Culture-Based Microbiological Assessment of Dentin Biomaterials Prepared from Long-Term Refrigerated Human Teeth: A Pilot Study Toward Evidence-Based Tooth Banking

Dentin derived from extracted human teeth has emerged as a promising autogenous biomaterial for bone regeneration owing to its structural and biological similarity to bone tissue. Growing clinical interest in dentin-derived graft materials has also stimulated the development of tooth-banking concepts, in which extracted teeth are preserved for future regenerative applications. However, microbiological contamination remains one of the principal concerns associated with long-term storage before clinical use. This in vitro study evaluated the microbiological status of dentin biomaterials prepared from human teeth stored under long-term refrigerated conditions (approximately 4 °C) and subsequently processed using three commercially available dentin-processing systems: BonMaker (BM), Tooth Transformer (TT), and Smart Dentin Grinder (SDG). A total of 72 extracted teeth collected between 2018 and 2025 were allocated to experimental groups and processed into 24 pooled dentin samples. Five freshly extracted teeth served as controls. Culture-based microbiological assessment was performed before and after dentin processing using Schaedler Broth K3 culture media under aerobic and anaerobic conditions. Microorganisms isolated from positive cultures were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Baseline microbiological assessment detected bacterial contamination in 2 of 24 pooled dentin samples (8.3%) and in 4 of 5 control teeth (80.0%). The identified microorganisms included Staphylococcus epidermidis, Actinomyces viscosus, Streptococcus anginosus, Streptococcus mutans, and Staphylococcus hominis. No microbiologically detectable bacterial growth by culture-based methods was observed following processing with BM, TT, or SDG, irrespective of storage duration, including teeth preserved under refrigerated conditions for up to seven years. Within the limitations of this culture-based investigation, the evaluated dentin-processing protocols effectively eliminated microbiologically detectable cultivable bacterial contamination from long-term refrigerated dentin samples. These findings provide preliminary culture-based microbiological evidence supporting the feasibility of long-term refrigerated tooth preservation under the investigated storage conditions as one component of future tooth-banking research. Since culture-based microbiological methods do not detect viable but non-culturable microorganisms or other non-cultivable pathogens, the present findings should not be interpreted as evidence of complete sterility, overall biological safety, or the superiority of refrigerated storage over alternative preservation methods. Further studies employing molecular microbiological techniques together with biological and physicochemical analyses are required to comprehensively validate long-term tooth-banking protocols.