Osteogenic Response of Dental Pulp Stem Cells (DPSCs) to an Alginate/Chitosan/Cannabidiol Hydrogel

This study evaluated the osteogenic differentiation and paracrine response of dental pulp stem cells (DPSCs) cultured within a 3D-printed alginate/chitosan/cannabidiol (CBD) hydrogel. DPSCs from a single donor (passage 6; n = 3 technical replicates per condition) were cultured as monolayers or on hydrogel constructs (CBD 0–12 mg/mL) in basal or osteogenic medium for 7, 14, and 21 days. Alizarin Red S staining confirmed retention of osteogenic competence. Supernatant osteoprotegerin (OPG) and RANKL were quantified by Luminex immunoassay. Monolayer cultures produced up to 7867.5 ± 676.8 pg/mL OPG, whereas hydrogel cultures yielded 9.7–69.3 pg/mL (98–99.8% reduction; p < 0.001). RANKL remained stable at 9–11 pg/mL across all conditions; neither analyte varied with CBD concentration. The OPG/RANKL ratio collapsed from 215–858 to 1.0–6.3 in hydrogel groups. Since RANKL—co-secreted by the same cells—was unaffected, a generalized cellular or secretory deficit is unlikely to explain the selective OPG decrease. The data are consistent with charge-selective retention of cationic OPG (pI ≈ 8.5–9.0) within the anionic alginate network, although this mechanism remains hypothetical pending direct adsorption and ζ-potential verification. These findings indicate that scaffold fixed charge constitutes an active variable in the apparent secretome of three-dimensional cultures.