This study was supported by a research grant from the Swiss Dental Association (SSO Research Grant 292-16).

The teeth used in this study were part of a project approved by the local Ethics Committee (EKNZ UBE-15/111). The participants provided written informed consent, and all data were de-identified to protect patient confidentiality.
1. Detection of tooth colored composite resin material using FIT
<ol>
	<li>Darken the room (natural and artificial light).</li>
	<li>Wear clear or yellow-tinted safety glasses with UV protection.</li>
	<li>Use a fluorescence-inducing light source to illuminate the tooth substance and tooth-colored composite resin restoration (Figure 1). 
	NOTE: The composite resin material will appear brighter than the tooth substance (Figure 2). Any device with a wavelength of 398 &#177; 5 nm might be used as a fluorescence-inducing light source. Headlamp systems appear to be particularly suitable since the light spot illuminates the entire oral cavity and enables simultaneous tactile examination. Obstructive factors such as saliva and plaque do not interfere with the FIT method; therefore, previous cleaning and repeated drying of the teeth is not necessary.</li>
</ol>
2. Removal of composite resin-bonded trauma splints using FIT
<ol>
	<li>Preoperative scan with an intraoral scanning device and appropriate software for experimental evaluation purposes
	<ol>
		<li>Start the intraoral scanning device and open the software (see the Table of Materials).</li>
		<li>Press Add New Patient to register your patient, fill in the gaps Last Name, First Name, Date of Birth, and Patient ID, and press Add Case.</li>
		<li>Choose Jaw Scan and Impressions under the section Indications. Then, press Next.</li>
		<li>Darken the room (natural and artificial light) and dry the field of operation to ease the scanning procedure.</li>
		<li>Start the intraoral scanner and perform a digital surface scan of the field of operation (Figure 3A).</li>
	</ol>
	</li>
	<li>Visualization of the composite resin material
	<ol>
		<li>Repeat steps 1.1-1.3.</li>
		<li>Remove the composite resin material using the common methods (e.g., a high-speed contra-angle handpiece with diamond burs and polishing devices) (Figure 4). 
		NOTE: Remove the composite resin remnants close to the enamel with a carbide bur designed for debonding.</li>
	</ol>
	</li>
	<li>Postoperative scan for experimental volumetric assessment
	<ol>
		<li>Repeat steps 2.1.1-2.1.5. 
		&#160; 
		&#160;</li>
	</ol>
	</li>
	<li>Experimental volumetric assessment
	<ol>
		<li>Press Export to export the pre- and postoperative scans as surface tessellation language (STL) in highest resolution.</li>
		<li>Open suitable software and press recombine.</li>
		<li>Upload the pre- and postoperative scans to the software by pressing Import.</li>
		<li>Press Arrangement to superimpose the pre- and postoperative scans by the best fit method.</li>
		<li>Press Analysis to visualize the volumetric changes from pre- to postoperative scans. Select the tooth sites where volumetric changes presumably occurred by choosing Region under the section Tools. Analyze volumetric changes by using the linear and volumetric measurement software tools, Distances tool and Volume Analysis tool, respectively.
		<ol>
			<li>Press Distances under the section Tools for linear quantification of tooth substance loss and composite resin remnants in color (e.g., unchanged areas: green, substance loss: blue and violet, excess material: yellow and red, Figure 3B). Use the color bar on the left to quantify linear volumetric changes. Additionally, locate the cursor on the relevant tooth sites; look for the exact cursor distance in the box on the left.</li>
			<li>Press Volume Analysis under the section Tools for volumetric quantification of tooth substance loss and composite resin remnants . Look for the volumetric change in the box on the left.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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All authors declare that they have no conflict of interest.

The conventional illumination (for example by a dental unit lamp) is an unsatisfactory diagnostic tool for the identification of composite resin restorations. For superior diagnostics with conventional illumination, a magnification aid, drying, or even effortful cleaning of the teeth is necessary. Even under ideal circumstances, conventional illumination seems to be insufficient. A study showed that conventional illumination may lead to misdetection of composite resin restorations and sound tooth substance33. The FIT method seems to be superior in many ways. FIT is a diagnostic tool with a high accuracy, reproducibility, and repeatability1,2. Even obstructive factors such as saliva or biofilm do not influence the result of FIT1.
FIT also shows excellent inter- and intraoperator agreement1. Studies have shown FIT to provide satisfying results when used by dentists with different levels of experience1,33. Even dental students showed comparable results with experienced dentists using FIT1,33. However, the visual acuity which shows individual variation is influenced by many factors. In people above the age of 40 years, a physiological loosening of accommodation (presbyopia) occurs34. In studies, younger examiners under 40 years showed a higher sensitivity in detecting a composite resin restoration than a group older than 40 years33.
Any fluorescence-inducing light source is applicable for the FIT method33. Expensive and tedious systems can be avoided, and straightforward and low-cost systems, such as head-lamps, hand-lamps, or a modified micromotor can be favored. Given the availability of a suitable system for the FIT method, FIT is a diagnostic tool with a wide range of applications. FIT can be used for diagnostic purposes and as an additional tool for the removal of composite resin materials in reconstructive dentistry, dental traumatology (trauma splint removal), and orthodontics (bracket debonding)24,25,26,27,28,29,30,31,32. FIT is also advantageous in dental forensics, as several studies have found that more restoration sites can be detected using FIT35,36,37,38,39.
The evaluation of composite remnants and sound tooth substance loss after trauma splint removal using software after overlapping pre- and postoperative scans illustrates the accuracy of the FIT method. Intraoral scanners are suitable for this purpose since they are accurate and reliable28. Minor volumetric changes can be detected with a high level of precision28. Many of the available composite resin materials show fluorescence properties. However, fluorescence luminosity varies significantly according to the manufacturer and the shade of contemporary composite resin materials39.
Notably, some materials fluoresce less or even not enough for the application of the FIT method3,36,37,39. Moreover, the fluorescence signal of the composite resin materials decreases with time6,7,8,9. It might be harder to identify older composite resin restorations with the FIT method. These factors are the main drawback of the FIT method and must be taken into account when applying FIT. In conclusion, FIT is a reliable, fast, and noninvasive approach for the detection of composite resins.

The application of FIT facilitates the distinction of composite resin materials from sound tooth substance compared to conventional illumination, for example, by a dental unit lamp1,2. Fluorescence occurs when a material emits the light at a higher wavelength than it has been absorbed. As a result of this illumination, the material appears brighter than the tooth3. The maximum fluorescence of composite resin materials occurs when illuminated by a wavelength of 398 &#177; 5 nanometers3. Fluorescence in composite resin materials appears due to rare earth oxides added to the glass fillers, some of the main components of composite resins4,5. The addition of these fluorescent substances intends to adapt the optical properties of composite resins to the tooth structure to improve the esthetic properties of composite resins4,5. FIT is applicable on many composite resin materials as they show these fluorescence properties3. However, fluorescence decreases with the ageing of the composite resin materials6,7,8,9.
The distinguishment of composite resin materials from tooth structure with conventional illumination is a challenge since modern composite resin materials match the optical properties of tooth substance almost perfectly10,11. The misdiagnosis of composite resin results in inaccurate dental charts, false caries risk assessment, and inappropriate treatment planning11. Moreover, epidemiological data is falsified12.
Composite resin is the material of choice for direct restorations due to its straightforward handling, esthetic properties, and clinical performance13. Nevertheless, many composite restorations must be renewed due to secondary caries, fractures, or other reasons14,15. However, the removal of residual composite resin materials can be demanding under conventional light conditions. Even with the application of a magnification aid and the use of tactile probes or extensive drying of the teeth, composite residues are sometimes difficult to distinguish from sound tooth structure. Leftovers of composite remnants during the removal of the adhesive restoration lower the quality of further restorations and have an esthetic impairment due to possible discoloration of the margins1,16,17,18,19,20,21,22. On the contrary, an overpreparation due to misdiagnosis of composite resin versus tooth structure may result in unnecessary substance loss1,2.
In dental traumatology, fixation of the injured teeth using trauma splints is frequent and mandatory in many cases23. The trauma splints are usually fixed on the teeth using a flowable composite resin material. Incomplete removal of the composite resin material in this scenario may lead to the impairments described above. Since dental trauma occurs mostly in front teeth, an impairment of the esthetics and sufficient adhesion of further reconstructions are crucial. Therefore, the aim of the article is to demonstrate the application of the FIT method as an efficient and straightforward approach for detecting and removing composite resin materials.

<table><tbody><tr><td>Bonding Resin Remover, H22ALGK 016</td><td>Komet Dental, Lemgo, Germany</td><td></td><td>Any other material/equpiment with the same function/purpose might be used.</td></tr><tr><td>Cerec Omicam, Connect SW 5.1.3</td><td>Dentsply Sirona, York, PA, USA</td><td></td><td>Any other material/equpiment with the same function/purpose might be used.</td></tr><tr><td>Diamant bur</td><td>Intensiv SA, Montagnola, Switzerland</td><td></td><td>Any other material/equpiment with the same function/purpose might be used.</td></tr><tr><td>Mandrell</td><td>3M, Saint Paul, MN, USA</td><td></td><td>Any other material/equpiment with the same function/purpose might be used.</td></tr><tr><td>MASTERmatic</td><td>KaVo Dental GmbH, Biberach, Germany</td><td></td><td>Any other material/equpiment with the same function/purpose might be used.</td></tr><tr><td>Occlubrush</td><td>Kerr, Orange, CA, USA</td><td></td><td>brush polishing system</td></tr><tr><td>OraCheck Software, Version 5.0.0</td><td>Cyfex AG, Zurich, Switzerland</td><td></td><td>Any other material/equpiment with the same function/purpose might be used.</td></tr><tr><td>SIROInspect</td><td>Dentsply Sirona, York, PA, USA</td><td></td><td>Any other material/equpiment with the same function/purpose might be used.</td></tr><tr><td>Sof-Lex</td><td>3M, Saint Paul, MN, USA</td><td></td><td>Contouring/polishing discs; any other material/equpiment with the same function/purpose might be used.</td></tr></tbody></table>

detection and removal of tooth-colored composite resin using the fluorescence-aided identification technique

The detection and removal of tooth-colored filling materials is a major challenge for every dentist. The Fluorescence-aided Identification Technique (FIT) is a noninvasive tool to facilitate the distinction of composite resin material from sound tooth substance. Compared to conventional illumination, FIT is a very accurate, reliable, and fast diagnostic method. When composite resin is illuminated with a wavelength of approximately 398 &plusmn; 5 nm, certain fluorescent components make the composite resin appear brighter than the tooth structure. Any fluorescence-inducing light source with the appropriate wavelength can be used for this method. Optimally, this technique is used without additional natural or artificial lighting. The application of FIT can be used for diagnostic purposes, for example, dental charts, and additionally for the complete and minimally invasive removal of composite resin restorations, bracket debonding, and trauma splint removal. The assessment of volumetric changes after composite removal can be provided by overlapping pre- and postoperative scans and subsequent calculation using suitable software.

Usage of the FIT method makes most composite resin materials appear brighter than sound tooth structure (Figure 2 and Figure 5). Therefore, FIT is applicable not only in the detection of composite resin material, but it also facilitates the removal of composite resin materials in general, and explicitly in posterior teeth, during orthodontic bracket debonding and in trauma splint removal1,2,24,25,26,27,28,29,30,31,32.
Figure 6 shows a tooth model after trauma splint removal under conventional illumination (teeth 13, 12, 11) and with the aid of FIT (teeth 21, 22, 23). Figure 6C shows the quantification of composite remnants and tooth substance loss in the software (unchanged areas: green, substance loss: blue and violet, excess material: yellow and red). The discrepancy of the pre- and postoperative scan measured by the Distance tool revealed composite remnants and substance loss of&#160;&#177;&#160;0.1 mm in teeth 13, 12, and 11. Teeth 21, 22, and 23 showed nearly no changes in the surface (&#177; 0.01 mm) after trauma splint removal. Moreover, the composite remnants are made visible by the FIT method (Figure 6B), whereas they remain undetected under conventional light illumination (Figure 6A).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63656/63656fig01.jpg" /> 
Figure 1: Fluorescence-inducing light source. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63656/63656fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63656/63656fig02.jpg" /> 
Figure 2: Tooth model with several composite resin restorations. (A) Conventional illumination, (B) FIT. Abbreviation: FIT = Fluorescence-aided Identification Technique. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63656/63656fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63656/63656fig03.jpg" /> 
Figure 3: Preoperative scan and visualization of the volumetric changes after trauma splint removal. (A) Preoperative surface scan. (B) Visualization of the volumetric changes from pre- to postoperative scans in color (unchanged areas: green, substance loss: blue and violet, excess material: yellow and red). The color bar on the left enables the quantification of tooth substance loss and composite resin remnants. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63656/63656fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63656/63656fig04.jpg" /> 
Figure 4: Suitable devices for the removal of composite resin during trauma splint removal. Left to right: diamond bur, bonding resin remover, mandrel, contouring and polishing discs, brush polishing system. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63656/63656fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63656/63656fig05.jpg" /> 
Figure 5: Tooth model with trauma splint fixed with composite resin. (A) Conventional illumination, (B) FIT. Abbreviation: FIT = Fluorescence-aided Identification Technique. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63656/63656fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63656/63656fig06.jpg" /> 
Figure 6: Tooth model after trauma splint removal under conventional illumination (teeth 13, 12, 11) and with the aid of FIT (teeth 21, 22, 23). (A) Under conventional light illumination, (B) illuminated by FIT (marked: composite remnants), (C) volumetric assessment (marked: composite remnants and substance loss). Abbreviation: FIT = Fluorescence-aided Identification Technique. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63656/63656fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Detection and Removal of Tooth-Colored Composite Resin Using the Fluorescence-Aided Identification Technique at JoVE.com

Detection and Removal of Tooth-Colored Composite Resin Using the Fluorescence-Aided Identification Technique

The Fluorescence-aided Identification Technique is a practicable, fast, and reliable approach for the differentiation of composite resin restorations from tooth substance, and facilitates the minimally invasive and complete removal of composite resin restorations and composite bonded trauma splints.

The detection and removal of tooth-colored filling materials is a major challenge for every dentist. The Fluorescence-aided Identification Technique (FIT) ...

detection-removal-tooth-colored-composite-resin-using-fluorescence

Nanyang Technological University

Research

JoVE Journal

Medicine

3.4K Views.  University of Basel. Het opsporen en verwijderen van tandkleurig vulmateriaal is een uitdaging. De fluorescentie-ondersteunde identificatietechniek, FIT, is een diagnostisch hulpmiddel dat de differentiatie van composiethars van tandsubstantie vergemakkelijkt. FIT maakt de minimaal invasieve verwijdering van composietharsrestauraties en composietgebonden traumaspalken mogelijk.Het is een eenvoudige, snelle en betrouwbare diagnostische aanpak. Draag om te beginnen een heldere of geel getinte veiligheidsbril...

Video: Detection and Removal of Tooth-Colored Composite Resin Using the Fluorescence-Aided Identification Technique

Qualitative Identification of Carboxylic Acids, Boronic Acids, and Amines Using Cruciform Fluorophores

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with electron-donors, and the other with electron-acceptors, cruciforms&#39; HOMO will localize along the electron-rich and LUMO along the electron-poor axis. This spatial isolation of cruciforms&#39; frontier molecular orbitals (FMOs) is essential to their use as sensors, since analyte binding to the cruciform invariably changes its HOMO-LUMO gap and the associated optical properties. Using this principle, Bunz and Miljani&#263; groups developed 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole cruciforms, respectively, which act as fluorescent sensors for metal ions, carboxylic acids, boronic acids, phenols, amines, and anions. The emission colors observed when these cruciform are mixed with analytes are highly sensitive to the details of analyte&#39;s structure and - because of cruciforms&#39; charge-separated excited states - to the solvent in which emission is observed. Structurally closely related species can be qualitatively distinguished within several analyte classes: (a) carboxylic acids; (b) boronic acids, and (c) metals. Using a hybrid sensing system composed from benzobisoxazole cruciforms and boronic acid additives, we were also able to discern among structurally similar: (d) small organic and inorganic anions, (e) amines, and (f) phenols. The method used for this qualitative distinction is exceedingly simple. Dilute solutions (typically 10-6 M) of cruciforms in several off-the-shelf solvents are placed in UV/Vis vials. Then, analytes of interest are added, either directly as solids or in concentrated solution. Fluorescence changes occur virtually instantaneously and can be recorded through standard digital photography using a semi-professional digital camera in a dark room. With minimal graphic manipulation, representative cut-outs of emission color photographs can be arranged into panels which permit quick naked-eye distinction among analytes. For quantification purposes, Red/Green/Blue values can be extracted from these photographs and the obtained numeric data can be statistically processed.

Cross-conjugated cruciform fluorophores based on 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole nuclei can be used to qualitatively identify diverse Lewis acidic and Lewis basic analytes. This method relies on the differences in emission colors of the cruciforms that are observed upon analyte addition. Structurally closely related species can be distinguished from each other.

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with ...

Chemistry

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and composition 1, 2. In general, biofilms develop from initial microbial attachment on a surface followed by formation of cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which occur in a complex extracellular matrix. The majority of biofilm matrices harbor exopolysaccharides (EPS), and dental biofilms are no exception; especially those associated with caries disease, which are mostly mediated by mutans streptococci 3. The EPS are synthesized by microorganisms (S. mutans, a key contributor) by means of extracellular enzymes, such as glucosyltransferases using sucrose primarily as substrate 3.
Studies of biofilms formed on tooth surfaces are particularly challenging owing to their constant exposure to environmental challenges associated with complex diet-host-microbial interactions occurring in the oral cavity. Better understanding of the dynamic changes of the structural organization and composition of the matrix, physiology and transcriptome/proteome profile of biofilm-cells in response to these complex interactions would further advance the current knowledge of how oral biofilms modulate pathogenicity. Therefore, we have developed an analytical tool-box to facilitate biofilm analysis at structural, biochemical and molecular levels by combining commonly available and novel techniques with custom-made software for data analysis. Standard analytical (colorimetric assays, RT-qPCR and microarrays) and novel fluorescence techniques (for simultaneous labeling of bacteria and EPS) were integrated with specific software for data analysis to address the complex nature of oral biofilm research.
The tool-box is comprised of 4 distinct but interconnected steps (Figure 1): 1) Bioassays, 2) Raw Data Input, 3) Data Processing, and 4) Data Analysis. We used our in vitro biofilm model and specific experimental conditions to demonstrate the usefulness and flexibility of the tool-box. The biofilm model is simple, reproducible and multiple replicates of a single experiment can be done simultaneously 4, 5. Moreover, it allows temporal evaluation, inclusion of various microbial species 5 and assessment of the effects of distinct experimental conditions (e.g. treatments 6; comparison of knockout mutants vs. parental strain 5; carbohydrates availability 7). Here, we describe two specific components of the tool-box, including (i) new software for microarray data mining/organization (MDV) and fluorescence imaging analysis (DUOSTAT), and (ii) in situ EPS-labeling. We also provide an experimental case showing how the tool-box can assist with biofilms analysis, data organization, integration and interpretation.

Biofilms formed on tooth surfaces are highly complex and exposed to constant innate and exogenous environmental challenges, which modulate their architecture, physiology and transcriptome. We developed a toolbox to examine the composition, structural organization and gene expression of oral biofilms, which can be adapted to other areas of biofilm research.

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and ...

Immunology and Infection

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

Advanced Compositional Analysis of Nanoparticle-polymer Composites Using Direct Fluorescence Imaging

The fabrication of polymer-nanoparticle composites is extremely important in the development of many functional materials. Identifying the precise composition of these materials is essential, especially in the design of surface catalysts, where the surface concentration of the active component determines the activity of the material. Antimicrobial materials which utilize nanoparticles are a particular focus of this technology. Recently swell encapsulation has emerged as a technique for inserting antimicrobial nanoparticles into a host polymer matrix. Swell encapsulation provides the advantage of localizing the incorporation to the external surfaces of materials, which act as the active sites of these materials. However, quantification of this nanoparticle uptake is challenging. Previous studies explore the link between antimicrobial activity and surface concentration of the active component, but this is not directly visualized. Here we show a reliable method to monitor the incorporation of nanoparticles into a polymer host matrix via swell encapsulation. We show that the surface concentration of CdSe/ZnS nanoparticles can be accurately visualized through cross-sectional fluorescence imaging. Using this method, we can quantify the uptake of nanoparticles via swell encapsulation and measure the surface concentration of encapsulated particles, which is key in optimizing the activity of functional materials.

Here we present a reliable method to monitor the incorporation of nanoparticles into a polymer host matrix via swell encapsulation. We show that the surface concentration of cadmium selenide quantum dots can be accurately visualized through cross-sectional fluorescence imaging.

The fabrication of polymer-nanoparticle composites is extremely important in the development of many functional materials. Identifying the precise ...

Engineering

Shrinkage of Dental Composite in Simulated Cavity Measured with Digital Image Correlation

Polymerization shrinkage of dental resin composites can lead to restoration debonding or cracked tooth tissues in composite-restored teeth. In order to understand where and how shrinkage strain and stress develop in such restored teeth, Digital Image Correlation (DIC) was used to provide a comprehensive view of the displacement and strain distributions within model restorations that had undergone polymerization shrinkage.
Specimens with model cavities were made of cylindrical glass rods with both diameter and length being 10 mm. The dimensions of the mesial-occlusal-distal (MOD) cavity prepared in each specimen measured 3 mm and 2 mm in width and depth, respectively. After filling the cavity with resin composite, the surface under observation was sprayed with first a thin layer of white paint and then fine black charcoal powder to create high-contrast speckles. Pictures of that surface were then taken before curing and 5 min after. Finally, the two pictures were correlated using DIC software to calculate the displacement and strain distributions.
The resin composite shrunk vertically towards the bottom of the cavity, with the top center portion of the restoration having the largest downward displacement. At the same time, it shrunk horizontally towards its vertical midline. Shrinkage of the composite stretched the material in the vicinity of the &#8220;tooth-restoration&#8221; interface, resulting in cuspal deflections and high tensile strains around the restoration. Material close to the cavity walls or floor had direct strains mostly in the directions perpendicular to the interfaces. Summation of the two direct strain components showed a relatively uniform distribution around the restoration and its magnitude equaled approximately to the volumetric shrinkage strain of the material.

In order to understand the spatial development of polymerization shrinkage stress in dental resin-composite restorations, Digital Image Correlation was used to provide full-field displacement/strain measurement of restored model glass cavities by correlating images of the restoration taken before and after polymerization.

Polymerization shrinkage of dental resin composites can lead to restoration debonding or cracked tooth tissues in composite-restored teeth. In order to ...

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The objective of this study was to use 3D CLSM time lapse confocal imaging to assess the in vitro biocompatibility of dental composites. This method provides an accurate and sensitive indication of viable cell rate in contact with dental composite extracts. The ELS extra low shrinkage, a dental composite used for direct restoration, has been taken as example. In vitro assessment was performed on cultured primary human gingival fibroblast cells using Live/Dead staining. Images were obtained with the FV10i confocal biological inverted system and analyzed with the FV10-ASW 3.1 Software. Image analysis showed a very slight cytotoxicity in the presence of the tested composite after 5 hours of time lapse. A slight decrease of cell viability was shown in contact with the tested composite extracts compared to control cells. The findings highlighted the use of 3D CLSM time lapse imaging as a sensitive method to qualitatively and quantitatively evaluate the biocompatibility behavior of dental composites.

The most studied aspect of the biocompatibility of dental composites is their cytotoxicity 3D CLSM time lapse imaging combined with fluorescent signal quantification allows sensitive evaluation of the cell fate over time. Utilizing this method could be an efficient tool to assess the cytocompatibility of dental composites.

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The ...

Detection and Quantitation of Label-Retaining Cells in Mouse Incisors using a 3D Reconstruction Approach after Tissue Clearing

The murine incisor is an organ that grows continuously throughout the lifespan of the mouse. The epithelial and mesenchymal stem cells residing in the proximal tissues of incisors give rise to progeny that will differentiate into ameloblasts, odontoblasts, and pulp fibroblasts. These cells are crucial in supporting the sustained turnover of incisor tissues, making the murine incisor an excellent model for studying the homeostasis of adult stem cells. Stem cells are believed to contain long-living quiescent cells that can be labeled by nucleotide analogs such as 5-ethynyl-2&#180;-deoxyuridine (EdU). The cells retain this label over time and are accordingly named&#160;label-retaining cells (LRCs). Approaches for visualizing LRCs in vivo provide a robust tool for monitoring stem cell homeostasis. In this study, we described a method for visualizing and analyzing LRCs. Our innovative approach features LRCs in mouse incisors after tissue clearing and whole-mount EdU staining followed by confocal microscopy and a 3-dimensional (3D) reconstruction with the imaging software. This method enables 3D imaging acquisition and non-biased quantitation compared to traditional LRCs analysis on sectioned slides.

The mouse incisor contains valuable label-retaining cells in its stem cell niche. We have a novel way to unbiasedly detect and quantify the label-retaining cells; our study used EdU labeling and a 3D reconstruction approach after PEGASOS tissue clearing of the mandible.

The murine incisor is an organ that grows continuously throughout the lifespan of the mouse. The epithelial and mesenchymal stem cells residing in the ...

Developmental Biology

Multimodal Analysis of Bone Formation Using Explant Models

Comprehensive Characterization of Tissue Mineralization in an Ex Vivo Model

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous modalities that are currently available for analysis. Here, we propose a workflow for a comprehensive evaluation of new bone formation using a relevant large animal osseous ex vivo explant. A bone defect (diameter = 3.75 mm; depth = 5.0 mm) is created in an explanted sheep femoral head and injected with a macroporous bone substitute loaded with a pro-osteogenic growth factor (bone morphogenetic protein 2 - BMP2). Subsequently, the explant is maintained in culture for a 28-day period, allowing cellular colonization and subsequent bone formation. To evaluate the quality and structure of newly mineralized tissue, the following successive methods are set up: (i) Characterization and high-resolution 3D images of the entire explant using micro-CT, followed by deep learning image analyses to enhance the discrimination of mineralized tissues; (ii) Nano-indentation to determine the mechanical properties of the newly formed tissue; (iii) Histological examinations, such as Hematoxylin/Eosin/Saffron (HES), Goldner's trichrome, and Movat's pentachrome to provide a qualitative assessment of mineralized tissue, particularly with regard to the visualization of the osteoid barrier and the presence of bone cells; (iv) Back-scattering scanning electron microscopy (SEM) mapping with internal reference to quantify the degree of mineralization and provide detailed insights into surface morphology, mineral composition, and bone-biomaterial interface; (v) Raman spectroscopy to characterize the molecular composition of the mineralized matrix and to provide insights into the persistence of BMP2 within the cement through the detection of peptide bonds. This multimodal analysis will provide an effective assessment of newly formed bone and comprehensive qualitative and quantitative insights into mineralized tissues. Through the standardization of these protocols, we aim to facilitate interstudy comparisons and improve the validity and reliability of research findings.

Author Spotlight: Advanced Techniques for Characterizing Tissue Mineralization in Bone Regeneration Research

The proposed protocol entails a global approach to assess bone formation in the context of bone regeneration using multimodal analyses. It aims to provide qualitative and quantitative information on new bone formation, enhancing the rigor and validity of basic and pre-clinical investigations.

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous ...

Bioengineering

Semi-Automated Planimetric Quantification of Dental Plaque Using an Intraoral Fluorescence Camera

Dental plaque accumulation is quantified using clinical indices or, otherwise, the planimetric plaque index (PPI),&#160;which measures the relative area of a tooth that is covered by plaque deposits. Compared to clinical indices, the PPI has a higher discriminatory power, but traditional planimetry is a time-consuming analysis, as the plaque-covered and clean tooth areas have to be determined manually for each image using image-processing software. Here, we present a method for the semi-automated planimetric quantification of dental plaque, which allows for the rapid processing of up to 1,000 images simultaneously. The method exploits the enhanced contrast between disclosed plaque, sound tooth surfaces, and soft tissues in fluorescence images acquired with an intraoral camera. Careful execution of the clinical procedures and accurate image acquisition are crucial steps for the successful semi-automated identification of the plaque-covered areas. The method is suitable for planimetry on sound facial and oral tooth surfaces, on most composite resin restorations, and on teeth with orthodontic brackets, but not on metallic restorations. Compared to traditional PPI recordings, semi-automated planimetry considerably reduces the amount of time spent on the analysis, as well as the subjective human input, thus increasing the reproducibility of planimetric measurements.

This study presents a semi-automated digital image analysis procedure for the planimetric quantification of disclosed dental plaque based on images acquired with an intraoral fluorescence camera. The method allows for the rapid and reliable quantification of dental plaque in the research environment.

Dental plaque accumulation is quantified using clinical indices or, otherwise, the planimetric plaque index (PPI),&#160;which measures the relative area ...

Fluorescence-Detection Size-Exclusion Chromatography: A Technique to Identify the Integrity of Fluorescent Membrane Proteins upon Detergent Solubilization

Source: Bird, L. E., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/52357">Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli</a>. J. Vis. Exp. (2015)
In this video, we demonstrate a fluorescence-detection size-exclusion chromatography technique to screen for green fluorescent protein (GFP)-fused membrane protein stability in Escherichia coli following detergent solubilization. Proteins exhibiting a symmetrical peak in the size-exclusion profile with little or no free GFP indicate purified proteins with minimum degradation and aggregation, which can subsequently be selected for structure-function analysis.

Source: Bird, L. E., et al. Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli. J. Vis. Exp. (2015)
In this ...

Detection and Removal of Tooth-Colored Composite Resin Using the Fluorescence-Aided Identification Technique

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