Osteogenic Differentiation Induced by Dental Pulp Stem Cells Secretome: A Dose-Dependent Comparative Study

To investigate the effects of dental pulp stem cell-derived conditioned medium (DPSC-CM) on the proliferation, migration, and osteogenic differentiation of periodontal ligament stem cells (PDLSCs). Primary goals were to (i) identify the optimal DPSC-CM' concentration and (ii) evaluate its effect both independently (under Growth Medium, [GM]) and synergistically (under Osteogenic Medium [OM]).

DPSC-CM was collected, and an initial screen was conducted to determine their maximum non-toxic concentration (MNTS). For functional assays, three non-cytotoxic concentrations (V/V) (10% [CM-10], 30% [CM-30], 50% [CM-50]) were selected. While cytokine release profile, cell proliferation, and migration were assessed under GM conditions, differentiation was evaluated over 14d under both GM and OM conditions (p < 0.05).

The MNTS was established as CM-50. DPSC-CM promoted all cellular activities in a non-linear, concentration-specific manner. CM-10 demonstrated the most beneficial effects; it yielded the highest levels of TGF-β1 and VEGF secretion (p < 0.05), and significantly increased proliferation, alkaline phosphatase activity, and expression of osteogenic/angiogenic markers than CM-50 and CM-30 over time. However, peak cell migration was observed on Day 2 for both CM-30 and CM-10. While DPSC-CM alone enhanced osteogenesis, the highest induction was achieved under OM conditions.

DPSC-CM is a potent osteoinductive agent, enhancing proliferation, migration, and differentiation in a non-linear concentration-specific manner. The lowest concentration proved optimal for their proliferative and osteogenic properties, suggesting a Hormesis-like effect where higher doses may become sub-optimal due to inhibitory factors.