2025 Theses Doctoral

A Composite Scaffold Approach for Enamel Remineralization

Chan, Ming Chau

Dental caries is one of the most prevalent diseases worldwide, affecting millions of children and adults. In the clinic and research field, it is characterized as a pathological condition when there is excessive demineralization of enamel and/or dentin in the primary and permanent teeth. The underlying cause is primarily by the acidic byproducts secreted by bacteria that form the dental plaque, which also prevents the reprecipitation of mineral ions on the tooth surface throughout the day. If the condition is left untreated, this may result in permanently removing the infected tooth and losing its function entirely in the oral cavity.

The enamel is the outermost layer of tissue on the tooth structure; if damaged due to dental caries, the underlying structures, including the dentin-pulp complex, may be infected. This could lead to the patient feeling nociceptive pain and discomfort. In more severe cases, the dental plaque can spread systematically due to the presence of vasculature in the dentin-pulp complex. The enamel is unable to self-regenerate due to the lack of cells and vasculature in the mature tissue. Therefore, remineralization and arresting caries progression are vital if injury and infection are detected in the enamel.

The current gold standard in the clinical field for treating cavities localized in the enamel is limited to synthetic resin-based composites used as sealants, which do not provide mechanical and structural integration with the surrounding native tissue. As a result, this may lead to microcracks at the interface between the dental implant and the enamel, which can cause secondary caries development and lead to revision surgeries. Currently, in the United States, the chances of revision surgeries due to dental fillings failures are approximately 60 – 90%.

Presently, enamel regeneration approaches have focused on reforming enamel through cellular and peptide-based strategies, with some success. Major challenges include the need to demonstrate the feasibility of preventing early development of dental plaque which takes place as quick as 24 hours and retaining mineralization potential in a cariogenic environment.

This dissertation work explores the potential of a bioactive nanomaterial platform for supporting remineralization of enamel and a probiotic-delivery platform to prevent re-infection. Specifically, a bioactive glass nanoparticle (nBG) - poly(lactic-co-glycolic) acid (PLGA) composite scaffold was developed, and we tested the hypothesis that PLGA-nBG can act as a mineralization vehicle and serve as a delivery vehicle for probiotic E. coli Nissle (EcN) to prevent cariogenic bacteria colonization. The objectives of this thesis are: (1) fabricate and analyze the mineralization potential of the PLGA-nBG composite in a simulated oral environment under neutral and acidic conditions, (2) characterize EcN as a probiotic of choice to impede S. mutans virulence during early re-infection including growth, acidogenicity and biofilm deposition, (3) Investigate the potential for PLGA-nBG composite as a vehicle for EcN delivery and prevention for S. mutans colonization. In addition, the potential of the EcN attached PLGA-nBG composite to remineralize enamel is studied using an explant enamel defect model.

The work of this thesis demonstrate that PLGA-nBG construct supported remineralization on the native enamel and preseeding with EcN prevented Streptococcus mutans (S. mutans) colonization in an in vitro model simulating the clinically relevant oral environment. We first fabricated and tested the ability of the PLGA-nBG composite to deposit hydroxyapatite crystals in a healthy environment (a starting neutral pH) or an acidic environment (a starting pH of 5.0) and discovered that the construct deposited hydroxyapatite-like crystals in a healthy condition mimicking the calcium-phosphorus stoichiometric ratio. Interestingly in acidic conditions, the PLGA-nBG scaffold increased the pH level of the saliva compared to that of healthy saliva within 4 days, resulting in a condition that is more favorable for mineralization.

After establishing a scaffold of choice, to address the second objective, we developed a co-culture model to study the probiotic properties of EcN in the presence of S. mutans at different co-culture ratios and with 0% and 1% additional sucrose source in a simulated oral environment for 24 hours. It was observed that EcN was able to reduce S. mutans viability, acidogenicity, and biofilm deposition in a low-sucrose environment. However, in a high-sucrose environment, by monitoring the colony-forming units (CFU), iwe found that EcN reduced S. mutans viability. RNAseq analyses reveal that the presence of EcN reduced biofilm expression (gtf genes) after 24 hours from S. mutans through gene expression changes, which indicates a further reduction of S. mutans virulence. EcN, in turn, showed upregulation of growth-related (Iro genes), acid tolerance (gad genes), and antimicrobial (clb genes) markers, suggesting EcN can survive in an acidic environment and secrete antimicrobials to compete with S. mutans. Taken together, the above findings served as the foundation for using the PLGA-nBG scaffold as a mineralization agent and EcN as a strain of choice for delivery on the scaffold. Thus, to address the last thesis objective, we tested and observed the ability of the PLGA-nBG scaffold to control the attachment of EcN by pre-soaking the biomaterial in culture media under different durations. In addition, we found that the presence of EcN on the PLGA-nBG composite prevented S. mutans colonization and retained mineral deposition on the native enamel.

Collective these findings in this thesis work provided new insight into bioactive strategies for guiding remineralization of the enamel while demonstrating the promise of biomaterial-enabled probiotice delivery for preventing re-infection post clinical treatment of dental caries.