Serum-Free Conditioning Duration Shapes the Proteomic and Regenerative Profile of Human Dental Pulp Stem Cell-Conditioned Medium

Abstract

Background: Mesenchymal stromal cell-derived conditioned medium (MSC-CM) represents a promising cell-free approach for regenerative medicine; however, variability in production conditions remains a major challenge for standardization. This study investigated how serum-free conditioning duration influences the proteomic composition and regenerative activity of human dental pulp stem cell-conditioned medium (DPSC-CM). Methods: DPSC-CM was collected after 24, 48, and 72 h of serum-free conditioning and characterized by label-free LC-MS/MS proteomics using donor-blocked differential abundance analysis. DPSC viability and apoptosis were assessed by Annexin V/PI flow cytometry. The angiogenic activity of CM 48 h and CM 72 h was evaluated using the ex ovo chick chorioallantoic membrane (CAM) assay, while all CM groups were assessed for their effects on human exfoliated deciduous teeth (SHED) metabolic activity and osteogenic differentiation. Osteogenesis was evaluated by alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining. PI3K/Akt signaling was investigated for CM 48 h by Western blotting. Results: Serum-free conditioning for up to 72 h did not significantly affect DPSC viability or apoptosis. Proteomic analysis identified 1562 proteins. Differential abundance analysis identified 914 differentially abundant proteins (DAPs) between CM 48 h and CM 24 h and 745 between CM 72 h and CM 24 h, whereas no DAPs were detected between CM 72 h and CM 48 h. Enriched biological processes included extracellular matrix organization, cell-substrate adhesion, cytoskeletal organization, protein folding, and angiogenesis-related pathways. In the CAM assay, CM 48 h in-creased total vessel area, whereas CM 72 h increased total vessel length. CM 48 h en-hanced SHED metabolic activity at day 3. Although ALP staining did not differ significantly among groups, CM 48 h produced greater matrix mineralization than CM 24 h. PI3K inhibition reduced Akt phosphorylation, whereas CM 48 h had no significant effect on the p-Akt/Akt ratio. Conclusion: Serum-free conditioning duration influences DPSC-CM composition and biological activity, with the major proteomic transition occurring between 24 and 48 h. Among the conditioning periods evaluated, 48 h demonstrated the most consistent combination of proteomic and functional effects, supporting further investigation as a candidate conditioning duration for DPSC-CM production.