3D bioprinted small extracellular vesicles from periodontal cells enhance mesenchymal stromal cell function

This study demonstrates a ‘cell-free’ periodontal-regeneration strategy by microextrusion 3D bioprinting primary periodontal-cell-derived small extracellular vesicles (sEVs) into 10% GelMA constructs and evaluating their ability to enhance human buccal fat pad MSC (hBFP-MSC) attachment and lineage differentiation. sEVs from primary human periodontal ligament cells (hPDLCs) and gingival fibroblasts (hGFs) were enriched by size exclusion chromatography and confirmed to be <200 nm with canonical EV markers (CD9/CD63/CD81). sEVs were incorporated into GelMA bioink (~10^10 particles/mL) and printed into small lattice scaffolds, then release was tracked by DiO labeling/confocal imaging and CD9 ELISA, showing time-dependent release reaching ~80% by day 7 with ~50% scaffold weight loss by 2 weeks. When hBFP-MSCs were seeded onto printed constructs, the hPDLC-sEV group produced stronger adhesion-related and mechanotransduction gene upregulation (FAK,Vinculin,Paxillin,RhoA,Rac1,YAP) than GelMA alone or hGF-sEVs. In short- and long-term differentiation assays, GelMA/hPDLC-sEVs enhanced ligament-associated markers (e.g., MKX,TNC,FSP), increased ALP activity, calcium mineralization, and cementogenic marker expression (CEMP1), supporting the use of bioprinted periodontal sEVs as a sustained-release, cell-free platform to promote ligamentous and osteogenic/cementogenic differentiation in vitro.