Dental Caries Cell Models for Research

Dental caries remains the most prevalent non-communicable disease worldwide, affecting an estimated 2.5 billion adults and 514 million children according to the WHO Global Oral Health Status Report 2022. The disease imposes a substantial global burden, with untreated caries in permanent teeth ranking as the most common health condition globally. Key risk factors include frequent consumption of fermentable carbohydrates, inadequate fluoride exposure, poor oral hygiene, reduced salivary flow, and specific cariogenic bacterial species such as Streptococcus mutans and Lactobacillus spp. Unlike many chronic diseases, dental caries is largely preventable through fluoride use, dietary modification, and regular dental care. However, disparities in access to oral healthcare persist, particularly in low- and middle-income countries. The clinical impact extends beyond pain and infection to include tooth loss, impaired nutrition, and reduced quality of life. Importantly, dental caries is not a malignant disease; therefore, traditional cancer metrics such as 5-year survival by stage are not applicable. Instead, outcomes are measured by caries incidence, progression, and restoration longevity. Research into caries prevention and management is critical given the high global prevalence and the limitations of current restorative approaches, which are not curative and often require replacement over time.

Dental caries serves as an excellent model for studying host-microbe interactions, biofilm formation, and biomineralization because it involves a well-characterized microbial etiology and host genetic susceptibility. The disease is ideal for mechanistic studies due to several factors: 1) it affects a accessible tissue (enamel and dentin) that can be sampled non-invasively; 2) it has a polygenic inheritance pattern with several candidate genes identified; 3) it can be modeled in vitro using oral epithelial cells, odontoblast-like cells, and organotypic cultures; and 4) it benefits from public datasets such as the NCBI Gene database, which catalogs genes like AMELX, MMP20, and TUFT1 associated with enamel formation and caries susceptibility. Open questions include the precise genetic variants that confer resistance or susceptibility to caries, the role of the oral microbiome in modulating host gene expression, and the development of biomimetic materials that promote enamel regeneration. These questions drive demand for robust, reproducible cell models that can be genetically manipulated to test hypotheses.

Dental caries is not a carcinogenic process, but its pathogenesis involves several key molecular pathways that can be studied using gene-edited cell models. The major pathways include:

1. Bacterial adhesion and biofilm formation: Cariogenic bacteria such as Streptococcus mutans utilize glucosyltransferases (Gtfs) to synthesize glucans from sucrose, enabling adherence to tooth surfaces. This process involves genes like gtfB, gtfC, and gtfD.

2. Acid production and tolerance: Bacterial fermentation of carbohydrates produces lactic acid, lowering plaque pH and causing enamel demineralization. Acid tolerance responses involve the F-ATPase system and two-component signal transduction systems.

3. Host immune response: Toll-like receptors (TLRs) and cytokines (e.g., IL-1β, TNF-α) mediate inflammation in dental pulp and periodontal tissues. Genetic variants in IL1B and TNF have been associated with caries susceptibility.

4. Enamel and dentin biomineralization: Genes such as AMELX, AMBN, ENAM, and MMP20 regulate enamel matrix formation and mineralization. Mutations in these genes lead to enamel defects and increased caries risk.

These pathways can be dissected using CRISPR knockout or knock-in cell models to identify therapeutic targets.

Unlike cancer, dental caries does not harbor somatic mutations with high frequency. However, host genetic variants and microbial genes contribute to disease. The table below summarizes key genes and their relevance, based on data from NCBI Gene and ClinVar.

Note: Frequency data are approximate and derived from candidate gene studies; no large-scale somatic mutation catalog exists for caries as it is not a neoplastic disease.

Several signaling networks are implicated in dental caries pathogenesis and host response:

• • Wnt/β-catenin signaling: Regulates odontoblast differentiation and dentin formation. Dysregulation can impair reparative dentinogenesis.
• • TGF-β/BMP signaling: Controls enamel matrix secretion and mineralization. Mutations in BMP receptors affect enamel quality.
• • NF-κB pathway: Mediates inflammatory responses to bacterial lipoteichoic acid and lipopolysaccharides. Activation leads to cytokine release and pulpitis.
• • MAPK/ERK pathway: Involved in odontoblast survival and response to stress. Modulates matrix metalloproteinase (MMP) expression.
• • PI3K/AKT/mTOR pathway: Regulates cell growth and survival in oral epithelial cells; may influence susceptibility to infection.

Key nodes in these networks include β-catenin, SMAD proteins, NF-κB p65, ERK1/2, and AKT. Gene-edited cell models can be used to interrogate these nodes and their crosstalk.

Several cell lines are commonly used to study dental caries and related processes. The table below lists key lines with their origin and notable mutations.

Organoids derived from dental pulp stem cells or epithelial stem cells offer advantages such as three-dimensional architecture, cell-cell interactions, and differentiation capacity. They can be genetically modified using CRISPR to study caries-related genes in a more physiological context.

Animal models are essential for studying dental caries in vivo. Examples include:

• • Rat caries models: Specific pathogen-free rats infected with Streptococcus mutans and fed a high-sucrose diet develop caries lesions. These models are used to test anti-caries agents.
• • Mouse models: Knockout mice for Amelx, Mmp20, or Enam exhibit enamel defects and increased caries susceptibility. These are genetically engineered models (GEMMs).
• • Hamster models: Used historically for caries research due to their susceptibility to S. mutans.
• • Non-human primates: Rarely used due to ethical concerns, but provide closest resemblance to human dentition.
• • Induced models: Application of acid or bacterial cultures to tooth surfaces in vivo or ex vivo.

Patient-derived xenografts (PDX) are not applicable to caries as it is not a neoplastic disease. However, human tooth slices can be implanted in immunodeficient mice for short-term studies.

CRISPR-Cas9 technology enables the creation of isogenic cell lines with precise genetic modifications to study dental caries. Examples include:

• • AMELX knockout line: Generated in HAT-7 or LS8 cells to model amelogenesis imperfecta and increased caries risk. These cells lack amelogenin, a key enamel matrix protein.
• • MMP20 knock-in line: Introduction of a disease-associated missense mutation (e.g., p.Thr130Ile) into the endogenous MMP20 locus in odontoblast-like cells to study enamel maturation defects.
• • TUFT1 knockout line: Created in oral epithelial cells to investigate its role in enamel protein composition and caries susceptibility.
• • IL1B reporter line: Knock-in of a luciferase or fluorescent reporter under the IL1B promoter to monitor inflammatory responses to cariogenic bacteria.

Commercially available, sequence-verified gene-edited cell models can accelerate research by providing reproducible, validated tools. These models are engineered using CRISPR and are quality-controlled for genomic integrity and functional activity. They enable researchers to focus on experimental questions rather than model generation, thereby speeding up target validation and drug discovery in the dental caries field.

Gene-edited cell models are powerful tools for functional genomics in dental caries research. For example:

• • Knockout of AMELX in ameloblast-like cells confirms its essential role in enamel crystallite formation and reveals compensatory changes in other enamel genes.
• • Knock-in of a TUFT1 risk variant (rs17640579) in oral epithelial cells can be used to test its effect on cell adhesion and bacterial binding.
• • CRISPR interference (CRISPRi) screens can identify genes that modulate susceptibility to acid stress in oral keratinocytes.

These approaches validate candidate genes from genome-wide association studies (GWAS) and provide mechanistic insights into caries pathogenesis.

Isogenic pairs of cell lines (e.g., wild-type vs. AMELX knockout) are ideal for high-throughput drug screening to identify compounds that rescue enamel defects or prevent bacterial adhesion. For instance:

• • Screening of small molecule libraries for enhancers of amelogenin expression in knockout cells.
• • Testing of antimicrobial peptides or probiotics for their ability to inhibit S. mutans biofilm formation on engineered oral epithelial cells.
• • Modeling of fluoride resistance: Gene-edited cells with altered fluoride transport (e.g., SLC26A4 knockout) can be used to study fluoride uptake and resistance mechanisms.

These applications facilitate the development of novel preventive and therapeutic strategies for dental caries.

CRISPR synthetic lethality screens can identify genetic dependencies in caries-associated pathways. For example:

• • A genome-wide CRISPR screen in oral epithelial cells under acid stress can reveal genes that are essential for survival only in low pH, representing potential biomarkers or drug targets.
• • Knockout of IL1B in macrophages can be used to identify downstream biomarkers of inflammation in pulpitis.
• • Proteomic profiling of gene-edited odontoblasts can uncover secreted biomarkers of dentin degradation.

These discoveries can lead to non-invasive diagnostic tests for early caries detection and monitoring.

Note: TCGA and COSMIC are not directly relevant to dental caries as it is not a neoplastic disease. However, they are included for completeness as resources for oral cancer research that may intersect with caries studies.