Poster N9 - Adapting development programs to promote salivary gland tissue engineering

Introduction: Radiation therapy, a common treatment for head and neck cancers, can cause irreversible damage to the saliva-producing acinar cells. As a result, patients experience xerostomia, a condition characterized by salivary gland hypofunction. This condition has a profound impact on a patient’s quality of life.1 Salivary gland tissue engineering holds great promise as a regenerative solution. Previously, we cultured primary human salivary stem/progenitor cells (hS/PCs) in RGD-decorated, hyaluronic acid (HA)-based matrices with varying proteolytic degradability. Encapsulated single hS/PCs grew into multicellular spheroids by day 15.2 In the presence of a ROCK inhibitor, Y-27632, polarized epithelial structures with a common lumen were developed. However, complex interconnected lobular structures seen in the native tissue were not developed. During development, positional or geometric information is sensed by a subgroup of salivary epithelial cells to loosen cell-cell connections and strengthen cell-matrix interactions, allowing the bud to expand into the surrounding stroma. Work from the Yamada Lab shows that the extracellular matrix protein fibronectin is essential for cleft formation during the initiation of epithelial branching. Transient deposition of fibronectin in the cleft regions induces cell-matrix adhesion accompanied by an adjacent loss of cell-cell adhesion to promote branching.3 To recapitulate the dynamic changes in ECM seen in the developing glands and to deliver an innovative solution to salivary gland tissue engineering, we cultured hS/PCs in a bioorthogoanlly constructed HA hydrogel whose composition and properties can be dynamically altered in real time during live cell culture to induce phenotypic changes and, ultimately, promote tissue morphogenesis.

Materials and

Methods: hS/PCs were isolated from human salivary parotid gland tissues obtained from consented patients undergoing parotidectomy. The hydrogels were established via tetrazine ligation with slow (norbornene, Nb) and fast dienophiles (trans-cyclooctene, TCO). Hydrogel precursors, including tetrazine modified HA (HA-Tz), norbornene (Nb) modified matrix metalloproteinase (MMP)-degradable peptide crosslinkers (slow-degrading: GIW-bisNb; fast degrading: SMR-bisNb) and TCO modified cell adhesive peptide derived from fibronectin (RGD-TCO) and laminin (YIGSR-Nb) were synthesized following established protocols.4 The HA-Tz/bisNb/YIGSR-Nb solution mixture (Tz/Nb: 2/1) containing suspended hS/PCs was incubated at 37°C for 1 h to allow complete gelation before the HepatoStim media was added. The cellular constructs were incubated for an additional 1 or 6 days and RGD-TCO was added to the media to allow for peptide conjugation via diffusion controlled interfacial tetrazine ligation to proceed for 24 h. Cultures were maintained for additional 21 days and with media refreshments every 3 days. In selected cultures, transforming growth factor beta 1 (TGFβ1) was supplemented in the media at 10 ng/mL. Constructs were characterized using real time reverse transcription-polymerase chain reaction (RT-qPCR), immunofluorescence and ELISA analyses.

Results, Conclusions, and Discussions: The hydrogel platform was established using modular building blocks employing bioorthogonal tetrazine (Tz) ligation with slow (Nb) and fast (TCO) dienophiles. The cell laden gel construct was created via the slow, off-stoichiometric Tz/Nb reaction. YIGSR was conjugated to the network via bulk reaction using YIGSR-Nb and RGD signals were introduced to the network through the rapid Tz/TCO reaction at the gel-liquid interface. Cells encapsulated in RGD-containing gels crosslinked with SMR-bisNb failed to form multicellular spheroids. Cells that were encapsulated in gels crosslinked with GIW-bisNb all formed spheroids. Cells encapsulated in gels only tagged with YIGSR (2.8 mM) grew at a slow rate and formed small spheroids. Cells in gels with both YIGSR (1.4 mM) and RGD (1.4 mM) signals grew faster, and spheroids were detected as early as day 6 (Figure 1). Cells in gels with RGD only (2.8 mM) grew the fastest. Work is underway to characterize cell phenotype and the ultrastructure and function of the engineered gland. Formation of multicellular spheroids from dispersed hS/PCs depends on the matrix adhesiveness and degradability. We hypothesize that delayed RGD tagging combined with TGFβ1 supplementation will led to the development of small processes around the spheroids.

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

[1] Jasmer+, J. Clin. Med. 2020, 9 (12), 4095. [2] Metkari+, ACS Appl. Mater. Interfaces 2023, 15 (27), 32148–32161. [3] Sakai+, Nature 2003, 423 (6942), 876–881. [4] Pol+, Biomaterials 2023, 299, 122180.