Rapid SDS/trypsin decellularization of rat submandibular gland yields an ECM scaffold supporting salivary gland tissue engineering.

INTRODUCTION: Salivary gland hypofunction caused by radiation, Sjögren's syndrome, or other insults leads to xerostomia and currently lacks effective regenerative treatments. Decellularized organ-specific extracellular matrix (ECM) scaffolds offer a promising strategy toward gland restoration by recapitulating the native microenvironment.

METHODS: In this study, we developed a rapid decellularization protocol for rat submandibular glands (SMGs) to create a biomimetic ECM scaffold for salivary gland tissue engineering. Following freeze-thaw pretreatment, detergent and enzyme incubations totaled 4 h and the overall detergent/enzyme processing time, including intermediate PBS washes, was approximately 5 h. The resulting scaffold was characterized by histology, immunostaining, biochemical analysis, and proteomic analysis. Recellularization of the decellularized SMG (dSMG) was performed via intraductal injection of human submandibular gland mesenchymal stem cells (hMSCs) or human submandibular gland stem cells (hSMG-SCs), followed by in vitro culture. In addition, hSMG-SC-seeded dSMG scaffolds were implanted subcutaneously in immunodeficient mice for in vivo evaluation.

RESULTS: The protocol achieved effective decellularization, reducing residual DNA to <50 ng/mg dry tissue while preserving essential matrix components. The resulting dSMG scaffold retained key structural proteins, including collagens I and IV, laminin, and fibronectin, as well as glycosaminoglycans, as confirmed by histology, immunostaining and proteomic analysis. Recellularization of the dSMG resulted in cell repopulation, viability and proliferation over 7 days in culture. The hMSCs remained viable and upregulated genes associated with matrix remodeling, whereas hSMG-SCs maintained expression of epithelial markers, e.g., Cytokeratin 7, within the scaffold microenvironment. When hSMG-SC-seeded dSMG scaffolds were implanted subcutaneously into immunodeficient mice for 8 weeks, they became well-vascularized and supported CK7-positive duct-like epithelial organization with persistence of human cell signal, together with weak focal AQP5 expression, whereas these features were not observed in acellular controls.

DISCUSSION: These findings demonstrate that the dSMG scaffold can provide a favorable niche for cell survival and early glandular tissue organization, highlighting its potential as a biomaterial platform for salivary gland tissue engineering.