Remineralization: Current State of Science and Future Directions Meeting Summary

April 13, 2015​

Executive Summary

This report presents a summary of the discussions and recommendations of the workshop on “Remineralization: Current State of Science and Future Directions” that was held on April 13, 2015. The workshop was convened to determine current efforts in the field of tooth remineralization with a goal of identifying what advancements, if any, are needed to further develop this field.  It is expected that advances in tooth remineralization can help reduce caries which is still a prevalent public health problem. Three sessions, titled Clinical Needs and Desired Outcomes, Overview of the State of Science and Technology, and Regulatory and Commercialization Perspectives, each began with individual presentations followed by group discussion. Additional dialogue online and from a follow-up teleconference is also included in this report.  

The attendees highlighted the continuing need for remineralization strategies which would complement and improve upon the action and delivery of fluoride that has been so successful. It was thought that a number of compounds presented could be optimized with new chemical and biological combinations to guide their placement and timing. This approach would require more complete knowledge of the biofilm and the factors influencing the local environment of the demineralized enamel. In order to accomplish this, the activity of the biofilm itself has to be assessed. Currently, there are no non-destructive in vivo assessment techniques. There is also a need for clinical studies with predefined endpoints among different populations. The complexity of the disease process, variability in predictably controlling apatite precipitation, combined with the desired features of simplicity in the clinical setting, would require multidisciplinary teams to collaboratively develop novel approaches to remineralize enamel.

Research recommendations include:

• Physicochemical 
  • Improving penetration/diffusion of agents through a biofilm matrix and into the lesion 
  • Controlling precipitation of agents with different solubilities at different depths of the lesion 
  • Optimizing fluoride and calcium phosphate interactions
  • Delivering complex combinations of agents in as few simple steps as possible
  • Sustaining release over time of compounds such as amorphous calcium phosphates (ACP)
• Physiological​
  • Controlling the pH of the enamel-biofilm interface
  • Limiting activity of caries to shift balance away from demineralization
  • Mimicking biological activity with sophisticated chemistries 
  • Continuing studies of the mineralization front with enamel proteins or ameloblasts
  • Expanding studies on the influence of saliva and biofilm under physiological conditions
  • Enhancing properties of fluoride with other materials including antibacterial agents
  • Conducting clinical studies comparing effectiveness of different agents in different populations
• Methodological
  • Improving real-time in vivo diagnostic imaging tools of caries and mineralized surfaces (with a focus on activity assessment)
  • Developing and validating surrogates for assessment of clinical caries outcomes
  • Promoting implementation and behavioral change 

Introduction
Teeth are constantly subjected to many forces and factors including caries which leads to loss of enamel mineral. Demineralization resulting from acid attack is reversible to some extent through remineralization by saliva. However, this delicate balance is often skewed towards demineralization with poor oral hygiene and a cariogenic diet. Once too much mineral has been lost, a cavitated lesion develops which must be removed and filled. 

The discovery of the benefit of fluoride in the last century helped contribute to declines in prevalence and severity of tooth decay. However, data from the 2011-12 National Health and Nutrition Examination Survey revealed that 91% of adults aged 20-64 still had dental caries. 

As people are living longer, caries will be an increasing problem. Prevention of caries is indeed in the forefront, however, the bulk of scientific interest remains centered on fluoride forms of delivery because of its effectiveness. 

The National Institute of Dental and Craniofacial Research (NIDCR) convened a workshop to obtain expert input from an interdisciplinary group of scientists and clinicians on directions and approaches to advance research on remineralization of teeth and translation to the clinic. Eighteen individuals participated in the workshop held on April 13, 2015. The goal of this workshop was to examine the landscape of mineralization research including state-of-the-science knowledge base, clinical needs, and regulatory and commercialization pathways. The expected outcome was to identify areas where a focused effort in developing new techniques and approaches could prevent worsening of caries lesions and reverse damage to the tooth structure. 

Prior to the workshop, a dialogue was started where participants brought attention to topics in different ways. Using an online forum, individuals uploaded a number of peer-reviewed publications illustrating current thinking about new approaches to remineralization, reference material such as Cochrane reviews, clinical research studies, clinical guidance recommendations and guidelines from professional organizations, case reports, and primary research articles. The full list of these documents is located at the end of this report.

The day-long workshop was divided into three sessions covering the following topics: 

• Clinical Needs and Desired Outcomes
• Overview of the State of Science and Technology
• Regulatory and Commercialization Perspectives

Individuals were asked to address specific questions as prelude to a group discussion for each of the topics. This report is divided into sections summarizing the presentations, discussions, and recommendations. 

Session 1: Clinical Needs and Desired Outcomes 
The workshop opened with a session defining clinical needs and desired outcomes that would guide efforts toward a common set of goals. The discussion started by acknowledging that there are multiple challenges associated with determining the effectiveness of remineralization strategies, such as the need to diffuse through a biofilm, the need to rapidly precipitate and stabilize in caries lesions, and the need to provide a benefit over and above that of fluoride and saliva. From the clinicians’ viewpoints, several factors that influence the location and characteristics of a demineralized lesion needed consideration. For example, the majority of carious lesions are found in the pits and fissures of permanent teeth as well as interproximal surfaces, with molars being the most susceptible. In addition, as the population ages, and the potential for secondary caries and root caries increases, higher levels of fluoride may be required to prevent caries. Dental professionals use a variety of compounds and products, including fluoride and sealants, guided by evidence-based clinical practice guidelines. 

Remineralization agents encounter not only biophysical barriers but also behavioral barriers to adoption. Their dissemination and implementation is determined by reimbursement, media, sales representatives, cost effectiveness, simplicity and speed of use in the dental chair, which was of paramount importance. Development of medical management strategies, to prevent or intervene to preserve tooth structure, will require randomized clinical trials. These trials should be focused on clinical efficacy using caries as the primary outcome variable. However, as these trials are very expensive, efforts should continue to develop and validate surrogate models (e.g., in situ models) and evaluate surrogate outcomes (e.g., measurement of pH, bacterial outcomes). Animal models as well as longitudinal studies may help research in disease etiology.

There are also some population groups that may require special consideration for caries management. Such groups include young children at risk of fluorosis, elderly patients with hyposalivation and root caries, patients opposed to using fluoride, patients undergoing orthodontic treatment, and patients at lower socio-economic status having difficulty accessing dental care. In addition, there are also other conditions that require consideration such as tooth sensitivity, dental erosion, and hypomineralization. Developmental defects that affect enamel maturation include rare diseases like amelogenesis imperfecta and molar incisor hypomineralization (MIH), the latter showing higher prevalence in certain countries. MIH treatment has many challenges and goals including increasing awareness and improving diagnosis and options for therapy. 

Even within the United States, differences in diagnosis and treatment exist between urban and medical center settings. Economic incentives might enable change management and evidence-based practice. Regardless, adopters need to include patients as important stakeholders; and more research needs to be targeted towards behavioral challenges associated with the implementation of remineralization strategies, and evaluation of the implementation of best evidence-based practice guidelines into clinical settings also needs to be done. 

Discussion
The first session’s discussion centered on the scope of the clinical conditions and touched upon the shortcomings of existing tools and methods. Because the range of clinical case definitions involving enamel is so large, an important distinction needed to be made between the terms remineralization and biomineralization. Remineralization involves replenishment of lost minerals from a preexisting natural scaffold where approaches using fluoride and various other compounds can reverse the early lesion. Biomineralization involves replacement of completely destroyed and missing enamel in cavitated lesions. Enamel will also have to be synthesized de novo in cases of dental erosion where many micrometers of enamel thickness need to be generated without an existing scaffold.  

There was general agreement that sealants and fluoride were underutilized, and efforts should be taken to assess strategies to increase usage. Considering that lesion arrest is a success, fluoride is highly beneficial and could be used to a much greater extent even if used off-label. Adoption of fluoride and sealant use depends heavily upon the simplicity of delivery and the speed of the procedure. Strategies to promote use of these preventive measures include monetary incentives for the provider, reimbursements for the patient, and introduction of those products directly to consumers by industry. Industry can play a vital role by developing improved sealants that are acceptable to both dentists and patients.

Better diagnostic measures were viewed as a major pressing need, especially where precision for monitoring progression of caries at certain tooth sites could aid risk assessment and longitudinal studies. Optical assessment of the biofilm was mentioned several times as there are existing technologies that are well-suited for this purpose. In addition to identifying specific anatomical sites, targeting the appropriate population will be important for dissemination and implementation. The availability of improved diagnostic instruments or products was agreed to be as important as having patients and dentists change behavior to adapt new measures. 

Session 2: Overview of the State-of-Science and Technology 
The second session involved a mixture of presentations showcasing current knowledge of the chemical processes that enamel undergoes, interactions with oral microflora, and tools to assess enamel. Enamel is a complex tissue with variable mechanical properties that make it hard to reproduce synthetically. However, several macromolecules and ions hold potential such as bioglass, calcium phosphates mixed with peptides or peptoids, and functionalized calcium phosphates with fluoride. Furthermore, peptide fragments of amelogenin have been used to initiate self-assembly into higher-ordered structures, and binding of these secreted proteins to specific faces of the enamel crystal is being explored.

Current challenges in this field include insufficient understanding of calcium-fluoride-ion interactions and the dynamic caries process. Enhancing and optimizing bioequivalence models that take an individual’s unique biofilm into consideration would lead to increased efficacy of anti-caries therapies. A range of complexities extend from growing enamel with native toughness and hardness properties, to capping with an enamel-like surface and adhering to the preexisting layer to restore lost tissue. Electrokinetic technology, where an electric field is applied to move bulk solvent containing ions and large particles, may be advantageous in removing inhibitory contaminants and accelerating repair time for remineralization systems.   

Recapitulation of the chemistry and structure of enamel could be achieved with techniques applied from the tissue engineering field. Click chemistry allows for temporal and spatial control of chemical moieties and growth factor placement at selective depths within a lesion. Creating these controlled gradients can fulfill a variety of needs such as establishing a bonding layer to existing enamel surfaces, a reservoir for minerals, and an aesthetically-appropriate surface coat. Chemistries can also be used to generate variations of ACP. Further improvements of currently available adhesives and enamel scaffolds can be made to avoid undesirable characteristics such as distinctive boundaries between new and existing layers. To accomplish these studies, there was agreement that larger sizes of enamel or enamel-like crystals are needed which could be achieved by combining existing scaffolds with spatiotemporal control of ion delivery.  

Fluoride is highly effective when it forms a protective precipitate, but has an inherent limitation in remineralizing deeper parts of a lesion because it is readily consumed and incorporated in the surface coating. Fluoride’s efficacy surpasses that of other soluble ions that could serve the same function. Compounds such as calcium, strontium, barium, iron, and lead can incorporate into the apatite lattice structure resulting in a mineral that differs in hardness, mechanical strength, solubility, size, and shape from fluorapatite. Opportunities for discovery include analyses of structure and composition of phases that precipitate during remineralization, and investigations of stabilizing compounds in the mouth under acidic conditions.

The biofilm is a microbial community enmeshed in a complex extracellular matrix that limits diffusion and contains acidic niches of different pH values where active bacteria reside. There exists a knowledge gap in how, why, and where biofilms form. pH-activated carrier molecules, such as farnesol-loaded nanocarriers, could be advantageous for acidic pH-triggered release of a variety of remineralizing or anticaries compounds, precisely when and where they are most needed. Future research will need to include approaches to modify the physiochemical properties of the matrix and microbial composition of the biofilm, to assess pathogenicity or alkalization, and to increase bioavailability of various agents at the tooth/biofilm interface. In vitro studies encounter hurdles such as highly variable lesions between serial sections of the same tooth and a non-native environment. Practical issues to consider for in vivo studies are ethics, feasibility, study costs, and cost-benefit ratio of specific therapies.  

Technologies developed over the past two decades for early caries detection include optical coherence tomography and Raman spectroscopy, among many others. Of the emerging light-based methods, fluorescence-based methods are more affordable. Technological challenges of these methods include difficulty distinguishing between active and inactive caries, subjectivity based on examiner expertise, difficulty visualizing certain surfaces, and lack of clinical validation studies. Also, there are very few nondestructive tools for longitudinally assessing the quality and quantity of newly-formed mineral. Clinical adoption of these technologies will depend on automation, simplicity of device operation, increased area of screening, and patient engagement. Partnerships with industry and additional studies for clinical validation are also needed.

Discussion
The second session’s discussion brought up a number of issues relating to chemistry of the agents and consideration of the local environment. One lingering question was whether the biofilm would interfere with processes such as electrokinetics and if it had to be removed for successful treatment. Removal of the biofilm, although time-consuming and costly in the dental office, could allow for unlimited remineralization potential. On the other hand, its presence could also sequester ions released from the demineralization process, in turn serving as a source of ions over long time frames for remineralization. 

It was acknowledged that a number of current agents with or without additional fluoride show evidence of efficacy; however, relevant models need to be validated. Although there are animal models for caries, standard in vivo models for remineralization are lacking and are needed in order to study specific conditions such as MIH. Many proteins are required in the complex process of forming a mineralization front and present a practical technical challenge.  Nevertheless, these proteins should be studied as a group, rather than in isolation, in order to fully understand mechanisms of enamel formation and maturation. Depending upon the goals of the study and the system involved, the models will be different. For example, in contrast to a study on caries, studies of tooth erosion do not necessarily need to include a biofilm since the loss of mineral is not due to the action of bacteria. For studies of biofilms, a simple in situ model reproducing characteristics of enamel, is needed. These may facilitate development of compatible techniques to detect active and arrested caries lesions. 

The need for a real-time in vivo dynamic visual tool to follow de- and remineralization was stressed. In order to analyze candidate remineralization compounds, nondestructive analytical tools are needed to definitively determine what phase of precipitate is formed on the tooth. Essential for any longitudinal study, this information would inform how to stabilize ACP in the mouth given that repair times may be lengthy. If this analysis can be performed with a reduced complexity of instruments and steps, combined with automation of image analysis, it would greatly increase utility in research as well as clinical use. 

In addition to optical tools to determine chemistries in newly-formed or remineralized enamel, other advanced tools to analyze material properties are available. Some of these techniques will require wet enamel samples and large surface areas or volumes and could be used to determine clinically important outcomes of toughness, hardness, and wear resistance. Judicious pairing of an instrument with a material property of interest could prove useful in the study of enamel. For example, it was suggested that changes in mechanical properties can potentially be analyzed by ultrasound as a diagnostic imaging modality of hard dental tissues. Following material properties over time will be useful for assessing progress and extent of remineralized regions.  

Session 3: Regulatory and Commercialization Perspectives
The workshop closed with a session on Regulatory and Commercialization Perspectives. Several gaps need to be filled before any drug or device can be approved by the Food and Drug Administration. Drug approval would require evidence of efficacy with validated endpoints that provide meaningful clinical improvement. Key questions concern the validity of remineralization as a therapeutic endpoint, reliability of models to provide high sensitivity and specificity for prediction of caries prevention, and quantification of the magnitude of the remineralization outcome for anticaries therapy. There is also considerable interest in other outcomes besides decayed, missing, filled surface/teeth (DMFS/T), as well as the need for high quality clinical evidence. Although the International Organization for Standardization Technical Committee 106 standards for dentistry are available, they do not address clinical trials.

Trends in the literature indicate there is movement toward minimally invasive or “interventional” dentistry. Several methods are available for caries detection including indicator dyes, visual examination without use of a dental explorer, fluorescence and transillumination imaging, optical coherence tomography, Raman spectroscopy, and radiography. Micro computed tomography is a more recently developed non-destructive method that has the potential to be more widely applied for in vitro and in situ studies. Transverse Microradiography has many advantages for use in vitro and in situ for measuring de- and remineralization and can give information on density, mineral loss and gain, and spatial information for which there is a great need. However, simple quantitative nondestructive testing procedures are still needed that can detect and confirm early caries. 

Market research data are also important determinants in guiding product development. For example, with the growing number of patients reporting dentin hypersensitivity, use of a calcium phosphate prophy paste reduced this hypersensitivity; these benefits are supported by two clinical studies. Future areas of focus by industry likely will include products which claim an anticaries effect or white spot lesion repair, and the quantification of the extent of remineralization. 

Discussion

The last session’s discussion repeatedly touched upon surrogate models. Currently, the well-established index of DMFT is a key measure of caries severity but there is a need for other methods to assess outcome; examples could be measurements of pH level and biofilm activity, or image analysis of the caries site. These endpoints should have a clinically accepted detection method to recognize and confirm early caries.  Furthermore, surrogate models need to be defined and validated, as they can reduce the burden and cost of clinical trials. Whether it is a drug, device, or combination product, utilization by the provider and compliance by the patient ultimately are the drivers of the business model. 

At the end of the workshop, input from participants was gathered for short- to long-term goals ranging from simple to complex challenges in the field. 

Research Recommendations: Short-term
Low complexity short-term goals include:

• understanding clinical challenges to developing new remineralization materials/technologies,
• developing in vivo models for remineralization,
• studying fluoride-calcium interactions, and 
• studying the mechanism of the mineralization process. 

Medium complexity goals that could be met in the short-term include:

• developing tools for assessing active versus arrested lesions, 
• conducting hypomineralization research, 
• finding remineralization surrogates, 
• discovering new caries detection/diagnostic techniques,
• developing root caries remineralization techniques, and 
• engaging stakeholders in public-private partnerships. 

High complexity goals that are within reach in the short-term include a) completing the determination of physical and mechanical properties of enamel, and b) determining how to enhance the effects of fluoride, inorganic agents, and antibiofilm technologies. 

Research Recommendations: Long-term
Simple goals thought to be achievable beyond 10 years were a) determining caries susceptibility and b) investigating the composition and concentration of inorganic compounds such as carbonates in enamel. 

Many medium complexity goals were identified including:

• precisely identifying targets and measurement of caries activities, 
• determining composition of low solubility precipitates with fluoride, 
• assessing remineralization using tools such as Raman spectroscopy, etc., 
• identifying proteins to promote remineralization, 
• studying dentin de- and remineralization, including root caries studies, and 
• classifying clinical targets for caries. 

Highly complex goals include:

• studying occlusal non-cavitated lesions and their remineralization, 
• growing enamel from 3-D scaffolds,  both in vivo and in vitro,
• advancing chemistry and material science of these scaffolds and materials, 
• validating bioequivalence models and in vivo models, 
• determining clinical outcomes for early caries detection and interproximal caries, and 
• standardizing caries detection protocols.  

Very complex long-term goals include: 

• conducting clinical studies and comparative effectiveness studies,
• disseminating medical management strategies,
• educating clinicians on new and available strategies, 
• reconstructing enamel to include the intricate naturally occurring rod and inter-rod geometries, and 
• addressing the biofilm in remineralization and demineralization. 

Follow-up Conference Call 
Following the workshop, an active online chat discussion about clinical needs continued which prompted a follow-up conference call that was held on May 8, 2015. Ten workshop participants discussed the following topics: challenges in biomineralization (in situ/ex vivo, utilizing ameloblast cells as an approach, effects of a biofilm barrier on biomineralization), etiology of caries (caries susceptibility based on heterogeneity of teeth, role of the biofilm, different types of lesions), role of saliva in remineralization, and extension to root caries. Many acknowledged that saliva and biofilm are the major factors affecting disease progression and outcome.

There was agreement that a biomineralization approach to large lesions would involve prefabrication of enamel ex vivo prior to adhesion to the existing tooth surface. Although work on pluripotent stem cell-derived ameloblasts is underway, a synthetic approach to producing enamel ex vivo was viewed as more feasible. Standard computer-aided design and manufacturing (CAD/CAM) would provide customization while appropriate bioresorbable materials would allow for a continuous interface joining the material to the endogenous enamel. In order to form and remain intact, these adhesives would need to disrupt or accommodate a heterogeneous biofilm which would be impossible to completely remove from the site. Consequently, it was also felt that there is a need to study the plaque itself, as the composition and pH of the plaque will strongly influence the success of biomineralization therapy.

Another challenge in building enamel is restarting growth of enamel crystal rods which have already terminated their elongation. The most biological solution to achieving properly extended and organized enamel rods is through guided apatite mineralization. However, this regenerative medicine approach with cells in situ may not be possible in the oral cavity. Additionally, it was pointed out that saliva composition, flow rate, and individual risk factors together contribute to varying caries susceptibility within populations and need to be considered in a precision medicine approach. Studying fluoride and calcium phosphate interactions under physiologically relevant conditions can advance the mechanistic understanding of these processes.

Lastly, there was further expansion of the discussion on clinical needs. This included discussions of the measures for caries risk assessment, disease models, caries biomarkers for host susceptibility or pathogen virulence, and additional potential remineralization agents that can be used as safely and effectively as fluoride. The next generation of remineralization agents will require additional clinical studies to optimize ratios, solubility, and kinetics, as well as comparative effectiveness studies to establish clinical benefits. 

Summary of the Workshop
The panel reached a consensus that fluoride and related sealants have been highly effective in slowing, or in certain instances preventing, tooth decay. Many clinical conditions can benefit from remineralization therapies including white spots and early non-cavitated caries lesions in high-risk individuals and adolescents with coronal and interproximal caries, as well as root caries in the geriatric population. Additional locations and conditions that can be targeted include pits and fissures, dental erosion and specific diseases such as MIH and xerostomia. 

Current state of the art technologies can be adapted to reproduce the highly complex ultrastructural organization of enamel. Remineralization agents with or without fluoride can be combined with proteins or peptides essential for amelogenesis to create a biomimetic approach for reforming enamel. Newer advanced techniques to improve ion penetration and diffusion through both biofilm and enamel should allow for acceleration of repair time and delivery of higher concentrations of favorable ions. In parallel, existing imaging modalities can be tailored to evaluate and assess remineralized areas with high sensitivity and specificity. Improving these real-time imaging tools is as crucial as identifying risk markers.

The panel agreed that in order to succeed with any drug or device approval, both fluoride and non-fluoride products and tools need to be validated to establish remineralization efficacy. This needs to be accomplished with realistic models that accurately mimic tooth topology and the oral microenvironment, and utilize methods that measure caries activity and remineralization. Real-time in vivo monitoring of the de- and re-mineralization cycle remains a gap that needs to be filled before evaluation of therapies can take place. Success of these therapies will be measured by adoption by individuals at all levels of socioeconomic status and by clear criteria judging clinical efficacy. Medical management reinforced with dissemination of evidence-based information and guidelines can also be improved as some preventive methods are underutilized or have not been adopted. Additionally, partnerships with industry can facilitate development of better materials and enhancement of product qualities attractive to both providers and consumers.

The effort to improve mineralization therapies to preserve existing tooth structure, arrest caries, or even reverse caries damage will require many interdisciplinary teams working together. Clinicians and scientists depend on each other to identify needs and provide solutions. Regulatory and commercial organizations will also be important players in bringing effective therapies for patients to the clinic. With this common goal, early intervention with remineralization strategies should benefit society at many levels.

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Participant List

Elliot Abt, DDS, MS, MSc; Abt & Willis family dentistry, Skokie, IL
Arif A. Baig, PhD; The Procter & Gamble Company, Mason, OH
Christopher N. Bowman, PhD; University of Colorado, Boulder, CO
Daniel C. N. Chan, DMD, MS, DDS; University of Washington, Seattle, WA
Lin-P’ing Choo-Smith, PhD; Composites Innovation Centre, Winnipeg, MB, Canada
Yong-Hee Patricia Chun, DDS, DR MED DENT, MS, PhD; University of Texas Health Science Center at San Antonio, TX
Roger Ellwood BDS, MDS, MSc, PhD, FFPH; Colgate-Palmolive Company, Piscataway, NJ
Margherita R. Fontana, DDS, PhD; University of Michigan, Ann Arbor, MI
Frederick N. Hyman, DDS, MPH; Food and Drug Administration, Silver Spring, MD
Hyun (Michel) Koo, DDS, MS, PhD; University of Pennsylvania, Philadelphia, PA
Chris Longbottom, DDS, PhD; King's College, London, UK
Grayson Marshall Jr, DDS, PhD, MPH; University of California, San Francisco, CA
Jill D. Pasteris, PhD; Washington University, St. Louis, MO
Susan Runner, DDS, MA; Food and Drug Administration, Silver Spring, MD
Gary E. Schumacher, DDS, MS; National Institute of Standards and Technology, Gaithersburg, MD
Wendy J. Shaw, PhD; Pacific Northwest National Laboratory, Richland, WA
Tom Simonton, MS; Dentsply International Professional Division, York, PA
Stacey K. Van Scoyoc, DDS; Bloomington Family Dental, Ltd, Bloomington, IL
​​​​​​​Domenick T. Zero, DDS, MS; Indiana University School of Dentistry, Indianapolis, IN