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.

Oral biofilm formation on different materials for dental implants. Bacterial biofilms are complex, functionally and structurally organized microbial communities, characterized by a diversity of microbial species that synthesize extracellular, biologically-active polymer matrix. Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation.

The use of materials with chemical composition and surface topography that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the overall environment complexity and oral biofilm heterogeneity, microscopic techniques that can enable the structural analysis of biofilm from teeth and dental material surfaces are needed. This article describes a series of protocols implemented for comparing oral biofilm formation on different materials for dental implants.

The steps involved in the usage of biofilm formation in this study were as follows:Record the maxillary arch by means of an alginate impression. Pour the alginate impression with Type IV stone to fabricate a model of maxillary arch. Fabricate retentive clips of micro chrome wire and locate them on the model.

Manipulate the self-curing acrylic resin according to the manufacturer's instructions and press the acrylic resin between two glass plates during the plastic phase to make a sheet three millimeters thick. Lay the acrylic sheet onto the palatal region of the model, according to the device's design, and trim off the surplus acrylic resin before polymerization. Fabricate wax discs using a matrix with 10 millimeters diameter and two millimeters thick.

Embed four wax discs into the acrylic resin, two of them between the premolars and the other two next to the second molars. Let the acrylic resin polymerize in a pressure pot under 20 pounds of compressed air for 20 minutes. Finish the intra-oral device using an electric handpiece and milling and polishing points.

Polish the specimen's surfaces with water-cooled sandpapers of decreasing abrasiveness. Clean the specimens with liquid detergent and tap water and isopropyl alcohol ultrasonic bath for 15 minutes. Then, dry with absorbent paper towels.

Fix the specimens in the intra-oral device with non-toxic hot-melt adhesive. Install the device containing the specimens in the oral cavity. The intra-oral device containing the specimens should be worn for 48 hours.

Prepare a dying solution by adding three microliters of SYTO 9 and three microliters of propidium iodide to one milliliter of sterile distilled water. Transfer the specimens to a 24-well plate and wash thoroughly with PBS to remove non-adherent cells. Add the appropriate volume of fluorescent dye solution to cover the biofilm-containing specimen.

Add the dye very carefully so as not to disorganize the biofilm. Incubate the specimen for 20 to 30 minutes at room temperature protected from light. Gently wash the biofilm sample with a sterile distilled water to remove excess dye.

Place the specimen in a glass-bottom dish for biofilm analysis, using multi-photon laser scanning microscopy. The size of the images obtained was 5.16 x 5.16 mm which corresponds to 26.64 square millimeters of the total area of each specimen or 33.94%of the total area. Images were analyzed using Fiji software.

Each cell was selected using selection tool and intensity of fluorescence was measured through the integrated density of pixels, subtracting the image background. Select three specimens of each test material and assess the chemical composition in two different areas of each specimen, using a scanning electron microscope coupled to a dispersive energy spectrometer. Fix the biofilm by immersing the specimens in 2.5%of glutaraldehyde diluted in 0.1 M sodium cacodylate buffer for 24 hours.

Wash in PBS buffer. Post-fixate with 1%osmium tetroxide for one hour. Wash in PBS buffer.

Dehydrate the biofilm samples carefully, keeping them immersed in increasing concentrations of ethanol solutions. Transfer the specimens to the critical point dryer and make several substitutions with carbon dioxide until the specimens are dried. Remove the dried specimens from the apparatus and mount them onto the scanning electron microscope holders.

Disperse a coating of 20 nanometers of gold layer onto the specimen surfaces for 120 seconds. The colonization density of the biofilm after 48 hours of in situ growth was represented in this study by the proportion of the colonized area to the titanium and Zirconia discs in relation to the total scanned area of the specimen using multi-photon microscopy. The figure shows bacteria adhered to the surfaces of the test materials.

A higher density of biofilm was observed on the surfaces of cast and machined titanium discs than in zirconia discs. This figure shows live and dead bacteria adhered to the surface of the specimens. In this protocol, the nucleic-acid-dyed propidium iodide penetrates only into bacteria with damaged membrane and therefore, emits a red fluorescent signal that is related to dead cells.

SYTO 9 penetrates the bacterial cells with intact or compromised membrane and emits green fluorescent signal from live microorganisms. Cellular bacterial viability was similar between the test materials with predominance of live microorganisms in all groups. Cracks, grooves, or abrasion defects produced during the process of polishing and/or machining were present on the surface of the different materials analyzed.

In the Zirconia discs, large areas were observed with absence of microorganisms and presence of small polymorphic microbial aggregates consisting mainly of cocci and bacilli and filamentous bacteria. The presence of cocci and bacilli was scattered on the surfaces of machined titanium discs. Cast titanium specimens presented colonies of microorganisms involved in a matrix with biofilm-like appearance on the surface.

Less matrix material was observed on the surface of the zirconia discs, compared to the machined titanium and cast titanium discs. The EDS analysis revealed in the zirconia discs 70.83%of Zirconia. In the cast titanium discs, 95.16 of titanium and in the machined titanium discs, 89.86%of titanium.

The protocol described in this study provided satisfactory preservation of the 48 hours biofilm, demonstrating therefore, methodological adequacy. The use of fluoro source and the processing of the images using multi-photon microscopy allowed the analysis of the bacterial integrity in a very heterogeneous population of microorganisms from a representative area of this specimen with minimal cellular damage. The preparation of biological specimens for electron microscopy promoted the structural preservation of the biofilm, which resulted in image with good resolution and no artifacts.

This allowed the visualization and morphological characterization of adhered microorganisms.