Name: oral biofilm formation on different materials for dental implants
Uploaded: 2018-06-24T14:00:00
Description: Watch this Scientific Journal Video about Oral Biofilm Formation on Different Materials for Dental Implants at JoVE.com

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 excl...

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.

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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 ...

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