Unraveling novel insights into dual-species cariogenic biofilm formation on aged teeth: a comparative analysis on natural vs artificial bioengineered dentin models

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ABSTRACT Dental caries is the most prevalent biofilm-associated disease affecting billions of people worldwide, including elderly individuals. Conventional biofilm study methods rely on human or animal-derived samples, posing challenges regarding accessibility, cost, and ethical considerations. While in vitro systems offer a promising alternative, they often fail to replicate the structural characteristics of dentin, which play a crucial role in bacterial adhesion. To bridge this gap, a bioengineered dentin construct has recently been developed as a reproducible and accessible model for studying biofilm formation specifically associated with dental aging. Therefore, this study aimed to assess dual-species Streptococcus mutans and Candida albicans biofilm formation on bioengineered dentin substrates and compare it to biofilm formation on natural human aged dentin. For this, S. mutans UA159 and C. albicans (ATCC 90028) were co-cultured on bioengineered and natural dentin slabs, and polymicrobial biofilm formation and extracellular polysaccharide matrix production were characterized via high-resolution confocal laser scanning microscopy. Following biofilm formation, image processing was conducted using COMSTAT software to determine biofilm growth parameters. Additionally, fluorescence intensity was quantified via microplate readings, and cell viability was assessed using a Live/Dead viability kit. Overall, results showed comparable biofilm formation patterns between the bioengineered and aged dentin, with no significant differences found in biofilm physical properties or viability. These findings suggest that this bioengineered dentin construct provides a reliable platform for studying biofilm formation in the context of dental aging, making it a valuable tool for investigating microbial adhesion and cariogenic biofilm development under controlled conditions, potentially facilitating future research in biofilm-related oral diseases. IMPORTANCE Dental caries is one of the most common chronic diseases worldwide and is driven by complex microbial biofilms formed on the tooth’s surface. However, existing models for studying these biofilms in the laboratory often rely on human or animal tissues, which are difficult to standardize and present ethical challenges. In this study, we validate a bioengineered dentin-like model that accurately mimics the microarchitecture of aged human dentin, a key site for root caries in the elderly. By comparing biofilms formed by the clinically significant Streptococcus mutans and Candida albicans on both artificial and natural substrates, we show that the engineered model supports biofilm development under comparable parameters and enables detection of changes in microbial virulence. Overall, this platform provides a reproducible and scalable alternative for studying oral biofilms with potential applications in understanding disease pathogenesis, novel treatment testing, and integration into next-generation organ-on-a-chip systems.

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