Poster AA20 - Integrating Vascular Cues to Promote Salivary Gland Tissue Repair and Regeneration

Introduction: The salivary gland comprises lumenized acini that produce saliva and interconnected ducts that modify and carry the saliva to the oral cavity. Salivary glands can be irreversibly damaged by high energy radiation commonly used to treat head and neck cancers. As a result, patients develop xerostomia (or dry mouth syndrome), and their quality-of-life is severely compromised. Currently, there are no curative solutions, and all the palliative treatments are ineffective. Salivary gland tissue engineering offers an alternative long-term solution to xerostomia. Salivary gland morphogenesis requires input and signalling from the integrated vasculature. To deliver an innovative engineering solution to salivary gland regeneration, human primary salivary gland stem/progenitor cells (hS/PCs) were cocultured with human umbilical vein endothelial cells (HUVECs) in a hyaluronic acid (HA)-based hydrogels in a tissue-mimetic configuration. Here, we report how endothelial cells affect the phenotype and function of salivary gland epithelial cells

Materials and

Methods: hS/PCs were isolated from patients undergoing parotidectomy with institutional approval and patients' consent. hS/PC spheroids were produced in agarose microwells following our established methods.1 HUVECs were purchased from ATCC and expanded in EMG2 media. Hydrogel building blocks, including tetrazine-modified HA (HA-Tz), a norbornene (Nb)-functionalized protease degradable crosslinker SMR-bisNb, trans-cyclooctene (TCO)-functionalized cell adhesive peptide (RGD-TCO), were synthesized and characterized following our previously reported methods.2 The HA-Tz/SMR-bisNb solution mixture containing suspended hS/PC spheroids was incubated at 37°C for 1 h to allow complete gelation before EGM2 was added. The epithelial constructs were incubated for an additional 4 h and RGD-TCO was added to the EGM2 media to allow for RGD conjugation via diffusion controlled interfacial tetrazine ligation to proceed for 24 h. On day 1, HUVECs were seeded on top of the construct and the coculture was maintained in EGM2 for 14 days, with media refreshments every 3 days. Constructs were characterized using real time reverse transcription-polymerase chain reaction (RT-qPCR), immunofluorescence and ELISA analyses. Cell-free hydrogels prepared in EGM2 were implanted in partially resected parotid glands of Long Evans rats (male and female, 3-month-old) following our reported procedures.3 Hydrogel degradation was monitored in vivo by tagging the HA-Tz gels with 5 µM Cy7-TCO and imaged using an in vivo imaging system (IVIS). Rats were sacrificed on day 7 and day 21 and the parotid glands with or without HA gels were harvested for histological analysis.

Results, Conclusions, and Discussions: Results and Discussions: hS/PC and HUVEC monocultures on 2D were screened cultured in HepatoSTIM4 or EGM2 media, designed specifically for epithelial and endothelial cell cultures, respectively. We found that EGM2 is most appropriate for the coculture studies as it permitted rapid cell growth and maintained the correct phenotype for both cell types. Under the 3D co-culture conditions, pre-assembled spheroids grew into lobular structures with interconnected nodes (Fig. 1). hS/PCs expressed significantly higher levels of stem/progenitor biomarkers (KRT5, KRT14, ETV5), acinar biomarkers (AMY, AQP3, SLC12A2) and ductal biomarkers (KRT7, TFCP2L1), as compared to the epithelial monoculture. To further evaluate the effect of endothelial cues on the regenerative properties of the salivary gland, endothelial cues/growth factors loaded hydrogels were implanted at the site of resection in rat parotid glands. Tissues with hydrogel implant showed reduced fibrosis, restored acinar structures, and increased regeneration of the glands.

Conclusions: Collectively, endothelial cues promote pro-acinar differentiation and reduce tissue fibrosis. This is significant because radiation therapy is known to cause tissue fibrosis, which in turn, leads to acinar cell damage, senescence, and apoptosis.

Acknowledgements (Optional): Acknowledgement: This work was supported by National Institutes of Health through R01 DE029655

References: 1.Ozdemir+ Biomaterials, 2017, 142, 124. 2.Pol+ Biomaterials, 2023, 299, 122180. 3.Pradhan-Bhatt+ Laryngoscope, 2014, 124, 456. 4.Metkari+ ACS Appl. Mater. Interfaces, 2023, 27, 32148.