gland cell cluster formation and organization. However, laminin-111 protein as a whole is not suitable for clinical applications as some protein domains may contribute to unwanted side effects such as degradation, tumorigenesis and immune responses. Conversely, the use of synthetic laminin-111 peptides makes it possible to minimize the immune reactivity or pathogen transfer. In addition, it is relatively simple and inexpensive as compared to animal-derived proteins. Therefore, the goal of this study was to demonstrate whether a 20 day treatment with laminin-111-derived peptide conjugated fibrin hydrogel promotes tissue regeneration in submandibular glands of a wound healing mouse model. In this study, laminin-111-derived peptide conjugated fibrin hydrogel significantly accelerated formation of salivary gland tissue. The regenerated gland tissues displayed not only structural but also functional restoration.

Fig 1. Procedure to create wounded SMG model.

(A) A skin incision of approximately 1 cm in length was made along the anterior surface of the neck, mSMG were exposed, (B) a 3-mm diameter biopsy punch was performed, surgical wounds were completed, (C) wounds were filled with or without L_1p-FH or FH and (D) the skin incision was sutured.

Fig 2. L_1p-FH successfully attach to mSMG and are degraded over time in vivo.

(A) Rheology measurements were performed for FH alone as well as L_1p-FH. Data represent the elasticity (G’) versus strain (%) of unmodified FH (○) and L_1p-FH (□). The in vivo stability of L_1p-FH was monitored using a Xenogen IVIS 100 Bioluminescent Imager at days (B) 1, (C) 3, (D) 8 and (E) 20. Radiant Efficiency: (p/sec/cm²/sr)/(μW/cm²).

Fig 3. L_1p-FH applied to mSMG increased body weight.

Changes in body weight (%) of FH alone (■) or L_1p-FH (▲) treated mice groups were compared with untreated mice group (●) and sham control group (○) over 20-day period. Data represent the means ± SD of n = 7 mice per condition and statistical significance was assessed by two-way ANOVA (p < 0.01) and Dunnett's post-hoc test for multiple comparisons to the untreated group.

Fig 4. L_1p-FH applied to mSMG improved saliva secretion over untreated and FH alone-treated mice.

Mice were anesthetized and stimulated with pilocarpine at day 20. Then, saliva was collected for 5 min. Data represent the means ± SD of n = 5 mice per condition and statistical significance was assessed by one-way ANOVA (p < 0.01) and Dunnett's post-hoc test for multiple comparisons to the untreated group. * = significant difference from the untreated group; n.s. = no significant difference from the untreated group.

Fig 5. L_1p-FH applied to mSMG restored saliva composition.

Fifteen microgram of saliva protein from each group was fractionated by SDS-PAGE. The gel was stained with (A) 0.25% Coomassie Brilliant Blue R-250 for total proteins and (B) 0.5% Alcian Blue 8GX for mucins. (C) The mucin compositions were analysed using ImageJ. The white bar indicates MUC5B and the gray bar indicates MUC7. Statistical significance was assessed by one-way ANOVA (p < 0.01) and Dunnett's post-hoc test for multiple comparisons to the sham group. * = significant difference from the sham group; n.s. = no significant difference from the sham group.

Fig 6. Surgical wounds treated with L_1p-FH displayed organized mSMG.

Rehydrated sections were stained with hematoxylin-eosin (A, C, E, G) or picrosirius red (B, D, F, H) stains and analyzed using a Leica DMI6000B at 10× magnifications. Shown are wounded mSMG without scaffold (A, B), wounded mSMG with FH alone (C, D), wounded mSMG with L_1p-FH (E, F), and sham control (G, H). (I) The ratio of acinar and ductal structures was analyzed using ImageJ. Red arrows indicate acinar structures and yellow arrows indicate ductal structures. Scale bars = 200 μm.

Fig 7. Acinar and ductal markers were expressed in the regenerating mSMG.

Salivary structural and functional marker organization in wounded mSMG without scaffold (A, E, I), wounded mSMG with FH alone (B, F, J), wounded mSMG with L_1p-FH (C, G, K), and sham control (D, H, L) was determined using Confocal microscopy as follows: (A-D; green) rabbit anti-aquaporin 5 and (A-D; red) mouse anti-cytokeratin 7, (E-H; green) rabbit anti-TMEM-16A and (E-H; red), mouse anti-Na⁺/K⁺-ATPase, (I-L; green) rabbit anti-PECAM-1 and (I-L; red) mouse anti-β-tubulin III. Scale bars = 50 μm.

Fig 8. L_1p-FH does not promote abnormal cell proliferation.

The proliferation marker Ki67 showed similar staining patterns in wounded mSMG without scaffold (A), wounded mSMG with FH alone (B), wounded mSMG with L_1p-FH (C), and sham control (D). Statistical significance was assessed by one-way ANOVA (p < 0.01) and Dunnett's post-hoc test for multiple comparisons to the sham group (E). Scale bars = 50 μm.