Sex-dependent Regeneration Patterns in Mouse Submandibular Glands.

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 L1p-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.

Using cell sheets to regenerate mouse submandibular glands.

Temperature-responsive polymer grafted tissue culture dishes release cells as confluent living sheets in response to small changes in temperature, with recovered cell sheets retaining cell-cell communications, functional extracellular matrices and tissue-like behaviors. These features promote tissue regeneration and improve transplantation efficacy in various tissues including cartilage, heart, kidney, liver, endometrium, cornea, middle ear, periodontium, and esophageal living sheet transplants. However, the functional effects of cell sheets for salivary gland regeneration to treat hyposalivation have not yet been studied. Thus, the present study aims to both establish the viability of thermoresponsive cell sheets for use in salivary glands and then explore the delivery option (i.e., single vs. multiple layers) that would result in the most complete tissue growth in terms of cell differentiation and recovered tissue integrity. Results indicate that single cell sheets form polarized structures that maintain cell-cell junctions and secretory granules in vitro while layering of two-single cell sheets forms a glandular-like pattern in vitro. Moreover, double layer cell sheets enhance tissue formation, cell differentiation and saliva secretion in vivo. In contrast, single cell sheets demonstrated only modest gains relative to the robust growth seen with the double layer variety. Together, these data verify the utility of thermoresponsive cell sheets for use in salivary glands and indicates the double layer form to provide the best option in terms of cell differentiation and recovered tissue integrity, thereby offering a potential new therapeutic strategy for treating hyposalivation.

Synergistic effects of laminin-1 peptides, VEGF and FGF9 on salivary gland regeneration.

Hyposalivation is associated with radiation therapy, Sjögren's syndrome and/or aging, and is a significant clinical problem that decreases oral health and overall health in many patients and currently lacks effective treatment. Hence, methods to regenerate salivary glands and restore saliva secretion are urgently needed. To this end, this study describes the modification of fibrin hydrogels with a combination of laminin-1 peptides (YIGSR and A99) and human growth factors (vascular endothelial growth factor and fibroblast growth factor 9) to enhance regeneration in a salivary gland injury mouse model. Our results indicate that these fortified hydrogels enhanced angiogenesis and neurogenesis while promoting formation of acinar structures, thereby leading to enhanced saliva secretion. Such functional recovery indicates salivary gland regeneration and suggests that our technology may be useful in promoting gland regeneration and reversing hyposalivation in a clinical setting. STATEMENT OF SIGNIFICANCE: We engineered Fibrin Hydrogels (FH) to contain multiple regenerative cues including laminin-1 peptides (L1p) and growth factors (GFs). L1p and GF modified FH were used to induce salivary gland regeneration in a wounded mouse model. Treatment with L1p and GF modified FH promoted salivary epithelial tissue regeneration, vascularization, neurogenesis and healing as compared to L1p-FH or FH alone. Results indicate that L1p and GF modified FH can be used for future therapeutic applications.