Comparative evaluation of 3D culture strategies for pulp-dentin models.

BACKGROUND: The development of dependable in vitro models that replicate the pulp-dentin complex is important for regenerative endodontics, biomaterials testing, and disease modelling. However, most existing approaches concentrate on isolated techniques, providing limited guidance regarding their comparative performance, practical limitations, or translational applicability. Furthermore, they often present only the final optimized method without elaborating on the decision-making process, including the challenges faced and the rationale for not pursuing alternative approaches. This study aimed to address this gap by evaluating multiple three-dimensional (3D) strategies for the development of pulp-dentin models. METHODS: Two principal assembly routes were explored: manual and digital. Manual assemblies used natural dentin rings combined with different 3D culture strategies to generate the pulp core. Scaffold-free spheroids were formed using ultra-low attachment plates (ULA) and non-adherent overlays (Agarose and Matrigel), while scaffold-based systems employed Matrigel encapsulation. These were manually integrated within dentin rings to establish pulp-dentin interfaces. Digital assemblies utilized extrusion-based 3D bioprinting to fabricate composite constructs composed of dentin powder-reinforced Gelatin Methacryloyl (GelMA) or alginate outer rings and collagen or alginate-based cellular cores. All constructs were evaluated for structural stability, reproducibility, cellular organization, and interaction with the dentin interface, primarily through morphological and histological analyses. RESULTS: Both manual and digital assembly strategies successfully produced 3D pulp-dentin constructs with distinct characteristics. In the manual assemblies, scaffold-free and scaffold-based approaches enabled spheroid formation and matrix-supported tissue organization, respectively. When integrated with natural dentin rings, these cultures established localized pulp-dentin interfaces with Dentin Sialophosphoprotein (DSPP) positive cells, indicating odontogenic differentiation. However, construct uniformity and stability were influenced by spheroid size, hydrogel degradation, and dentin ring geometry. Digital bioprinting enabled precise fabrication of biphasic constructs comprising a dentin powder-reinforced outer phase and a cell-laden inner core. Stable and reproducible dentin-mimetic outer rings were achieved at dentin powder concentrations ≤20% (w/w), with GelMA exhibiting slower degradation and greater mass retention than alginate. The inner pulp-like core was bioprinted using cell-laden bioinks containing 5 × 10⁵ cells, with LifeInk 220 collagen providing consistent print fidelity and homogeneous cell distribution. The resulting dual-layered architecture enhanced the structural and biological resemblance of the model to native pulp-dentin tissue, with dentin powder incorporation contributing to dentin-like features and supporting odontogenic cell organization. CONCLUSION: This study establishes a methodological framework for developing in vitro pulp-dentin models by comparing manual and digital assembly strategies. Rather than identifying a single optimal approach, the work highlights how each method contributes unique advantages and challenges. Documenting both successful outcomes and technical limitations provides valuable guidance for optimizing 3D pulp-dentin constructs and advancing their application in regenerative endodontics, biomaterial evaluation, and translational research.