Dental diseases such as caries and sensitivity necessitate the application of biomaterials directly onto tooth cavities. This process involves attaching the biomaterial to dentin, the calcified tissue underlying the dental enamel. The dentin matrix forms a unique interface with biomaterials, indirectly affecting the dental pulp through the dentinal tubules. Understanding this biomaterial-dentin-pulp interface is crucial for developing effective dental treatments. Traditional model systems, while useful, have limitations in directly observing and controlling the cellular and metabolic events at this interface. This gap can potentially be addressed by organ-on-a-chip technology. However, organ-on-a-chip for dental research, particularly for studying the dentin-pulp interface, remains underdeveloped.

Dental researchers have long been striving to find new technologies and breakthroughs for dental research. In 2019, they achieved a significant victory: the first creation of a "tooth-on-a-chip," providing a new level of insight into the biological complexity of the tooth-biomaterials interface. One day, the "tooth-on-a-chip" could enable more personalized dental treatments, allowing dentists to determine the most effective and long-lasting dental filling materials based on the patient's own teeth and oral microbiome.

Materials and Methods

The design of the tooth-on-a-chip was modeled using computer-aided design (CAD) software (Autodesk Fusion 360) and the template was created by laser cutting on a polymethyl methacrylate (PMMA) sheet. Subsequently, the template was installed at the bottom of a molding container and molded using PDMS (polydimethylsiloxane) prepolymer, which was then cured overnight at 80°C. The cured PDMS mold was removed, and four reservoirs were prepared at the ends of the channels using an 8mm punch. This device features two parallel channels, two chambers for perfusion, and a central slot designed to hold dentin fragments. Extracted third molars were cut into specified sizes of dentin fragments using a low-speed saw, then after plasma treatment of the PDMS components and cover glass, the dentin fragments were inserted into each mold to assemble a sealed and leak-free microdevice. The fabricated "tooth-on-a-chip" replicates the interface between dentin and pulp, as well as dentin and dental restorative materials, forming two chambers that simulate the "pulp side" and the "cavity side." Subsequently, stem cells from apical papilla (SCAPs) were cultured to assess cell attachment and differentiation, and living cell imaging, cytotoxicity testing, and gelatin degradation activity analysis were performed to evaluate the responses of pulp cells to various dental materials.

Fig. 1 Fabrication of the tooth-on-a-chip.

Results

Morphogenesis of SCAP on Dentin-Pulp Interface

The tooth-on-a-chip system was developed with a 500 μm thick dentin barrier and a monolayer of differentiated SCAPs to mimic a deep dental cavity. Time-lapse imaging showed complete monolayer formation on the dentin wall in 24 hours. Cell death was tracked using Helix NP NIR dye after treatment with 20 mM 2-hydroxyethyl methacrylate (HEMA), revealing increasing non-viability over 60 minutes. The system demonstrated real-time cell responses to phosphoric acid, visible through contraction towards the dentin wall and bubble formation, showcasing the system's potential to explore the biological intricacies of tooth–biomaterial interactions.

Fig. 2 Live-cell imaging on-chip.

Effect of Dental Materials on SCAP Morphology and Proliferation

After 24 hours, all tested dental materials caused noticeable cellular damage both on-chip and off-chip. HEMA resulted in poorly connected, rounded cells with pyknotic nuclei, and a reduction in cell number. PA caused monolayer disorganization and cytoplasmic injury, while SB led to dim actin staining and increased intercellular spaces. By day 7, untreated cells maintained their morphology, whereas treated cells showed significant morphological changes and reduced numbers. On-chip cultures had higher cell counts compared to off-chip samples for all treatments, with HEMA and SB showing significant reductions in cell number relative to untreated controls.

Fig. 3 SCAP morphology after biomaterials treatment on-chip and off-chip.

Metabolic Activity

All tested dental materials exhibited cytotoxicity in both on-chip and off-chip systems, with HEMA showing the highest toxicity, followed by SB and PA. On-chip cells demonstrated consistently higher metabolic activity compared to off-chip samples. The dentin barrier's presence appeared to mitigate some adverse effects observed in off-chip controls.

Fig. 4 Comparison of metabolic activity between cultures on-chip and off-chip.

Gelatinolytic Activity

Metalloproteinase (MMP) activity was higher in chips seeded with cells, indicating greater hybrid layer degradation. Intense green fluorescence from conjugated gelatin was observed in the cell cytoplasm and the hybrid layer, demonstrating the contribution of cellular gelatinases to hybrid layer degradation.

Fig. 5 Gelatinolytic activity in the hybrid layer on-chip with and without cells after 48 h.

Conclusion

Overall, the "tooth-on-a-chip" represents a significant advancement in dental research, potentially translating into improved dental treatments, personalized care, and better preventive strategies. The real-time insights it provides could revolutionize traditional dentistry practices, leading to a future where dental care is more precise, effective, and tailored to individual patients.

• Gelatin
• Metalloproteinase
• EDTA