Assessing Bioactivity and Biointegration of Engineered Salivary Tissue Constructs in a Preclinical Unilateral Fractionated Irradiated Rat Model

Human salivary stem/progenitor cell (hS/PC)-loaded hyaluronic acid (HA)-based hydrogels, termed 3D-salivary tissue constructs (3D-ST), hold great promise as a regenerative platform for repairing radiation-damaged salivary glands. Here, we developed a next-generation 3D-ST using heparin-modified HA and bioactive peptide-modified hydrogels. This formulation provides upcoming opportunities for controlled pre-loading and localized presentation of heparin-binding growth factors prior to surgical implantation to enhance in vivo hS/PC bioactivity. To model clinically relevant radiation injury, we established an athymic rat model subjected to computed tomography (CT)-guided fractionated radiation, resulting in hallmark features of radiation-induced salivary dysfunction. Over 60-days post-irradiation, glands exhibited progressive loss of acini, increased fibrosis, and disruption of endothelial, neuronal, and myoepithelial compartments. Within this injured environment, a surgical pocket was created to precisely implant 3D-STs to assess graft performance. Fluorescent labeling of the 3D-STs enabled post-mortem localization post-implantation. Over 14 days, implanted 3D-STs remained structurally stable within irradiated glands, and hS/PCs remained viable without evidence of local inflammatory responses. Compared to non-injured glands, the irradiated microenvironment suppressed hS/PC proliferation and phenotype, indicating alterations in the irradiated tissue negatively impact hS/PC bioactivity. In addition, host neurovascular migration into the 3D-ST was majorly restricted in irradiated glands, providing opportunities to enhance biointegration. Overall, this work establishes a reproducible preclinical framework for assessing hydrogel biocompatibility and stability, cell bioactivity, and host-graft biointegration prior to scale up into preclinical large animal models. This study has established a tractable approach for improving 3D-ST formulations to enhance hS/PC expansion, differentiation, and future biointegration following implantation into radiation-injured beds. STATEMENT OF SIGNIFICANCE: Our study evaluates the biostability, bioactivity, and biointegration of hydrogel-encapsulated human epithelial stem/progenitors transplanted in healthy versus irradiated parotid gland beds. These findings provide preclinical evidence supporting the translational development of this cell-containing hydrogel for the repair of irradiated glands in patients suffering from xerostomia.