Available online 5 May 2023

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We recently developed a salivary gland tissue mimetic (SGm), comprised of salivary gland cells encapsulated in matrix metalloproteinase (MMP)–degradable poly(ethylene glycol) hydrogels within arrays of ∼320 μm diameter spherical cavities molded in PDMS. The SGm provides a functional and physiologically relevant platform well-suited to high-throughput drug screening for radioprotective compounds. However, the utility of the SGm would benefit from improved retention of acinar cell phenotype and function. We hypothesized that tuning biochemical cues presented within the PEG hydrogel matrix would improve maintenance of acinar cell phenotype and function by mimicking the natural extracellular matrix microenvironment of the intact gland. Hydrogels formed using slower-degrading MMP-sensitive peptide crosslinkers showed >2-fold increase in sphere number formed at 48 h, increased expression of acinar cell markers, and more robust response to calcium stimulation by the secretory agonist, carbachol, with reduced SGm tissue cluster disruption and outgrowth during prolonged culture. The incorporation of adhesive peptides containing RGD or IKVAV improved calcium flux response to secretory agonists at 14 days of culture. Tuning the hydrogel matrix improved cell aggregation, and promoted acinar cell phenotype, and stability of the SGm over 14 days of culture. Furthermore, combining this matrix with optimized media conditions synergistically prolonged the retention of the acinar cell phenotype in SGm.

Statement of Significance

Salivary gland (SG) dysfunction occurs due to off-target radiation due to head and neck cancer treatments. Progress in understanding gland dysfunction and developing therapeutic strategies for the SG are hampered by the lack of in vitro models, as salivary gland cells rapidly lose critical secretory function within 24 hours in vitro. Herein, we identify properties of poly(ethylene glycol) hydrogel matrices that enhance the secretory phenotype of SG tissue mimetics within the previously-described SG-microbubble tissue chip environment. Combining slow-degrading hydrogels with media conditions optimized for secretory marker expression further enhanced functional secretory response and secretory marker expression. These results should be of broad interest to the scientific, engineering, and medical communities, and are therefore wellsuited for publication in Acta Biomaterialia.

Section snippetsINTRODUCTION

Approximately 80% of the 700,000 head and neck cancer patients diagnosed each year globally will receive radiation therapy[1,2]. Chronic dry mouth or xerostomia occurs in >50% of patients due to off-target salivary gland damage, leading to loss of acinar cell secretory function[3], [4], [5]. Xerostomia causes chronic sore throat, difficulty eating and talking, dry, painful oral and nasal passages, and chronic oral infections[6,7]. Existing treatments are palliative, and no cure exists[8]. Thus,

Animals

Female C57BL/6J (Jackson Laboratory, ME, USA) mice were used in this study at 6-10 weeks of age. Mouse salivary gland tissue was chosen due to the variability in age, sex, and disease state of surgically discarded human salivary gland tissue. Due to the sexual dimorphism in adult murine salivary glands, only female mice were used due to greater similarity with human tissue. Human tissue does not contain granular convoluted tubules (GCT), while female mice do contain a poorly developed GCT and

Quantification of hydrogel degradation properties

Gels formed using each of the three crosslinkers showed equivalent initial compressive moduli and swelling ratios during degradation (Fig. 1a). Peptide crosslinkers were synthesized with biotinylated lysine residues in regions flanking the MMP-degradable sequence. PEG hydrogels synthesized using these crosslinkers could be labeled by incubation with streptavidin, followed by fluorescently conjugated biotin. Fluorescent and confocal microscopy visualized the resulting fluorescent gels within the

DISCUSSION

The initial SGm tissue chip platform retained acinar cells responsive to agonist signals, making it suitable for high-throughput radioprotective drug screening[23]. However, improvements in the platform could yield augmentation of the acinar phenotype and more consistent cultures over a longer duration, which may broaden the screening capabilities. Therefore, the focus of this study was to engineer tissue mimetics that best preserve the acinar phenotype to provide a suitable platform for

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors gratefully acknowledge the support of the National Institute of Dental and Craniofacial Research (NIDCR), and the National Center for Advancing Translational Sciences (NCATS) of the National Institutes of Health, under award numbers UH3 DE027695 and UG DE027695 to D.S.W.B., C.E.O., and L.A.D., T32 ES007026 to J.A.M., F31 DE029658 to L.P, and F30 CA183320 to A.D.S. The authors would like to thank Dr. Matthew Ingalls and M. Azmeer Sharipol for training and many helpful conversations,

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© 2023 Published by Elsevier Ltd on behalf of Acta Materialia Inc.