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

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.

Sox9+ Cell is Required for Salivary Gland Regeneration after Radiation Damage via Wnt/β-Catenin Pathway

Background: Radiotherapy for head and neck cancer can cause serious side effects, including severe damage to the salivary glands, resulting in symptoms such as xerostomia, dental caries, oral infectious and so on. Due to lack of long-term treatment for the symptoms of saliva barren, current research has focused on finding endogenous stem cells that can differentiate into various cell lineage to replace lost tissue and restore function. Results: In our study, we identified Sox9+ cells can differentiate into various salivary epithelial cell lineages under homeostatic conditions. After ablating Sox9+ cells, the salivary glands of irradiated mice showed more severe phenotypes and reduced proliferative capacity. Analysis of online single cell RNA-sequencing data revealed enrichment of the Wnt/β-catenin pathway in Sox9+ cell population. Furthermore, treatment of Wnt/β-catenin inhibitor to irradiated mice inhibited the regenerative capability of Sox9+ cells. Finally, we showed that Sox9+ cells were able to form organoids in vitro and transplanting these organoids into salivary glands after radiation restored part of salivary gland function. Conclusions: In short, our research indicated that regenerative therapy targeting Sox9+ cells is a promising method to solve the radiation induced salivary gland injury.

Cell Sheets Restore Secretory Function in Wounded Mouse Submandibular Glands

Thermoresponsive cell culture plates release cells as confluent living sheets in response to small changes in temperature, with recovered cell sheets retaining functional extracellular matrix proteins and tight junctions, both of which indicate formation of intact and functional tissue. Our recent studies demonstrated that cell sheets are highly effective in promoting mouse submandibular gland (SMG) cell differentiation and recovering tissue integrity. However, these studies were performed only at early time points and extension of the observation period is needed to investigate duration of the cell sheets. Thus, the goal of this study was to demonstrate that treatment of wounded mouse SMG with cell sheets is capable of increasing salivary epithelial integrity over extended time periods. The results indicate that cell sheets promote tissue organization as early as eight days after transplantation and that these effects endure through Day 20. Furthermore, cell sheet transplantation in wounded SMG induces a significant time-dependent enhancement of cell polarization, differentiation and ion transporter expression. Finally, this treatment restored saliva quantity to pre-wounding levels at both eight and twenty days post-surgery and significantly improved saliva quality at twenty days post-surgery. These data indicate that cell sheets engineered with thermoresponsive cell culture plates are useful for salivary gland regeneration and provide evidence for the long-term stability of cell sheets, thereby offering a potential new therapeutic strategy for treating hyposalivation.