BDNF-loaded oxidized alginate hydrogel enhances neurogenic expression in two-dimensional/three-dimensional dental pulp stem cell cultures

Objectives: Three-dimensional (3D) culture systems provide more physiologically relevant environments than traditional two-dimensional (2D) cultures for studying dental pulp stem cells (DPSCs) in regenerative applications. This study aimed to evaluate the neurogenic potential of brain-derived neurotrophic factor (BDNF) delivered through an oxidized alginate hydrogel (OAH) for sustained release. The effects of BDNF-OAH were compared between magnetic 3D bioassembled (M3DB) spheroids and 2D DPSC cultures. Methods: OAH was synthesized by partial oxidation of sodium alginate with sodium periodate, and successful oxidation was confirmed via FTIR. DPSCs were preconditioned with all-trans retinoic acid (ATRA) for five days, followed by exposure to BDNF-loaded OAH for three days in both 2D monolayer and M3DB spheroid cultures. Cell viability was assessed using MTT and Live/Dead assays. Neurogenic differentiation was analyzed by qPCR and immunofluorescence staining for doublecortin (DCX) and βIII-tubulin, quantified using ImageJ by integrated fluorescence intensity normalized to Hoechst nuclear staining. Data (n = 3) were analyzed using one-way ANOVA or Kruskal–Wallis tests with significance set at p < 0.05. Results: All DPSC cultures maintained high viability across treatment groups. Immunofluorescence analysis revealed significantly increased expression of DCX and βIII-tubulin in both BDNF and BDNF-OAH groups within M3DB spheroids compared to controls, with more uniform and stable fluorescence signals in 3D. In 2D cultures, only the BDNF-OAH group demonstrated a significant increase in both markers, whereas the BDNF-only group showed elevated DCX but not βIII-tubulin expression. Conclusions: Combining 3D DPSC culture with sustained neurotrophic factor delivery via oxidized alginate hydrogel enhances neurogenic differentiation, as indicated by upregulated DCX and βIII-tubulin expression. The BDNF-OAH system demonstrates potential as a bioactive scaffold for promoting neuroregeneration in pulp tissue engineering, supporting restoration of sensory function, neuroimmune defense, and pulp vitality in regenerative endodontics.