Treatment of acini-like structures with the -adrenergic agonists norepinephrine or isoproterenol increased granule production and -amylase staining in treated structures, demonstrating regain of protein secretion. seeded on 2.5D, as well as those encapsulated in 3D HA hydrogels, self-assembled into acini-like structures and expressed functional neurotransmitter receptors. Structures in 3D hydrogels merged to form organized 50?m spheroids that could be maintained in culture for over 100 days and merged to form structures over 500?m in size. Treatment of acini-like structures with the -adrenergic agonists norepinephrine or isoproterenol increased granule production and -amylase staining in treated structures, demonstrating regain of protein secretion. Upon treatment with the M3 muscarinic agonist acetylcholine, acini-like structures activated the fluid production pathway by increasing intracellular calcium levels. The increase in intracellular calcium seen in nicein-125kDa structures in the 3D hydrogel culture system was more robust and prolonged than that in 2.5D. To compare the long-term survival and retention of acini-like structures that regain both fluid and protein secreting functions and are suitable for tissue restoration. Introduction Saliva is an essential oral lubricant containing an array of vital proteins for maintaining oral health. Radiation therapy (RT) for head and neck cancer often results in irreversible salivary gland damage, leading to salivary hypofunction, dental caries, and fungal infections from loss of protective saliva.1C3 Although advanced radiation techniques such as intensity-modulated radiation therapy (IMRT) significantly reduce radiation to the salivary glands compared to conventional radiation, a large percentage of patients develop xerostomia post-IMRT.4,5 Postradiotherapy palliative therapies remain largely ineffective for long-term resolution of xerostomia. 3 Salivary tissue engineering potentially offers LEP (116-130) (mouse) permanent relief of xerostomia. We previously reported the isolation of acinar-like cells from human salivary tissues, and their self-assembly into three-dimensional (3D) acini-like structures when cultured on 2.5-dimensional (2.5D) hyaluronic acid (HA) hydrogels modified with extracellular matrix (ECM)-derived bioactive peptide fragments.6 HA is a naturally derived, biocompatible matrix that can be chemically functionalized for use as a crosslinkable tissue engineering scaffold.7,8 Unlike other hydrogels, HA is uniquely recognized by its cellular receptors, CD44 and RHAMM. We recently employed a 3D HA-based culture system to organize salivary acinar-like cells into spherical structures. Creating functional implantable tissues with these structures would enable autologous restoration strategies to remove healthy tissue before RT, expand the necessary cell populations and and continued to produce -amylase. Open in a separate window FIG. 6. Survival and retention of acini-like LEP (116-130) (mouse) structures in 2. 5D and 3D hydrogels. The 2 2.5D implants wrapped in gelatin membranes removed from athymic rats at day 11 (A) and at 3 weeks (B). The 3D implants removed from athymic rats at day 11 (D) and at 3 weeks (E). Dispersed single cells positive for -amylase (red) in rat tissue stained with vimentin (green) are seen in (C). Intact spheroid structures (boxed) in the 3D hydrogel implant stained for -amylase (red) and -catenin (green) are seen in (F). Arrows point to blood vessels. White dashed lines mark the hydrogelCtissue interface. Discussion A biodegradable and human-compatible scaffold able to support growth and differentiation of human salivary gland cells into functional salivary units that secrete fluid and protein upon stimulation is a key step toward creation of an artificial salivary gland. Previously, we reported the isolation of human salivary acinar cells that self-assemble into acini-like structures and express LEP (116-130) (mouse) salivary biomarkers when cultured on ECM derived human-compatible biomimetic peptides.30 To better mimic the environment, we developed an HA-based, 2.5D hydrogel culture system that can support the growth and partial differentiation of 3D acini-like structures as well as in vitro. It should be noted that the 3D gels we used to encapsulate the cells are softer (G 68?Pa) than the 2.5D gels (G 1490?Pa) and they contain polyethylene glycol, but they allow full encapsulation of primary cells. Groups that reported salivary acini-like structures or spheroids growing in 2D or 2.5D34C38 obtained less well-organized salivary structures compared to those seen in 3D.