Regenerative processes occur at various levels in all organisms, yet their complexity continues to raise new questions about their mechanisms. It has been demonstrated that small extracellular vesicles (sEVs), secreted by all cells and influencing their function, play a significant role in regeneration. In the context of regenerative processes, oral mucosal tissues consistently receive interest, as they are among the most rapidly healing tissues in the human body. In this study, we utilized spatial transcriptomics to map gene expression to specific spatial locations within the gingiva tissue section, using publicly available transcriptomic data. This analysis revealed new insights into this tissue and the biogenesis of sEVs within it. The identified clusters encompassed two main regions-the epithelium and lamina propria-as well as minor niches within them. Using Gene Ontology (GO) analysis, we identified two clusters most enriched in extracellular vesicle-related GO processes. These included the superficial and deeper layers of the sulcular epithelium, one of the most peripheral regions of the gingiva. Of the 43 genes identified in the literature as having a potential or documented role in sEVs biogenesis, 12 were selected for further analysis. MUC1, SDCBP2, and VPS37B showed clear specificity and the highest expression in the superficial layer of the sulcular epithelium. CHMP4C also exhibited high expression in this layer, though its levels were comparable to the outer layer of the oral epithelium. Other well-established sEVs marker genes, such as ANXA2, CD9, CD63, CD81, FLOT1, RAB22A, RAB27B, and RAB5A, were also expressed in the examined tissue; however, their expression was not specifically exclusive to the sulcular epithelium. Our study is the first to perform a meta-analysis of available gingival transcriptomic data in the specific context of sEVs biogenesis. The presented data and conclusions provide new insights into the role of different structures within healthy human gingiva and shed new light on both known and potential markers of sEVs biogenesis. These findings may contribute to the development of regeneration-targeted research, especially on oral tissues.

Center of Assisted Reproduction Department of Obstetrics and Gynecology University Hospital and Masaryk University 625 00 Brno Czech Republic

Department of Anatomy and Histology Collegium Medicum University of Zielona Gora 65 046 Zielona Góra Poland

Department of Diagnostics and Clinical Sciences Institute of Veterinary Medicine Nicolaus Copernicus University in Torun 87 100 Toruń Poland

Department of Diagnostics Poznan University of Medical Sciences 60 812 Poznań Poland

Department of Histology and Embryology Poznan University of Medical Sciences 60 781 Poznań Poland

Department of Immunology Poznan University of Medical Sciences 60 806 Poznań Poland

Department of Preventive Dentistry Collegium Medicum in Bydgoszcz Faculty of Medicine Nicolaus Copernicus University in Torun 85 067 Bydgoszcz Poland

Department of Veterinary Surgery Institute of Veterinary Medicine Nicolaus Copernicus University 87 100 Toruń Poland

Division of Anatomy Department of Human Morphology and Embryology Faculty of Medicine Wroclaw Medical University 50 368 Wrocław Poland

Doctoral School Poznan University of Medical Sciences 60 812 Poznań Poland

Prestage Department of Poultry Science College of Agriculture and Life Sciences North Carolina State University Raleigh NC 27695 7608 USA

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