The authors thank Jos&#233; Augusto Maulin from Microscopy Multiuser Laboratory (School of Medicine of Ribeir&#227;o Preto) for his generous assistance with the EDS and SEM analyses&#160;and Hermano Teixeira Machado for his generous technical assistance in the video edition.

This study was approved by the Institutional Review Board of the School of Dentistry of Ribeir&#227;o Preto, and the volunteer participant signed the written consent (Process 2011.1.371.583).
1. Biofilm Formation in Situ
<ol>
	<li>Selection of participants
	<ol>
		<li>Select patients based on the following inclusion criteria: a healthy individual with a complete dentition and no clinical signs of oral diseases.</li>
		<li>Exclude patients based on the following exclusion criteria: pregnancy, lactation, dental caries, a periodontal disease or antibiotic treatment in the last 3 months, smoker, or any systemic disease that could influence the periodontal status.</li>
	</ol>
	</li>
	<li>Preparation of intraoral device
	<ol>
		<li>Record the maxillary arch by means of an alginate impression.</li>
		<li>Prepare type IV stone by adding 19 mL of water to 100 g of stone powder. Pour the stone into the alginate mold to make a model of the maxillary arch.</li>
		<li>Design and fabricate an acrylic intraoral device to support the test specimens (titanium and ceramic disks).
		<ol>
			<li>Fabricate retentive clasps from NiCr wire (0.7 mm in diameter) using orthodontic pliers #139 and position them on the model. To position the clamp between the upper premolars, bend one end of each wire so that they form a loop.
			<ol>
				<li>Adapt the loop in the region of the interdental papilla. In the occlusal, insert a gentle curvature so as not to interfere with the points of contact and with the occlusion. Make a 90&#176; fold at the end of the clamp for a retention in the acrylic.</li>
				<li>To fit the clamp in the upper second molar, adapt the curvature of the wire in the cervical third (vestibular) to contour the tooth (distal) and make a 90&#176; fold at the end of the clamp for a retention in the acrylic. 
				NOTE: In order to provide adequate retention/stability to the intraoral device, the retentive clasps were positioned between the upper premolars and distal aspect of the second molar on both sides of the dental arch.</li>
			</ol>
			</li>
			<li>Manipulate the self-curing acrylic resin according to the manufacturer&#39;s instructions and press the acrylic resin between 2 glass plates (with a 3 mm thick spacer interposed) during the plastic phase, to make a 3 mm thick sheet.</li>
			<li>Lay the acrylic sheet on the palatal region of the model, according to the device&#39;s design, and trim off the surplus acrylic resin with a carver, before polymerization.</li>
			<li>Fabricate wax disks using a metallic matrix [10 mm in diameter, 2 mm in thickness (surface area of 78.5 mm2)] by placing molten wax in the matrix and waiting for the wax to solidify. Manually embed 4 wax disks into the acrylic resin, 2 of them between the premolars and the other 2 next to the second molars.</li>
			<li>Let the acrylic resin polymerize in a pressure pot under 20 psi of compressed air for 20 min.</li>
			<li>Finish the intraoral device with an electric dental lab handpiece and cutter and polish it with abrasive rubber.</li>
			<li>Adjust the device for a proper fit in the mouth using the equipment described above.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of the titanium and zirconia specimens 
	Note: Specimens (n = 14) are 10 mm in diameter and 2 mm in thickness and have a surface area of 78.5 mm2.
	<ol>
		<li>Polish the specimens&#39; surfaces with water-cooled sandpapers of decreasing abrasiveness (600, 1200, and 2000 grit), for approximately 20 min. 
		NOTE: The polishing of the specimens was performed to standardize the surface roughness at 0.2 &#181;m.</li>
		<li>Clean and disinfect the specimens and intraoral device with liquid detergent and tap water, and then use an isopropyl alcohol ultrasonic bath for 15 min. Dry them with absorbent paper towels.</li>
		<li>Fix the specimens in the intraoral oral device with about 0.1 mL of non-toxic hot melt adhesive (Figure 1).</li>
		<li>Install the device containing the specimens in the oral cavity. 
		NOTE: The intraoral device containing the specimens should be worn for 48 h. The device was removed and stored in phosphate buffered saline (PBS) while the patient was eating and performing oral cleaning.</li>
	</ol>
	</li>
</ol>
2. Assessment of Bacterial Viability
Note: The sample size n = 10.
<ol>
	<li>Prepare a dyeing solution by adding 3 &#181;L of SYTO9 (green fluorescent nucleic acid dye) and 3 &#181;L of propidium iodide (red fluorescent nucleic acid dye) to 1 mL of sterile distilled water. 
	NOTE: Prepare solutions protected from light.</li>
	<li>Transfer the specimens to a 24-well plate and wash them thoroughly with PBS to remove any non-adherent cells.</li>
	<li>Add the appropriate volume (1 mL) of fluorescent dye solution to cover the biofilm-containing specimen. Add the dye very carefully so as not to disorganize the biofilm.</li>
	<li>Incubate the specimen for 20 - 30 min at room temperature, protected from light.</li>
	<li>Gently wash the biofilm sample with sterile distilled water to remove any excess dye.</li>
	<li>Place the specimen in a glass bottom dish and perform multiphoton laser scanning microscopy for the biofilm analysis. 
	NOTE: The analysis of the bacterial cells viability was carried out using a multiphoton microscopy system. The fluorescence of propidium iodide was detected using a filter with an excitation/emission wavelength of 546/680 nm and 477/600 nm for SYTO9. The size of the images obtained was 5.16188 x 5.16188 mm, which corresponds to 26.64 mm2 of the total area of each specimen (78.5 mm2) or 33.94% of the total area. The images were made from the most central portion of the sample, with a resolution of 1,024 x 1,024 pixels. The red channel and green channel images were analyzed separately using Fiji software31. Each cell was selected using a selection tool and the intensity of the fluorescence was measured through the integrated density of the pixels, subtracting the image background.</li>
</ol>
3. Analysis of the Specimens&#39; Chemical Composition by Energy Dispersive Spectroscopy (EDS)
Note: The sample size n = 3.
<ol>
	<li>Select 3 specimens of each biofilm-free test material, and assess the chemical composition in 2 different areas of each specimen using a scanning electron microscope coupled to a dispersive energy spectrometer, with an electron beam voltage of 10 kV32.</li>
</ol>
4. Morphological Analysis of the Bacterial Biofilm by Scanning Electron Microscopy
Note: The sample size n = 1.
<ol>
	<li>Fix the biofilm by immersing the specimens in 2.5% glutaraldehyde diluted in 0.1 M sodium cacodylate buffer, pH 7.0 - 7.3, for 24 h.</li>
	<li>Wash the sample in PBS buffer (pH = 7.6).</li>
	<li>Postfix the biofilm with 1% osmium tetroxide for 1 h.</li>
	<li>Wash the sample in PBS buffer (pH = 7.6).</li>
	<li>Dehydrate the biofilm samples carefully, keeping them immersed in increasing concentrations of ethanol solutions (50%, 70%, 90%, 95%, and 100%). 
	NOTE: Perform 3 exchanges of ethanol at a 100% concentration. The total dehydration step takes about 2 h.</li>
	<li>Transfer the specimens to the critical point dryer and make several substitutions with carbon dioxide (CO2) until the specimens are dry.</li>
	<li>Remove the dried specimens from the apparatus and mount them onto the scanning electron microscope holders.</li>
	<li>Sputter-coat a 20 nm layer of gold onto the specimen&#39;s surface for 120 s.</li>
	<li>Insert the gold-coated discs in the scanning electron microscope chamber. Obtain images from 5 different areas of each specimen, at 20 - 30 kV under variable pressure and at 650X magnification12.</li>
</ol>

The authors have nothing to disclose.

The protocol described in this study was developed to evaluate the biofilm formation on titanium and zirconia materials for prosthetic abutments, including the analysis of bacterial cell viability and morphological characteristics. In order to accomplish this, an in situ model of biofilm formation was designed, consisting of an intraoral device capable to accommodate samples of the test materials and keep them exposed to the dynamic oral environment for 48 h. The device was considered comfortable and easy to ins...

Bacterial biofilms are complex, functionally and structurally organized microbial communities, characterized by a diversity of microbial species that synthesize an extracellular, biologically active polymer matrix1,2. The bacterial adhesion to biotic or abiotic surfaces is preceded by a formation of the acquired pellicle, mainly consisting of salivary glycoproteins1,3,4. Weak physicochemical interactions between the microorganisms and the pellicle are initially established and followed by stronger interactions between bacterial adhesins and glycoprotein receptors of the acquired pellicle. Microbial diversity gradually increases through the coaggregation of secondary colonizers to the receptors of the already attached bacteria, forming a multispecies community1,3,4,5.
Homeostasis of the oral microbiota and its symbiotic relationship with the host is important in maintaining oral health. The dysbiosis within oral biofilms may increase the risk for the development of caries and periodontal disease2,5. Clinical studies demonstrate a cause-and-effect relationship between the accumulation of biofilm on teeth or dental implants and the development of gingivitis or peri-implant mucositis6,7. The progression of the inflammatory process leads to peri-implantitis and the consequent loss of the implant8.
Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation9. The use of materials with a chemical composition and surface topography that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases9,10. Titanium is the most-used material for the manufacture of prosthetic abutments for implants; however, ceramic materials were recently introduced and are gaining popularity as an alternative to titanium because of their aesthetic properties and biocompatibility11,12. Also importantly, ceramic materials have been associated with a supposedly reduced potential to adhere to microorganisms, mainly due to their surface roughness, wettability, and surface free energy10,13.
In vitro studies have contributed to significant advances in the understanding of microbial adhesion to prosthetic abutment surfaces9,14,15,16,17. However, the dynamic environment of the oral cavity, characterized by its varying temperature and pH and nutrient availability, as well as by the presence of shear forces, is not reproducible in in vitro experimental protocols18,19. To overcome this problem, an alternative is the use of in situ models of biofilm formation, which advantageously preserves its three-dimensional structure for ex vivo analysis10,20,21,22,23,24.
The analysis of the complex structure of the biofilm formed on oral substrates requires the use of microscopy techniques capable of displaying optically dense matter25. Multiphoton laser scanning microscopy is a modern option for biofilm structural analysis26. It is characterized by the use of nonlinear optics with an illumination source close to the infrared wavelength, pulsed to femtoseconds27. This method is indicated for the image acquisition of autofluorescence materials or materials marked by fluorophores, in addition to images generated by non-linear optical signals derived from a phenomenon known as Second Harmonic Generation. Among the advantages of multiphoton microscopy is the great image depth obtained with minimum cell damage caused by the intensity of the excitation light27.
For a viability analysis of biofilm on abiotic surfaces by multiphoton microscopy, the use of fluorescent nucleic acid dyes with different spectral characteristics and a penetration capacity in bacterial cells is required28. Fluorophores SYTO9 (green-fluorescent) and propidium iodide (red-fluorescent) can be used for a visual differentiation between live and dead bacteria28,29,30. Propidium iodide penetrates only bacteria with damaged membranes, while SYTO9 enters bacterial cells with an intact and compromised membrane. When both dyes are present inside a cell, propidium iodide has a greater affinity for nucleic acids and displaces SYTO9, marking it red28,30.
In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable the biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels.

<table><tbody><tr><td>Hydrogum 5</td><td>Zhermack Dental</td><td>C302070</td><td></td></tr><tr><td>Durone IV</td><td>Dentsply</td><td>17130500002</td><td></td></tr><tr><td>NiCr wire&amp;nbsp;</td><td>Morelli</td><td>55.01.070</td><td></td></tr><tr><td>JET auto polymerizing acrylic</td><td>Cl&amp;aacute;ssico</td><td></td><td></td></tr><tr><td>Dental wax&amp;nbsp;</td><td>Cl&amp;aacute;ssico</td><td></td><td></td></tr><tr><td>Pressure pot&amp;nbsp;</td><td>Essencedental</td><td></td><td></td></tr><tr><td>Sandpapers 600 grit</td><td>NORTON</td><td>T216</td><td></td></tr><tr><td>Sandpapers 1200 grit</td><td>NORTON</td><td>T401</td><td></td></tr><tr><td>Sandpapers 2000 grit</td><td>NORTON</td><td>T402</td><td></td></tr><tr><td>Metallographic Polishing Machine</td><td>Arotec</td><td></td><td></td></tr><tr><td>Isopropyl alcohol</td><td>SIGMA-ALDRICH</td><td>W292907</td><td></td></tr><tr><td>Hot melt adhesive</td><td>TECSIL</td><td>PAH M20017</td><td></td></tr><tr><td>Filmtracer LIVE/DEAD Biofilm Viability Kit</td><td>Invitrogen</td><td>L10316</td><td></td></tr><tr><td>Pipette Tips, 10 &amp;micro;L</td><td>KASVI</td><td>K8-10&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Pipette Tips, 1,000 &amp;micro;L</td><td>KASVI</td><td>K8-1000B&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>24-well plate&amp;nbsp;</td><td>KASVI</td><td>K12-024</td><td></td></tr><tr><td>Glass Bottom Dish</td><td>Thermo Scientific</td><td>150680</td><td></td></tr><tr><td>AxioObserver inverted microscope&amp;nbsp;</td><td>ZEISS</td><td></td><td></td></tr><tr><td>Chameleon vision ii laser</td><td>Coherent</td><td></td><td></td></tr><tr><td>Objective EC Plan-Neofluar 40x/1.30 Oil DIC</td><td>ZEISS</td><td>440452-9903-000</td><td></td></tr><tr><td>SDD sensors - X-Max 20mm&amp;sup2;</td><td>Oxford Instruments</td><td></td><td></td></tr><tr><td>Glutaraldehyde solution</td><td>SIGMA-ALDRICH</td><td>G5882</td><td></td></tr><tr><td>Sodium cacodylate Buffer&amp;nbsp;</td><td>SIGMA-ALDRICH</td><td>97068&amp;nbsp;</td><td></td></tr><tr><td>Osmium tetroxide</td><td>SIGMA-ALDRICH</td><td>201030</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>SIGMA-ALDRICH</td><td>S9638</td><td>Used for preparation of phosphate buffered saline</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>SIGMA-ALDRICH</td><td>P9791&amp;nbsp;</td><td></td></tr><tr><td>NaCl</td><td>MERK</td><td>1.06404</td><td></td></tr><tr><td>Kcl</td><td>SIGMA-ALDRICH</td><td>P9333&amp;nbsp;</td><td></td></tr><tr><td>Ethanol absolute for analysis EMSURE</td><td>MERK</td><td>1.00983</td><td></td></tr><tr><td>CPD 030 Critical Point Dryer</td><td>BAL-TEC</td><td></td><td></td></tr><tr><td>JSM-6610 Series Scanning Electron Microscope</td><td>JEOL</td><td></td><td></td></tr><tr><td>SCD 050 Sputter Coater</td><td>BAL-TEC</td><td></td><td></td></tr></tbody></table>

oral biofilm formation on different materials for dental implants

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable a biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels. The in situ model to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments as described in this study provides a satisfactory preservation of the 48 h biofilm, thereby demonstrating methodological adequacy. Multiphoton microscopy allows the analysis of an area representative of the biofilm formed on the test materials. In addition, the use of fluorophores and the processing of the images using multiphoton microscopy allows the analysis of the bacterial viability in a very heterogeneous population of microorganisms. The preparation of biological specimens for electron microscopy promotes the structural preservation of biofilm, images with good resolution, and no artifacts.

The colonization density of the biofilm after 48 h of in situ growth was represented in this study by the proportion of the colonized area on the titanium and zirconia disks in relation to the total scanned area of the specimen using multiphoton microscopy (26.64 mm2). Figure 2 represents the bacterial colonization density on the surface of the 3 tested materials. A higher density of biofilm was observed on the surfaces of the cast and on ...

Watch this Scientific Journal Video about Oral Biofilm Formation on Different Materials for Dental Implants at JoVE.com

Oral Biofilm Formation on Different Materials for Dental Implants

Here, we present a protocol to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments, including the analysis of bacterial cells viability and morphological characteristics. An in situ model associated with powerful microscopy techniques is used for the oral biofilm analysis.

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low ...

oral-biofilm-formation-on-different-materials-for-dental-implants

Research

JoVE Journal

Medicine

11.4K Views.  University of São Paulo. Here, we present a protocol to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments, including the analysis of bacterial cells viability and morphological characteristics. An in situ model associated with powerful microscopy techniques is used for the oral biofilm analysis.

Video: Oral Biofilm Formation on Different Materials for Dental Implants

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 ...

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Bioengineering

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 &#181;m thickness, but is limited to the pH range between 4.5 and 7.0.

A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

Oral Biofilm Sampling for Microbiome Analysis in Healthy Children

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition leads to a better understanding of cariogenic and periopathogenic mechanisms. Microbial changes taking place in the oral cavity during childhood are of interest for several reasons. The evolution of the child oral microbiota and shifts in its composition need to be analyzed further to understand and possibly prevent the onset of disease. At the same time, advanced knowledge of the natural composition of oral biofilm is needed. Early stages of caries-free permanent dentition with healthy gums provide a widely unaffected subgingival habitat that can serve as an in situ baseline for studying features of oral health and disease. Analysis of children's oral biofilm during different stages in life is thus an important theme in the field. Modern molecular analysis methods can provide comprehensive information about the bacterial diversity of such biofilms. To enable microbiota data comparison, it is important to standardize each step in the procedure for molecular data generation. This procedure spans from clinical sampling, Next Generation Sequencing (NGS), bioinformatic data processing, to taxonomic interpretation. One of the most critical factors here is biofilm sampling. Sampling in children is even more challenging in particular due to limited space in subgingival areas. We thus focus on the use of paper points for subgingival sampling. This article provides a detailed protocol for oral biofilm sampling of the subgingival sulcus, the mucosa, and saliva in children.

Changes in the oral microbiome throughout childhood are of growing interest. Comparison of different microbiome studies reveals a lack of standardized sampling protocols. Limited space makes sampling the sound subgingival sulcus of children challenging. Paper point sampling is presented here in detail as the method of choice for this area.

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition ...

Direct and Indirect Culture Methods for Studying Biodegradable Implant Materials In Vitro

Over the past several decades, biodegradable materials have been extensively explored for biomedical applications such as orthopedic, dental, and craniomaxillofacial implants. To screen biodegradable materials for biomedical applications, it is necessary to evaluate these materials in terms of in vitro cell responses, cytocompatibility, and cytotoxicity. International Organization for Standardization (ISO) standards have been widely utilized in the evaluation of biomaterials. However, most ISO standards were originally established to assess the cytotoxicity of nondegradable materials, thus providing limited value for screening biodegradable materials. 
This article introduces and discusses three different culture methods, namely, direct culture method, direct exposure culture method, and exposure culture method for evaluating the in vitro cytocompatibility of biodegradable implant materials, including biodegradable polymers, ceramics, metals, and their composites, with different cell types. Research has shown that culture methods influence cell responses to biodegradable materials because their dynamic degradation induces spatiotemporal differences at the interface and in the local environment. Specifically, the direct culture method reveals the responses of cells seeded directly on the implants; the direct exposure culture method elucidates the responses of established host cells coming in contact with the implants; and the exposure culture method evaluates the established host cells that are not in direct contact with the implants but are influenced by the changes in the local environment due to implant degradation. 
This article provides examples of these three culture methods for studying the in vitro cytocompatibility of biodegradable implant materials and their interactions with bone marrow-derived mesenchymal stem cells (BMSCs). It also describes how to harvest, passage, culture, seed, fix, stain, characterize the cells, and analyze postculture media and materials. The in vitro methods described in this article mimic different scenarios of the in vivo environment, broadening the applicability and relevance of in vitro cytocompatibility testing of different biomaterials for various biomedical applications.

Direct and Indirect Culture Methods for Studying Biodegradable Implant Materials In Vitro

We introduce three methods of direct culture, direct exposure culture, and exposure culture for evaluating the in vitro cytocompatibility of biodegradable implant materials. These in vitro methods mimic different in vivo cell-implant interactions and can be applied to study various biodegradable materials.

Over the past several decades, biodegradable materials have been extensively explored for biomedical applications such as orthopedic, dental, and ...

Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active compounds from plants are challenging because of the complex plant matrix and time-consuming isolation and identification procedures. Therefore, a stepwise approach for screening natural compounds from plants, including the isolation and identification of potentially active molecules, is presented. It includes the collection of the plant material; preparation and fractionation of crude extracts; chromatography and spectrometry (UHPLC-DAD-HRMS and NMR) approaches for analysis and compounds identification; bioassays (antimicrobial and antibiofilm activities; bacterial &#34;adhesion strength&#34; to the salivary pellicle and initial glucan matrix treated with selected treatments); and data analysis. The model is simple, reproducible, and allows high-throughput screening of multiple compounds, concentrations, and treatment steps can be consistently controlled. The data obtained provide the foundation for future studies, including formulations with the most active extracts and/or fractions, isolation of molecules, modeling molecules to specific targets in microbial cells and biofilms. For example, one target to control cariogenic biofilm is to inhibit the activity of Streptococcus mutans glucosyltransferases that synthesize the extracellular matrix&#8217; glucans. The inhibition of those enzymes prevents the biofilm build-up, decreasing its virulence.

Natural products represent promising starting points for the development of new drugs and therapeutic agents. However, due to the high chemical diversity, finding new therapeutic compounds from plants is a challenging and time-consuming task. We describe a simplified approach to identify antimicrobial and antibiofilm molecules from plant extracts and fractions.

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active ...

Biology

Effects of Mechanical Methods Used in Peri-implantitis Treatment on Implant Surface Decontamination and Roughness

Various mechanical methods have been proposed for decontaminating dental implant surfaces with varying success. This in vitro study evaluated the decontamination efficiency of an air abrasion (AA) system with erythritol powder, a polyether-ether-ketone (PEEK) ultrasonic tip, and titanium curettes (TIT) and their effects on implant surface topography using scanning electron microscopy (SEM). A total of 60 implants were stained with permanent red ink and placed in 3D-printed Class 1A and Class 1B peri-implantitis defects, forming six groups (n=10 per group) based on defect type and treatment protocol. Additionally, one positive and one negative control implant was used. Erythritol powder, PEEK ultrasonic tips, and titanium curettes were applied for 2 min in Class 1A defects and 3 minutes in Class 1B defects. Residual red ink areas were quantified with digital software, and implant surface changes were analyzed using SEM and EDS. None of the methods achieved complete decontamination. However, erythritol powder was significantly the most effective, leaving a residual ink rate of 24% &#177; 6% (p &#60; 0.001). PEEK ultrasonic tips resulted in 41% &#177; 4% residual ink, while titanium curettes left 55% &#177; 3%. Significant differences were observed among all methods. No significant difference in decontamination efficacy was found between Class 1A and Class 1B defects. SEM analysis showed minimal surface damage with erythritol powder and PEEK tips, whereas titanium curettes caused moderate to severe damage. Based on both decontamination efficiency and surface preservation, erythritol powder and PEEK tips are safe and effective options for peri-implantitis treatment, while titanium curettes are less effective and cause considerable surface damage. These findings may assist clinicians in peri-implantitis treatment planning.

The present protocol describes an experimental model based on ink-staining which can be used for in vitro implant surface decontamination and roughness research to contribute to clinical decision-making.

Various mechanical methods have been proposed for decontaminating dental implant surfaces with varying success. This in vitro study evaluated the ...

Assessment of Acinetobacter Biofilm Formation and Characterization of Biofilms

Quantification, Viability Assessment, and Visualization Strategies for Acinetobacter Biofilms

Acinetobacter causes nosocomial infections and its biofilm formation can contribute to the survival on dry surfaces such as hospital environments. Thus, biofilm quantification and visualization are important methods to assess the potential of Acinetobacter strains to cause nosocomial infections. The biofilms forming on the surface of the microplate can be quantified in terms of volume and cell numbers. Biofilm volumes can be quantified by staining using crystal violet, washing, destaining using ethanol, then measuring the solubilized dye using a microplate reader. To quantify the number of cells embedded in the biofilms, the biofilms are scrapped off using cell scrapers, harvested in the saline, vigorously agitated in the presence of glass beads, and spread on Acinetobacter agar. Then, the plates are incubated at 30 &#176;C for 24-42 h. After incubation, the red colonies are enumerated to estimate the number of cells in biofilms. This viable count method can also be useful for counting Acinetobacter cells in mixed-species biofilms. Acinetobacter biofilms can be visualized using fluorescent dyes. A commercially available microplate designed for microscopic analysis is employed to form biofilms. Then, the bottom-surface attached biofilms are stained with SYTO9 and propidium iodide dyes, washed, then visualized with confocal laser scanning microscopy.

Author Spotlight: A Comprehensive Protocol for Acinetobacter Biofilm Quantification, Assessment, and Visualization 

This protocol describes the preparation of the inoculum, the biofilm quantification on microtiter plates using crystal violet dye, the viable count in biofilms, and the visualization of biofilms of Acinetobacter.

Acinetobacter causes nosocomial infections and its biofilm formation can contribute to the survival on dry surfaces such as hospital environments. Thus, ...

Oral Biofilm Formation on Different Materials for Dental Implants

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Direct and Indirect Culture Methods for Studying Biodegradable Implant Materials In Vitro

Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries

Effects of Mechanical Methods Used in Peri-implantitis Treatment on Implant Surface Decontamination and Roughness

Quantification, Viability Assessment, and Visualization Strategies for Acinetobacter Biofilms