Fibrin hydrogels fortified with FGF-7/10 and laminin-1 peptides promote regeneration of irradiated salivary glands

Abstract

Ionizing radiation, commonly used for head and neck cancer treatment, typically damages the salivary glands, resulting in hyposalivation. The development of treatments to restore this lost function is crucial for improving the quality of life for patients suffering from this condition. To address this clinical need, we have developed an innovative hydrogel by chemically conjugating laminin-1 peptides (A99 and YIGSR) and growth factors, FGF-7 and FGF-10, to fibrin hydrogels. Our results demonstrate that FGF-7/10 and laminin-1 peptides fortified fibrin hydrogel [enhanced laminin-1 peptides fibrin hydrogel (Ep-FH)] promotes salivary gland regeneration and functionality by improving epithelial tissue organization, establishing a healthy network of blood vessels and nerves, while reducing fibrosis in a head and neck irradiated mouse model. These results indicate that fibrin hydrogel-based implantable scaffolds containing pro-regenerative signals promote sustained secretory function of irradiated salivary glands, offering a potential alternative treatment for hyposalivation in head and neck cancer patients undergoing radiation treatment. These unique findings emphasize the potential of fibrin hydrogel-based implantable scaffolds enriched with pro-regenerative signals in sustaining the secretory function of irradiated salivary glands and offer a promising alternative treatment for addressing hyposalivation in head and neck cancer patients undergoing radiation therapy. STATEMENT OF SIGNIFICANCE: Radiation therapies used to treat head and neck cancers often result in damaged salivary gland, leading to severe dryness of the oral cavity. In this study, we engineered FGF-7 and FGF-10 and immobilized them into L_1p-FH. The resulting hydrogel, Ep-FH, restored irradiated salivary gland functionality by enhancing epithelial tissue organization, promoting the development of a healthy network of blood vessels and nerves as well as reduction of fibrosis.

Keywords: Fibrin hydrogels; Growth factor; Saliva; Submandibular glands; Tissue engineering.

Fig. 1.

(a) Schematic representation of the fusion proteins. (b) Predicted 3D structure of the MBP-fused (green), NQEQVSP conjugated (activated) and native FGF-7 and FGF-10. The structure of the fusion protein was predicted using ColabFold v1.5.2, with the resulting model indicating that the NQEQVSP-conjugated proteins (red) are likely to resemble the native structure of FGF-7 and FGF-10 (yellow).

Fig. 2.

Treatment with Ep-FH preserves epithelial integrity when applied after radiation exposure. Hematoxylin and eosin staining was performed and tissue morphology was analyzed using a Leica DMI6000B microscope. Arrows indicate the following: white arrows for fatty replacement, red arrows for interstitial fibrosis, green arrows for acinar cell atrophy, blue arrows for vacuolization and yellow arrows for inflammatory cells. Black scale bars represent 1 mm and white scale bars represent 500 μm. SMG indicates submandibular gland, and SLG indicates sublingual gland, respectively.

Fig. 3.

Treatment with Ep-FH prevents fibrosis in irradiated salivary glands. Picro-Sirius Red staining was performed and tissue morphology was analyzed using a Leica DMI6000B microscope. Fibrosis is indicated by blue arrows in the images. Black scale bars represent 1 mm.

Fig. 4.

Treatment with Ep-FH significantly prevents fibrosis in irradiated salivary glands. Results from Picro-Sirius Red staining were quantified using the AVIA software as detailed in Materials and Methods. Statistical analysis was performed with GraphPad Prism 6 using one-way (for Fig. 4e) and two-way analysis of variance (ANOVA; for Fig. 4a–d, f–h) and Dunnett’s post hoc test for multiple comparisons (p < 0.05, n = 4). White scale bars represent 1 mm.

Fig. 5.

Treatment with Ep-FH promotes CD31 and β-tubulin III expression in irradiated salivary glands. Blood vessels and neuronal structures were detected with CD31 (highly expressed on the surface of endothelial cells; green) and β-tubulin III (microtubule exclusively found in neurons; red) antibodies, respectively. Nuclei were counter-stained in blue with DAPI and images were analyzed using STELLARIS confocal microscope. Images represent n = 3 mice/group, where scale bars = 100 μm.

Fig. 6.

Treatment with Ep-FH promotes neuronal and vascular connections in irradiated salivary glands. The images from Fig. 5 were processed with the use of AIVIA software to render 3D constructs. Images represent n = 3 mice/group. The scale bars represent 25 μm.

Fig. 7.

Treatment with Ep-FH maintains AQP5 and CK7 expression in irradiated salivary. Acinar and ductal structures were detected with AQP5 (green) and CK7 (red) antibodies, respectively. Nuclei were counterstained in blue with DAPI and images were analyzed using STELLARIS confocal microscope. Images represent n = 3 samples, where scale bars = 25 μm.

Fig. 8.

Treatment with Ep-FH increases saliva secretion after radiation exposure. Saliva was collected for 20 min following pilocarpine stimulation (2.5 mg/kg). Results represent data from n = 9 mice per condition and data presented as means ± SD. Statistical significance was assessed using one-way ANOVA, followed by Dunnett’s post-hoc test for multiple comparisons to the irradiated group. Symbols are defined as follows: circles represent D30, squares represent D60, and triangles represent D90. Symbol notation is as follows: black for (non-IR), white for (IR-untreated), and gray for (IR-Ep-FH treated) with *p < 0.05; **p < 0.01; ****p < 0.0001 indicating significant differences compared to irradiated control group.