Research

CLINICAL ASSESSMENT OF TOOTH DEMINERALIZATION & REMINERALIZATION WITH PS-OCT

(Daniel Fried, PI)

The overall objective of this proposed research is to develop polarization sensitive optical coherence tomography (PS-OCT) as a non-invasive optical device for the accurate and early clinical assessment of tooth demineralization and remineralization. The central hypothesis underlying this proposal is that the depth-resolved changes in the magnitude of light scattering of dental hard tissues at 1310-nm, that occur upon demineralization and remineralization, can be imaged and quantified in vivo using PS-OCT and that those changes can be correlated with the structural changes and relative mineral loss measured using polarized-light microscopy and microradiography. The overall objectives of this proposal will be achieved through the following specific aims: (1) To test the hypothesis that the lesion volume and reflectivity from that volume can be automatically assessed in simulated in vitro caries models and natural lesions on extracted teeth using a high speed PS-OCT scanning system; (2) To test the hypothesis that PS-OCT can be used to longitudinally monitor demineralization and the inhibition of demineralization by fluoride in an extended clinical study; (3) To test the hypothesis that PS-OCT can be used to detect changes in the structure and severity of existing lesions after exposure to a remineralization agent in vivo. These studies will further validate PS-OCT as a clinical tool for the accurate and early clinical assessment of tooth demineralization and remineralization. This is expected to lead to the clinical implementation of PS-OCT for the assessment and monitoring of the activity and severity of early carious lesions without the use of ionizing radiation, leading to an increased efficiency of caries clinical trials and a marked reduction in their scope and cost.


EARLY CARIES DETECTION WITH NEAR-IR LIGHT

(Daniel Fried, PI)

The overall objective of this proposed research is to develop near-infrared imaging for the detection and diagnosis of early dental caries (dental decay). New, more sophisticated diagnostic tools are needed for the detection and characterization of caries lesions in the early stages of development. If carious lesions are detected early enough, before cavitation, then they can be arrested/remineralized by non-surgical means through fluoride therapy, anti-bacterial therapy, dietary changes, or by low intensity laser irradiation. The principal factor limiting optical transmission through the tooth i the visible range from 400-700-nm is light scattering in sound enamel and dentin. The magnitude of light scattering decreases markedly at longer wavelengths in the near infrared due to the size of the principal light scatterers in enamel. The central hypothesis of this proposal is that the near-IR (NIR) region between 1300 and 1700-nm offers the greatest potential for new optical imaging modalities due to the weak scattering and absorption in sound dental hard tissue. The overall objectives of this proposal will be achieved through the following specific aims: (1) To test the hypothesis that the maximum contrast between sound and demineralized enamel in NIR reflectance measurements lies in the wavelength regions coincident with weak light scattering in enamel and strong water absorption, (2) To test the hypothesis that NIR imaging on proximal and occlusal surfaces can used for improved assessment of caries lesions for clinical screening, (3) To test the hypothesis that a NIR imaging system can be used for improved discrimination of composite sealants and restorations from sound and demineralized tooth structure. It is likely that if these studies and future clinical trials are a success, that his novel technology for imaging dental hard tissue will be employed for the detection and monitoring of early carious lesions without the use of ionizing radiation, thereby enabling conservative non-surgical intervention and the preservation of healthy tissue structure.


SELECTIVE ABLATION OF DENTAL CARIES AND COMPOSITES 

(Daniel Fried, PI)

The overall objective of this proposed research is to increase our fundamental understanding of the interaction of high intensity laser radiation with dental hard tissues and apply that knowledge to the development of laser based methods that selectively remove dental caries and composite restorative materials using an integrated approach of computer-controlled laser beam scanning coupled with optical methods of feedback. In addition to eliminating the noise and vibration associated with the dental hand-piece, lasers offer several unique advantages over current surgical technology for conservative dentistry. Under the appropriate irradiation conditions, lasers are capable of achieving markedly higher precision and selectivity, are well-suited for electronic control, can induce beneficial chemical and morphological changes in the walls of the drilled cavity that can render the tissues more resistant to further dental decay with enhanced adhesive properties to restorative materials. The specific aims of this proposal are to test the following hypotheses: 1) that caries lesions can be safely and selectively removed from tooth surfaces at clinically relevant rates using image guided laser ablation, and 2) that composite can be safely and selectively removed from tooth surfaces at clinically relevant rates using laser ablation in conjunction with spectral feedback. These fundamental studies will significantly advance our overall knowledge of laser-tissue interactions and facilitate the development of safer and more efficient laser systems for the removal of dental caries and restorative materials. The use of highly selective laser systems for the removal of dental caries and dental composites is likely to lead to the practice of more conservative dental procedures that will reduce the amount of healthy tissue loss that is generally associated with conventional cavity preparations and the repair of existing composite restorations.


A NEW CONCEPT OF AMELOGENIN-GUIDED MINERALIZATION IN ENAMEL

(S Habelitz, PI)

Enamel, the hardest and most mineralized tissue in the human body, is comprised of a unique organization of apatite nanofibers of only 50 nm width but several micrometers to millimeters in length. Its structure is the result of a protein-guided uniaxial growth process of apatite crystals along their c-axes in a three-dimensional organic framework that hydrolyzes in coordination with advancing mineralization to transform into a tissue almost entirely comprised of mineral. While the role of self-asembly of enamel matrix proteins, in particular amelogenin, has widely been recognized as a crucial factor in controlling structure development of enamel, the current model based on the formation of amelogenin nanospheres has significant limitations with regards to the ability of a spherical structure guidin the anisotropic growth of initially ribbon-like apatite crystals and their transformation into a compact mineralized structure. Amelogenin is a hydrophobic protein which comprises about 90% of the enamel matrix proteins. Recently we discovered that the recombinant human full-length amelogenin protein (rH174) forms ribbons of 17 nm width, which grow over a period of days to several micrometer in length. Such ribbons have the ability to self-align and to form bundles which resemble the appearance of aligned apatite crystallites in an enamel rod. Ribbon formation requires the presence of both calcium and phosphate ions suggesting that ion bridges develop and drive the self-assembly process. Synthesis of such ribbons was kinetically enhanced in a water-oil emuslion system but ribbons were also generated in an oil-free environment and commonly formed within 3 to 5 days of incubation in calcium phosphate solutions. While nanoribbons of rH174 contain calcium and phosphate, they do not directly promote apatite crystllization, but instead appear to stabilize an amorphous mineral. Oriented apatite formed however on nanoribbons made from an amelogenin cleavage product, rH146, indicating that the processing of the full- length protein might induce a transformation from amorphous to crystalline apatite. This proposal is based on the hypothesis that amelogenin nanoribbons are the biologically relevant supramolecular structures in developing enamel and hydrolysis of nanoribbons is required to enable oriented calcium phosphate mineralization. This hypothesis will be tested through the following two specific aims: 1. To induce oriented calcium phosphate crystal growth on amelogenin nanoribbons in-vitro; 2. To demonstrate that amelogenin nanoribbons, as observed in-vitro, are the predominant supramolecular structure of developing enamel in-vivo.


MIMICKING THE DENTIN-PULP COMPLEX IN-VITRO

(S Habelitz, PI)

This projects attempts to create the dentin-pulp complex in-vitro using microfabrication and dental pulp stem cells (DPSC). The dentin pulp interface is characterized by a lining of specialized cells of unique morphology that produce the collagenous dentin matrix and control its mineralization. Highly polarized odontoblasts with diameters of 5 to 10 ¿m are tightly packed along the pulpal wall. Their unique feature is the odontoblastic process that extends up to 10 mm deep along tubules into the mineralized dentin. The cells are anchored in this position for the lifetime of the tooth. Hence these cells can live fr decades and only respond to external insult by producing additional dentin matrix (reparative dentin). The surrounding dentin pulp contains progenitor cells that can differentiate into odontoblast cells and produce dentin matrix or form a dentin crown when implanted with epithelial or ameloblast-like stem cells into an immunocompromised mouse. This application proposes to reconstitute this highly organized arrangement of cells in-vitro using tissue engineering approaches. In collaboration with the bioengineering laboratory of Dr. Tejal Desai and the microfabrication facilities at the University of California at Berkeley, micropatterned polymer scaffolds will be generated that permit the positioning of cells in precise locations. In our design these locations will be aligned with an open cylindrical pore allowing for one cell being positioned on top of each pore. Chemotactic approaches will be used to encourage protrusion of odontoblastic processes into the pores resulting in a cellular configuration of polarized cells that is comparable to the dentin-pulp interface. These studies will produce a unique construct to investigate gene expression and cellular interactions in-vitro and improve our understanding of tooth development. Furthermore such pre-assembled mimicries of dental tissue will advance current approaches towards tooth regeneration and pulp revitalization.

 


FUNCTIONAL INTERFACES, ATTACHMENT SITES AND ORGAN BIOMECHANICS IN VERTEBRATES

(Sunita P. Ho,PI)   

Mechanical loads manifest into strains within tissues and interfaces of an organ. Strains within tissues are transduced by the cells to produce the needed extracellular matrix proteins to meet functional demands. This is the general philosophy of research in my laboratory which is within the Division of Biomaterials and Bioengineering. Our lab has a strong focus on mechanics, materials, and investigating adaptation of tissues/interfaces through spatiotemporal mapping of “mechano-responsiveness”. This is done by correlating mechanical strain induced biochemical signals at soft-hard tissue interfaces using several model systems including the bone-ligament-tooth fibrous joint.

Due to the interdisciplinary nature of research, my laboratory is extended to the Molecular Foundry of Lawrence Berkeley National Laboratory, and Stanford Synchrotron Radiation Lightsource (SSRL) at Stanford Linear Accelerator Center (SLAC) with the help of NIH and DOE funded/peer-reviewed proposals. Mapping of biochemical expressions, physical properties of load bearing tissues, and biomechanics of organs are performed at UCSF, and as a guest scientist at the national laboratories/facilities.


DENTIN STRUCTURE, DEMINERALIZATION AND REMINERALIZATION

(Grayson Marshall and S Habelitz, Co-PIs)

Current trends in conservative and minimally invasive dentistry (MID) emphasize the reversal and repair of the active caries process as a first step to restoring the diseased tissue. Enamel remineralization is an accepted phenomenon with established mechanisms, but dentin remineralization strategies are at an early stage of development. Results from the prior period have shown substantial recovery of the hydrated carious tissues mechanical properties, which we have termed "functional remineralization." If functional remineralization can be clinically achieved, it would become a key strategy in MID with the eventual outcome of improved oral health care and lower costs. In support of this goal, , new knowledge on basic biomineralization mechanisms has emerged, and has inspired new approaches that achieve appropriate remineralization within collagen fibrils (intrafibrillar) and between the fibrils (extrafibrillar) to improve the functional remineralization of carious dentin structures. We propose to continue the UCSF-Univ. of Florida collaboration established in the prior period, which will include: Aim 1 a) enhancing the polymer-induced liquid-precursor (PILP) process that has successfully mineralized a variety of collagen matrices, and shown significant functional remineralization of artificial caries lesions. b) extending PILP by potential synergisti approaches including other polyanionic polymers and constant composition methods. c) studying the structure of carious dentin zones to determine their role in limitations of the remineralization process. d) defining microstructural variations in normal and functionally remineralized dentin. Aim 2 evaluates dentin collagen mineralization in model systems including mouse models that lack critical non-collagenous proteins to gain insight into mineralization mechanisms of the collagen scaffold. Aim 3 a) applies the improvements from Aims 1 and insights from Aim 2 to applications in two in vitro models of natural human caries that progressively move towards clinical application. Aim 4 establishes that functional remineralization as indicated by AFM-based nanoindentation testing of hydrated tissue also reflects properties at clinically relevant sizes by use of 4-point bending tests combined with Micro X- ray Computed Tomography. To carry out this work we have established a talented team of SF Bay area investigators from UCSF, LBNL and SSRL, as well as our Florida collaborators who developed the PILP process. The proposed studies will build on our progress and translate the emerging understanding of mechanisms in biomineralization so that we can optimize remineralization kinetics and restoration of dentin caries and move toward a clinically relevant delivery system. Establishing methods to functionally remineralize dentin caries thereby restoring the mechanical properties of the hydrated tissue will minimize conventional restorative treatment and maximize conservation of tooth structure. PUBLIC HEALTH RELEVANCE: Dental caries (tooth decay) is the most common infectious disease and untreated disease ranges from about 20% in children 2-5 years to 26% in adults age 20-64 (http://www.cdc.gov/nchs/FASTATS/dental.htm). Although enamel caries may be remineralized in its early stages, once caries has reached the dentin, the tissue that forms the bulk of the tooth, standard conservative treatments require restoration (drill and fill). This projct develops methods to functionally remineralize dentin caries, restoring the mechanical properties of the hydrated tissue and thereby minimizing conventional restorative treatment and maximizing conservation of tooth structure.


 CLINICAL , MECHANICAL AND CHEMICAL EVALUATION OF AMALGAM AND ART RESTORATIONS

(Lilliam Pinzon, PI)

Dental caries (tooth decay) remains a significant national and international health problem, especially in low- income and disadvantaged populations. Results of several clinical trials conducted outside the US (US studies have not been conducted) have suggested that glass ionomer cement (GIC) restorations used in a simplified and economic procedure known as atraumatic restorative treatment (ART) technique may be more successful than conventional dental treatments for large carious lesions with respect to increased restoration longevity and decreased caries recurrence. The reasons for the success of ART have not been clearly established, but it has been suggested that the GIC may interact with the caries affected tissue to restore a portion of its mineral content. Dental caries occurs when bacterial dental plaque combines with consumed, fermentable carbohydrates. This combination produces acids that penetrate and dissolves the tooth surfaces. GIC's are biocompatible materials having low technique sensitivity and release fluoride (F) which creates internal remineralization of a carious lesion. ART is an "atraumatic" technique as no drilling of teeth or anesthesia is required, thus making it advantageous in certain settings and situations. Amalgam is an alloy of mercury with another metal (silver) used traditionally by dentists to fill cavities in teeth. In Aim 1, we plan to conduct a prospective cohort study of initially 5 to 11 year-old children with at least one carious primary molar (1 to 3 surfaces cavitated) that has been restored with ART or amalgam. The children, patients at the Asian Health Services Dental Clinic in Oakland, CA and the CARECEN dental clinic in the primarily Hispanic Mission neighborhood of San Francisco, will receive initial dental assessments including pre-treatment radiographs, and treatment by staff dentists. Both clinics have agreed to participate and use both materials as part of their usual care. Patients that fit the child and tooth-level selection criteria will be recruited. Baseline clinical and radiographic evaluation of the ART and amalgam restorations, and 6-month and 12-month follow-up assessments (without radiographs) will be take place to assess clinical outcomes: a) caries recurrence, b) restoration retention, and c) restoration marginal integrity over time. In aims 2 and 3, analyses of the two restorative materials will be undertaken with restored teeth that naturally exfoliate during the study period. In Aim 2 we will examine the mechanical properties, microstructural characteristics of the restoration/tooth interfaces (Nanoindentation, and SEM-EDS) to identify changes induced in the tissues by the restorative materials and to provide insight to the mechanisms that could induce such changes. In Aim 3 we will evaluate the chemical changes of the restoration/tooth interfaces (RMS) to identify the presence of remineralization caused by the interaction of the dental materials (GIC and amalgam) with the dental tissues (enamel and dentin). A better understanding of the similarities and differences in the clinical, mechanical and chemical properties of these two materials when placed in primary teeth will help guide future recommendations for their use in the US. Public Health Relevance: This clinical translational observational study will allow us to evaluate the effectiveness of two dental restorative materials in two populations of high caries-risk children from lower socio-economic status households, many of whom rely on public assistance. One population is treated at the Asian Health Services Dental Clinic (AHSDC) in Oakland, California and the other at the Central American Resource Center (CARECEN) dental clinic in primarily Hispanic, Mission Neighborhood of San Francisco, California. This research will advance our understanding of the clinical outcomes of atraumatic restorative treatment (ART) vs. traditional amalgam techniques. ART has the advantage of being low cost, relatively easy to apply, especially to young children, and not requiring anesthesia and drilling of teeth. Additionally, the restored teeth will be collected after exfoliation to examine the remineralization process, and mechanical and chemical properties. If the results are favorable for ART with regard to restoration retention, marginal integrity, decreased caries recurrence, remineralization and other interactions with dental tissues, this type of treatment has the potential to impact dental practice, patient management policies, and public health.


EVALUATION OF AMALGAM AND ART RESTORATIONS IN THE POPULATION OF NUQUI, COLOMBIA

(Lilliam Pinzon, PI)

This study is supported by a UCSF School of Dentistry Global Oral Health Pair Fellowship. In that study we are evaluating ART vs. amalgam restorations in families with participants 5 - 83 years in Nuqui, Colombia. Dr. Pinzon is collecting the primary teeth of participants that still have primary teeth and that were treated with ART or amalgam in order to evaluate the remineralization process of those teeth using laboratory instruments that show mechanical changes as well as chemical changes of the different substrates of the teeth.  The aims of this study are: 1. Recruit and enroll 60 participants from 5 to 90 year-old (20 participants 5 – 11 years old; 20 participants 18 – 40 years old, and 20 participants 50 – 90 years old) in Nuqui, Choco, Colombia at the Club Deportivo Talentos del Pacifico, Gobernacion del Choco with at least one carious primary or permanent molar and clinically evaluate single and multiple-surface atraumatic restorative treatment  (ART) and amalgam restorations at baseline, 6-months and 12-month, 18-month and 24-month follow-up assessments using a quality evaluation protocol to assess the clinical outcome of each procedure. A) Perform visual and tactile clinical evaluation of the retention and marginal integrity of the restorations. B) Assess the postoperative presence of recurrent caries of the participants with ART or amalgam. 2. Collect and evaluate the mechanical properties and interfacial microstructure of single- and multiple-surface ART and amalgam restorations in exfoliated primary teeth from children participating in Aim 1, to determine the differences in the tissue characteristics at the tooth/restoration interface. A) Collect naturally exfoliated primary molar teeth. B) Measure the mechanical properties of ART and amalgam restorations using nano-indentation. C) Evaluate microstructural differences at the restoration/tooth surface interface of ART and amalgam restorations under Environmental Scanning Electron Microscopy (SEM).  3.Determine the chemistry of the restoration/tooth interface to evaluate differences of the two different procedures in particular in regards to remineralization. A) Evaluate chemical differences of the tissue. If the results are favorable for ART with regard to restoration retention, marginal integrity, decreased caries recurrence, remineralization and other interactions with dental tissues, this type of treatment has the potential to impact dental practice, patient management policies, and public health.


EVALUATION OF DENTAL RESTORATIONS AT THE BINATIONAL FREE-CLINIC IN TIJUANA, MEXICO

(Lilliam Pinzon, PI)

This study is supported by the UC GloCal Fellowship is a career development fellowship sponsored by the National Institutes of Health (NIH) Fogarty International Center (FIC), as well as a consortium at the UCGHI. This consortium is comprised of UCSF, UCSD, UCLA, USD, 27 affiliated international sites across 16 countries, and institutes and centers at the National Institutes of Health (NIH). With this fellowship Dr. Pinzon is obtaining deeper knowledge and experience working in clinical international studies, and to understand other aspects to which the vulnerable population of Tijuana, Mexico is exposed so that in the future she might be able to initiate further studies. Her mentors in this study are Dr. Greenspan from the University of California and Dr. Zamudio from Universidad Autonoma de Baja California, Mexico The aims of the study are: 1. Recruit and enroll 100 participants (5 to 90 years old) in Tijuana, Mexico, at the Binational Student-Run Free-Clinic with at least one carious primary or permanent molar and clinically evaluate single and multiple-surface atraumatic restorative treatment (ART) and amalgam restorations at baseline, 5-month and 10-month assessments using a quality evaluation protocol to assess the clinical outcomes. A) Perform visual and tactile clinical evaluation of the retention and marginal integrity of the restorations. B) Assess the postoperative presence of recurrent caries of the participants. 2. Collect and evaluate the mechanical properties and interfacial microstructure of single- and multiple-surface ART and amalgam restorations in exfoliated primary teeth from children participating in Aim 1 to determine the differences in the tissue characteristics at the tooth/restoration interface. A) Collect naturally exfoliated primary molar teeth. B) Measure the remineralization process (mechanical properties using nano-indentation, and chemical changes under Environmental Scanning Electron Microscopy, and using Raman microspectroscopy). If the results are favorable for ART with regard to restoration retention, marginal integrity, decreased caries recurrence, remineralization and other interactions with dental tissues, this type of treatment has the potential to impact dental practice, patient management policies, and public health.