Imaging in vivosecondary caries and ex vivodental biofilms using cross-polarization optical coherence tomography

Abstract

Objective

Conventional diagnostic methods frequently detect only late stage enamel demineralization under composite resin restorations. The objective of this study is to examine the subsurface tooth–composite interface and to assess for the presence of secondary caries in pediatric patients using a novel Optical Coherence Tomography System with an intraoral probe.

Methods

A newly designed intraoral cross polarization swept source optical coherence tomography (CP-OCT) imaging system was used to examine the integrity of the enamel–composite interfaces in vivo. Twenty-two pediatric subjects were recruited with either recently placed or long standing composite restorations in their primary teeth. To better understand how bacterial biofilms cause demineralization at the interface, we also used the intraoral CP-OCT system to assess ex vivo bacterial biofilm growth on dental composites.

Results

As a positive control, cavitated secondary carious interfaces showed a 18.2 dB increase ( p < 0.001), or over 1–2 orders of magnitude higher, scattering than interfaces associated with recently placed composite restorations. Several long standing composite restorations, which appeared clinically sound, had a marked increase in scattering than recently placed restorations. This suggests the ability of CP-OCT to assess interfacial degradation such as early secondary caries prior to cavitation. CP-OCT was also able to image ex vivo biofilms on dental composites and assess their thickness.

Significance

This paper shows that CP-OCT imaging using a beam splitter based design can examine the subsurface interface of dental composites in human subjects. Furthermore, the probe dimensions and acquisition speed of the CP-OCT system allowed for analysis of caries development in children.

Introduction

Acid producing bacteria from a dental biofilm (dental plaque) can demineralize dental enamel on the tooth’s outer surface (incipient caries). Extensive demineralization is surgically removed by a dental drill and a biocompatible material, such as a resin composite, is placed. The outer interface between the dental material and the tooth’s enamel is a site for re-colonization of the dental biofilm and ‘secondary demineralization’ (secondary caries) can often occur. Visual inspection, such as marginal staining, and tactile probing ( e.g. dental explorer) that examines for the presence of surface cavitation, are unreliable in identifying early secondary caries prior to local enamel breakdown .

Optical coherence tomography (OCT) has seen broad applications in medicine and biology and has also been used to image dental hard and soft tissue . Significant ex vivo research efforts have studied the use of OCT to detect early enamel demineralization and secondary caries ; however, more work is needed to determine if in vivo carious lesions can be detected under intraoral conditions by OCT systems. OCT is a non-destructive imaging system that can utilize near-infrared (NIR) light to produce depth resolved images in dental enamel. NIR illumination, especially near 1310 nm, significantly improves the axial imaging depth over wavelengths in the visible range, since dental enamel has been shown to be nearly transparent to NIR light . Enamel demineralization highly scatters NIR light, and OCT measures this increased backscattering intensity . The advancement of Fourier Domain acquisition methods such as Swept Source OCT , where the depth resolved signal is extracted by measuring the interference spectrum of the tissue signal, has made clinical applications of OCT more feasible. Swept Source OCT (SS-OCT) has increased the acquisition speed, providing near real-time video rate imaging, while improving the overall signal to noise ratio of the acquired images .

Previous studies have shown that Polarization Sensitive (PS)-OCT can detect and quantify surface demineralization by utilizing linearly polarized incident light and measuring the backscattered signal in two orthogonal axes . By using polarization-maintaining (pm) fiber in the system, one detector can measure the signal parallel to the incident polarization axis and a second detector can measure the signal in the perpendicular or cross polarization axis. The high refractive index of enamel ( n = ∼1.63) and dental materials such as resin composite ( n = ∼1.5) causes a significant surface reflection, which can confound the imaging. However, the reflected light maintains the incident polarization state. Therefore, by measuring the signal in the cross polarization axis, the surface reflection signal is reduced significantly depending on the degree of crosstalk within the pm fiber . Since dental enamel is slightly birefringent (Δ n = −0.002) , a high degree of linear polarity of the incident light is maintained during the initial propagation into the subsurface tooth structure. Incipient and secondary demineralization initially affects the subsurface tooth structure before spreading deeper into the dental enamel or along the tooth–material interface. Demineralization causes an irregular arrangement of porosities in enamel with an increase in both the number and size of defects which are filled with water and organic material. An increase in both the irregularity and pore size cause a greater depolarization of the incident light. With demineralization causing higher degree of backscattering and depolarization than healthy tissue, the cross-polarization image provides excellent contrast between demineralized and healthy tooth structure .

In addition to contrasting demineralization with healthy sound enamel, cross polarization OCT imaging has the benefit of identifying demineralized tooth structure next to dental resin composites. OCT imaging of resin composites materials has been shown to be strongly influenced by the index of refraction differences between the resin and the reinforcement material . Fortunately, in dentistry, esthetic qualities are enhanced when composite compositions closely match the resin matrix ( e.g. methacrylate-based or urethane-based, n = 1.40–1.48) with different combinations of glass fillers. For esthetic dental composites that do not possess titanium dioxide, the degree of scattering and depolarization is significantly less than demineralized tooth enamel . This is especially important for clinicians diagnosing demineralization adjacent to these materials (secondary caries).

This study investigates an intraoral cross polarization swept source OCT (CP-OCT) system with a Micro-Electro-Mechanical System ( MEMS ) Scanning Mirror. This system is designed to illuminate the sample with linearly polarized light and isolate the perpendicular axis through a polarizing beam splitter based design. This design isolates and measures only the cross polarization axis signal. This straight forward approach allows the clinician to read and assess a single image in order to diagnosis secondary caries adjacent to resin restorations. While detecting secondary caries is important to a clinician, there is also a growing need to understand how material properties affect the growth of dental biofilms in order to prevent secondary caries. The nature of complex multi-species biofilms at the enamel–tooth interface is close to unknown. In this paper, we also present our initial phase in using CP-OCT to assess the growth of ex vivo oral biofilm microcosms. These multi-species oral biofilm microcosms are derived from sampling dental plaque from pediatric dental subjects with a history of Early Childhood Caries and are at risk of developing secondary caries. By growing these oral biofilm microcosms rather than single species bacteria, we are creating a laboratory model system that better replicates the intraoral environment. The overall plan in using this model system is to understand the interaction between resin composites and oral biofilms and eventually elucidate the unique process that leads to secondary caries.

The aim of this paper is to illustrate that CP-OCT can be used to assess the enamel under the margins of composite restorations in vivo but can also be used to assess the growth of multi-species oral biofilm microcosms on these materials in our developing laboratory model.

Methods

Cross-polarization optical coherence tomography

A custom cross-polarization swept source OCT (CP-OCT) system with an intraoral probe ( Fig. 1 ) was developed (IVS-200-CPM, Santec Co. Komaki, Japan) for a pediatric dental application. The swept source system utilized a high swept rate (30 kHz) continuous wavelength scanning laser centered near 1310 nm with a bandwidth of 104 nm. Interferometric concepts of swept source OCT imaging are described elsewhere . The interferometer component of the system ( Fig. 2 ) was housed in the intraoral probe body. This critical design was required so that the sample and reference arm paths experienced similar vibrations and significantly reduced the motion artifact caused from the device being used freehanded during intraoral imaging. The output beam from the swept source travels in single mode fiber and then was split to a sample and reference arm. In the sample arm, the output signal traveled through a collimator system and traveled through a polarizing beam splitter. The output wave was linearly polarized in the P-polarization state. Light then traveled through a fixed focusing lens ( f = 60) and was reflected onto a two axis tilt Micro-Electro-Mechanical System ( MEMS ) scanning mirror in the body of the probe. The MEMS mirror could collect B-scans (two dimensional images at 20 frames/second) in both an x and y direction. In order to accommodate the narrow spaces of the oral cavity, the linearly polarized output beam is reflected at the probe end to illuminate (∼8 mW) the tissue sample. The backscattered signal from the tissue sample traveled back through the probe and the polarizing beam splitter. At this point, the S-polarization state (cross-polarization of the incident beam) was diverted to recombine with the reference signal. The signals from the sample and reference arms were recombined and measured by balanced detection. The resulting interference pattern signal was recorded in time but can also be plotted in k-space (wavenumber) due to the time encoded wavenumber scanning of the output laser. The Fourier transform of this wavenumber spectrum produced the spatial information along the axial direction of the sample. The free space axial resolution for the source was experimentally measured to be 11 μm when a single reflective peak was measured at the −3 dB level. The dimensions of the probe was designed to be used freehanded and for a pediatric dental patient as young as 3 years old. Based on this constraint, the probe was designed with a fixed focal point lens. With the system focus fixed at 2.5 mm from the probe window, we choose a low numeric aperture (NA) design to maximize a Rayleigh range (depth of focus) of approximately 4 mm in order to accommodate imaging the complex tooth morphology. The trade off was an 80 μm lateral resolution (1/ e 2 ) that was confirmed using a digital caliper.

Fig. 1
Intraoral Probe with lateral and inferior views shown. (A) The maximum height of the probe body is 82 mm. (B) The height of the probe tip is 15 mm. (C) The length of the entire probe is 265 mm. (D) The width of the probe tip is 16 mm.

Fig. 2
Cross-Polarization OCT. Housed in the probe casing was a Mach-Zehnder type interferometer that used a polarization beam splitter (PBS) to illuminate a two axis tilt MEMS scanning mirror with linearly polarized light (P). Light from a swept source near infrared laser (HSL) was coupled into single mode fiber and then was split into a reference and sample arm. In the sample arm, a PBS isolated the cross polarization state (S) in the backscattered light. A collimator system (C) was used between the fiber and free space paths. In order to control the polarization states of the light in the reference and sample arms and produce an optimum interference pattern, polarization controllers (PC) were used. The interference signal was measured by two balanced detectors. The resulting electrical signal was then digitized by a high speed data acquisition board (DAQ) and image processed for reconstruction of the spatial information in the tooth sample.

Polarization suppression measurements

Parallel polarization suppression ratio of the CP-OCT system was measured by comparing two signal intensities with and without a quarter wave plate. An achromatic quarter wave plate was used (AHWP05M-1600, Thorlabs, Newton, NJ) with the fast axis aligned 45° to the linearly polarized incident beam. With a quarter wave plate (QWP) placed between the probe and a mirror, the illuminating linearly polarized light becomes circular polarized light and incident on the mirror. After reflecting from the mirror, the circular polarized light returns through the QWP where the light becomes orthogonally polarized (perpendicular axis or S-polarized) linear light. This S-polarized light is then reflected at the PBS and directed to recombine with the reference signal. By using the QWP, the total back reflected intensity was measured. Without a quarter wave plate (normal design) , unperturbed parallel polarized light was reflected back by the mirror to the incident path. In an ideal system, all parallel axis reflected light would not be directed to recombine with the reference arm. We measured the residual reflected light in the CP-OCT system that was detected by the balanced detectors.

Human subjects and image analysis

Serial CP-OCT images of anterior and posterior composite resin restorations (fillings) in primary teeth were obtained from pediatric subjects. Pediatric subjects ( n = 22) from an ongoing study examining oral biofilms associated with secondary caries are presented. One tooth was imaged per subject. Children were recruited from various clinics in the Minneapolis/Saint Paul metropolitan area and represented an ethnically diverse population. Particular efforts were made to recruit children with recently placed composite restorations in addition to those children who had restorations placed over 6 months prior to the study visit. Prior to enrollment, informed consent was obtained and human subject protection followed protocols approved by the Institutional Review Board at the University of Minnesota and by the National Institute for Health. The mean age of these patients was 8.5 years old. Only slight air drying (<3 s) or cotton roll drying was performed, prior to CP-OCT imaging, to remove any debris or excess pooling of saliva. The teeth were moist during imaging with no other isolation method utilized. Surface moisture and tissue hydration maintains the optical translucency of enamel ; therefore, enamel hydration improves the depth of CP-OCT imaging over excessive drying. A clinical disposable polyvinyl barrier covered the intraoral probe. To be included in the study, four through twelve year old children had to have one primary tooth restored with an anterior or posterior resin composite. The exclusion criteria included children with significant medical conditions, chronic medication use leading to xerostomia, recent antibiotic use, and congenital tooth anomalies. Each of the children enrolled had a chronic history of early dental decay and considered at risk of developing secondary caries under their restorations. A dental explorer was used to assess the enamel adjacent to the composite restorations (Class II or III). By gently probing the adjacent enamel, the presence (tactile positive) or absence (tactile negative) of localized enamel cavitation was assessed for each restoration. The presence of tactile cavitation (tactile positive) was chosen as a definitive positive control since many visual signs ( e.g. marginal staining) have been shown to be unreliable in determining the presence of secondary caries .

Raw CP-OCT images were image processed by a median filter to reduce the speckle noise inherent in OCT imaging. In addition, a small artifact produced by the internal reflections within the probe body was removed from a few images using an Exemplar based inpainting method . This method was programmed in Matlab™ and Python™. For image analysis, the mean backscattered intensity of the subsurface enamel below the composite restoration up to 500 μm below the cavosurface margin was assessed using Matlab™. This was similar to the method employed previously for ex vivo evaluation . The interface between the composite restoration and enamel was clearly demarcated due to the fundamental differences in the optical properties of the composite material and the underlying birefringent enamel .

Ex vivo oral biofilm microcosms

In order to initially understand how intraoral bacteria colonize composite–tooth interfaces, we developed an ex vivo model to assess the attachment and growth of dental biofilms. The ex vivo oral biofilm model system was based on the CDC Biofilm Reactor (Biosurface Technologies Corporation, Bozeman, Montana). This biofilm reactor type allowed growth media (BMM, basal mucin medium) to flow (∼6.5 ml/min) through a vessel container that was stirred (125 rpm). Within the vessel container are circular coupons ( e.g. resin composite discs, Filtek™ LS, 3 M and hydroxyapatite, Clarkson Chromatography) mounted on rods. These discs acted as colonization sites for bacterial biofilms. In our design, these discs were disinfected by 70% ethanol and then are pre-coated with sterilized (0.2 μm filter) diluted (Gibbon’s buffer 1:1) saliva from pediatric subjects with a history of Early Childhood Caries. Next, fresh dental plaque (inoculums) sampled from tooth surfaces of matched subjects were added to the vessel. The inoculums within the vessel are initially incubated overnight (@37 °C) in 350 ml of BMM. After incubation, media flow began and within a few hours the inoculums grew (@37 °C) as a multispecies biofilm within the vessel and colonized the discs. Biofilms were allowed to grow on the hydroxyapatite and composite material for up to 2 days. We have confirmed, through DNA microarray analysis (HOMIM, Forsyth Institute) , that these colonizing multispecies biofilms preserve ∼60% of the bacterial species in the original dental plaque sample. This confirmed that our approach more closely models the intraoral biofilm interaction with different surfaces than single species biofilms. CP-OCT imaging was done within 30 min after removal from the vessel with no histological preparation. Discs were also imaged using scanning electron microscopy (Hitachi TM-300) after the biofilms were fixed and stained with Alcian blue and osmium tetroxide, critical point dried, and coated with palladium alloy.

Methods

Cross-polarization optical coherence tomography

A custom cross-polarization swept source OCT (CP-OCT) system with an intraoral probe ( Fig. 1 ) was developed (IVS-200-CPM, Santec Co. Komaki, Japan) for a pediatric dental application. The swept source system utilized a high swept rate (30 kHz) continuous wavelength scanning laser centered near 1310 nm with a bandwidth of 104 nm. Interferometric concepts of swept source OCT imaging are described elsewhere . The interferometer component of the system ( Fig. 2 ) was housed in the intraoral probe body. This critical design was required so that the sample and reference arm paths experienced similar vibrations and significantly reduced the motion artifact caused from the device being used freehanded during intraoral imaging. The output beam from the swept source travels in single mode fiber and then was split to a sample and reference arm. In the sample arm, the output signal traveled through a collimator system and traveled through a polarizing beam splitter. The output wave was linearly polarized in the P-polarization state. Light then traveled through a fixed focusing lens ( f = 60) and was reflected onto a two axis tilt Micro-Electro-Mechanical System ( MEMS ) scanning mirror in the body of the probe. The MEMS mirror could collect B-scans (two dimensional images at 20 frames/second) in both an x and y direction. In order to accommodate the narrow spaces of the oral cavity, the linearly polarized output beam is reflected at the probe end to illuminate (∼8 mW) the tissue sample. The backscattered signal from the tissue sample traveled back through the probe and the polarizing beam splitter. At this point, the S-polarization state (cross-polarization of the incident beam) was diverted to recombine with the reference signal. The signals from the sample and reference arms were recombined and measured by balanced detection. The resulting interference pattern signal was recorded in time but can also be plotted in k-space (wavenumber) due to the time encoded wavenumber scanning of the output laser. The Fourier transform of this wavenumber spectrum produced the spatial information along the axial direction of the sample. The free space axial resolution for the source was experimentally measured to be 11 μm when a single reflective peak was measured at the −3 dB level. The dimensions of the probe was designed to be used freehanded and for a pediatric dental patient as young as 3 years old. Based on this constraint, the probe was designed with a fixed focal point lens. With the system focus fixed at 2.5 mm from the probe window, we choose a low numeric aperture (NA) design to maximize a Rayleigh range (depth of focus) of approximately 4 mm in order to accommodate imaging the complex tooth morphology. The trade off was an 80 μm lateral resolution (1/ e 2 ) that was confirmed using a digital caliper.

Nov 28, 2017 | Posted by in Dental Materials | Comments Off on Imaging in vivosecondary caries and ex vivodental biofilms using cross-polarization optical coherence tomography
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