AIM: This study aimed to evaluate whether human dental pulp stem cells (DPSCs), after long-term biobanking (7-8 years), retain their pro-angiogenic properties and can be used to engineer vascularised tissues, addressing their potential for clinical translation in regenerative dentistry. METHODOLOGY: Cryopreserved DPSCs from adolescent donors were recovered from biobanking and characterised for chromosomal integrity, MSC immunophenotype and multipotency. After conditioning in pro-angiogenic conditions in vitro, gene and protein expression were analysed by RT-qPCR array, flow cytometry and high-throughput immunophenotyping. Functional angiogenic capacity was assessed via in vitro tube formation, ex ovo CAM implantation assay, organ-on-chip perfusion model and long-term culture (45 days) in clinical-grade GelMA hydrogels, with and without HUVECs. RESULTS: Biobanked DPSCs retained MSC identity and multi-lineage differentiation potential. Pro-angiogenic/endothelial conditioning enhanced the expression of angiogenic/endothelial genes (PECAM1, VEGFR2, NRP1, ACE), yet most cells maintained a pericyte-like phenotype. Both naive and endothelial-conditioned DPSCs (i.e., naiveDPSCs and endoDPSCs, respectively) significantly enhanced vascular ingrowth in the CAM model. In the organ-on-chip system, naiveDPSCs formed perfusable vasculature with HUVECs and differentiated into perivascular cell types. Most notably, endoDPSCs alone successfully generated vascularised tissue with both CD31(+) and αSMA(+) cells present in GelMA hydrogels after prolonged stimulation. CONCLUSION: Long-term biobanked DPSCs preserve their angiogenic potential and, following extended endothelial induction, can independently generate vascularised tissue in 3D in vitro culture models. This is the first report demonstrating the comprehensive pro-angiogenic characterisation and the feasibility of using biobanked DPSCs for vascularised tissue engineering, highlighting their strong clinical applicability for future regenerative therapies.

Center for Advanced Stem Cell and Regenerative Research Tohoku University Graduate School of Dentistry Miyagi Japan

Center of Translational Oral Research Tissue Engineering Department of Clinical Dentistry University of Bergen Bergen Norway

Centre for Craniofacial and Regenerative Biology Faculty of Dentistry Oral and Craniofacial Sciences King's College London London UK

Department of Histology and Embryology Faculty of Medicine Masaryk University Brno Czechia

Division of Molecular and Regenerative Prosthodontics Tohoku University Graduate School of Dentistry Miyagi Japan

Institute of Animal Physiology and Genetics Czech Academy of Sciences Brno Czechia

Laboratory of Molecular Materials Division of Biophysics and Bioengineering Department of Physics Chemistry and Biology Linköping University Linköping Sweden

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