Objective: This study investigates the potential of post-mortem Gingival Stromal Cells (GSCs), derived from the neural crest, as a novel, accessible source for craniofacial tissue regeneration. A secondary aim explores the feasibility of using gingival tissue for post-mortem interval (PMI) estimation based on protein analysis and degradation products.

Methods: Murine and human post-mortem gingival tissues were collected at defined time points (up to 100h in mice and 5 days in humans). Tissue integrity, HIF-1α expression, and stromal cell markers were assessed using qPCR, Western blotting, immunohistochemistry, and flow cytometry. Isolated GSCs were cultured under 2D conditions for osteo/adipogenic differentiation and in 3D conditions for neurogenic differentiation and co-cultured with HUVECs (endothelial cells). Two in vivo mice models were then developed: (1) a subcutaneous angiogenesis model using spheroids containing post-mortem GSCs and HUVECs to evaluate vascular structure formation and anastomosis; (2) a calvarial critical-size defect model using hydrogels seeded with GSCs to assess bone regeneration via µCT and histology. Additionally, mass spectrometry on human samples was performed to identify protein degradation or stability patterns relevant for PMI estimation.

Results: Post-mortem gingival tissue remained histologically intact up to 5 days in humans and 100 hours in mice. GSCs isolated post-mortem preserved stromal stromal cell markers, demonstrated high proliferation, and retained multilineage differentiation capacity. In 3D cultures, they formed spheroids and vascular-like structures with HUVECs, and expressed early neurogenic markers. In vivo experiments are underway to assess the angiogenic and osteogenic potential of post-mortem GSCs in mouse models. Proteomic analyses revealed a large panel of stable proteins post-mortem, supporting their potential use as PMI biomarkers.

Conclusion: Post-mortem gingiva represents a non-invasive source of viable neural crest-derived stromal cells. These GSCs exhibit encouraging regenerative potential, supporting applications in craniofacial repair. Additionally, gingival tissue offers promising biomarkers for PMI estimation. The study redefines the limits of stromal cell viability post-mortem and supports the dual utility of gingival tissue in both forensic science and regenerative medicine.