A Standardized In Vitro Protocol for Evaluating Human Dental Pulp Stem Cell Differentiation and Proliferation on Nanomaterial Scaffolds

Reliable assessment of osteogenic and odontogenic differentiation, as well as stem cell proliferation, is fundamental to evaluating the biological performance of scaffold-based regenerative strategies. However, variability in experimental protocols, assay selection, and data interpretation often limits reproducibility and inter-study comparison. The protocol described in this article presents a standardized, reproducible workflow for the in vitro evaluation of osteogenic and odontogenic differentiation, as well as proliferative capacity, of stem cells cultured on biomaterial scaffolds. Osteogenic commitment is evaluated by alkaline phosphatase (ALP) enzymatic activity and Receptor Activator of Nuclear Factor κB Ligand (RANKL) immunofluorescence, while odontogenic commitment is assessed by DMP-1 (dentin matrix protein-1) immunofluorescence; both protein endpoints are quantified using standardized H-score methodology. Critical experimental parameters, including cell seeding density, induction timelines, assay conditions, normalization strategies (including protein-normalized ALP activity, cell-normalized 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability, and immunofluorescence H-score), and data analysis approaches, are detailed, along with troubleshooting guidance to minimize technical variability. The methodology has been applied as a standardized framework across multiple scaffold-based experimental studies, demonstrating robustness and reproducibility. By providing a comprehensive, validated framework, this article aims to facilitate standardized assessment of stem cell-scaffold interactions and improve comparability across preclinical regenerative studies.