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.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Hydrogum 5</td>
			<td style="width:135px;">Zhermack Dental</td>
			<td style="width:123px;">C302070</td>
			<td style="width:341px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Durone IV</td>
			<td style="width:135px;">Dentsply</td>
			<td style="width:123px;">17130500002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NiCr wire&#160;</td>
			<td style="width:135px;">Morelli</td>
			<td style="width:123px;">55.01.070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">JET auto polymerizing acrylic</td>
			<td style="width:135px;">Cl&#225;ssico</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dental wax&#160;</td>
			<td style="width:135px;">Cl&#225;ssico</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Pressure pot&#160;</td>
			<td style="width:135px;">Essencedental</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Sandpapers 600 grit</td>
			<td style="width:135px;">NORTON</td>
			<td style="width:123px;">T216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Sandpapers 1200 grit</td>
			<td style="width:135px;">NORTON</td>
			<td style="width:123px;">T401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Sandpapers 2000 grit</td>
			<td style="width:135px;">NORTON</td>
			<td style="width:123px;">T402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Metallographic Polishing Machine</td>
			<td style="width:135px;">Arotec</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Isopropyl alcohol</td>
			<td style="width:135px;">SIGMA-ALDRICH</td>
			<td style="width:123px;">W292907</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Hot melt adhesive</td>
			<td style="width:135px;">TECSIL</td>
			<td style="width:123px;">PAH M20017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Filmtracer LIVE/DEAD Biofilm Viability Kit</td>
			<td style="width:135px;">Invitrogen</td>
			<td style="width:123px;">L10316</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Pipette Tips, 10 &#181;L</td>
			<td style="width:135px;">KASVI</td>
			<td style="width:123px;">K8-10&#160;&#160;</td>
			<td style="width:341px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Pipette Tips, 1,000 &#181;L</td>
			<td style="width:135px;">KASVI</td>
			<td style="width:123px;">K8-1000B&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well plate&#160;</td>
			<td style="width:135px;">KASVI</td>
			<td>K12-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Glass Bottom Dish</td>
			<td style="width:135px;">Thermo Scientific</td>
			<td style="width:123px;">150680</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AxioObserver inverted microscope&#160;</td>
			<td style="width:135px;">ZEISS</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Chameleon vision ii laser</td>
			<td style="width:135px;">Coherent</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Objective EC Plan-Neofluar 40x/1.30 Oil DIC</td>
			<td style="width:135px;">ZEISS</td>
			<td>440452-9903-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">SDD sensors - X-Max 20mm&#178;</td>
			<td style="width:135px;">Oxford Instruments</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Glutaraldehyde solution</td>
			<td style="width:135px;">SIGMA-ALDRICH</td>
			<td style="width:123px;">G5882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Sodium cacodylate Buffer&#160;</td>
			<td style="width:135px;">SIGMA-ALDRICH</td>
			<td style="width:123px;">97068&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Osmium tetroxide</td>
			<td style="width:135px;">SIGMA-ALDRICH</td>
			<td style="width:123px;">201030</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:339px;">Na2HPO4</td>
			<td style="width:135px;">SIGMA-ALDRICH</td>
			<td style="width:123px;">S9638</td>
			<td rowspan="4">Used for preparation of phosphate buffered saline</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:339px;">KH2PO4</td>
			<td style="width:135px;">SIGMA-ALDRICH</td>
			<td style="width:123px;">P9791&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">NaCl</td>
			<td style="width:135px;">MERK</td>
			<td style="width:123px;">1.06404</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Kcl</td>
			<td style="width:135px;">SIGMA-ALDRICH</td>
			<td style="width:123px;">P9333&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Ethanol absolute for analysis EMSURE</td>
			<td style="width:135px;">MERK</td>
			<td style="width:123px;">1.00983</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">CPD 030 Critical Point Dryer</td>
			<td style="width:135px;">BAL-TEC</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">JSM-6610 Series Scanning Electron Microscope</td>
			<td style="width:135px;">JEOL</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">SCD 050 Sputter Coater</td>
			<td style="width:135px;">BAL-TEC</td>
			<td style="width:123px;"></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

11.3K 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

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Two Techniques to Create Hypoparathyroid Mice: Parathyroidectomy Using GFP Glands and Diphtheria-Toxin-Mediated Parathyroid Ablation

Hypoparathyroidism (HP) is a disorder characterized by low levels of PTH which lead to hypocalcemia, hyperphosphatemia, and low bone turnover. The most common cause of the disease is accidental removal of the parathyroid glands during thyroid surgery. Novel therapies for HP are needed, but testing them requires reliable animal models of acquired HP. 
Here, we demonstrate the generation of two mouse models of acquired HP. In the GFP-PTX model, mice with green fluorescent protein (GFP) expressed specifically in the parathyroids (PTHcre-mTmG) were created by crossing PTHcre+ mice with Rosa-mTmGfl/fl mice. Green fluorescing parathyroid glands are easily identified under a fluorescence dissecting microscope and parathyroidectomy is performed in less than 20 min. After fluorescence-guided surgery, mice are profoundly hypocalcemic. Contrary to the traditional thyro-parathyroidectomy, this precise surgical approach leaves thyroid glands and thyroid function intact. The second model, which does not require surgery, is based on a diphtheria-toxin approach. PTHcre-iDTR mice, which express the diphtheria toxin (DT) receptor specifically in the parathyroids, were generated by crossing the inducible DTR mouse with the PTHcre mouse. Parathyroid cells are thus rendered sensitive to diphtheria toxin (DT) and can be selectively destroyed by systemically injecting mice with DT. The resulting hypocalcemic phenotype is stable.

Mice with acquired hypoparathyroidism would be useful for studying novel drug therapies for hypoparathyroidism. Two procedures to create such mice are demonstrated. The GFP-PTX mouse is generated by surgical parathyroidectomy guided by green fluorescing parathyroid glands. A second, non-surgical approach is based on parathyroid-specific expression of the diphtheria toxin receptor.

Hypoparathyroidism (HP) is a disorder characterized by low levels of PTH which lead to hypocalcemia, hyperphosphatemia, and low bone turnover. The most ...

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 ...

Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result from altered activity of cells responsible for enamel synthesis named ameloblasts, which present in enamel organ. During amelogenesis, ameloblasts follow a specific and precise sequence of events of proliferation, differentiation, and death. A rat continually growing incisors is a suitable experimental model to study ameloblast activity and differentiation stages in physiological and pathological conditions. Here, we describe a reliable and consistent method to micro-dissect enamel organ of rats exposed to environmental toxicants. The micro-dissected dental epithelia contain secretion- and maturation-stage ameloblasts that may be used for qualitative experiments, such as immunohistochemistry assays and in situ hybridization, as well as for quantitative analyses such as RT-qPCR, RNA-seq, and Western blotting.

Understanding enamel formation and possible alterations requires the study of ameloblast activity. Here, we describe a reliable and consistent method to micro-dissect enamel organs containing secretion- and maturation-stage ameloblasts that may be used for further quantitative and qualitative experimental procedures.

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result ...

Oral Health Assessment by Lay Personnel for Older Adults

Oral health is an often-undervalued contributor to overall health. The literature, however, underscores the myriad of systemic diseases influenced by oral health, including type II diabetes, heart disease, and atherosclerosis. Thus, assessments of oral health, called oral screenings, have a significant role in assessing risk of disease, managing disease, and even improving disease by oral care. Here we present a method to assess oral health quickly and consistently across time. The protocol is simple enough for non-oral health professionals such as students, family, and caregivers. Useful for any age of patient, the method is particularly key for older individuals who are often at risk of inflammation and chronic disease. Components of the method include existing oral health assessment scales and inventories, which are combined to produce a comprehensive assessment of oral health. Thus, oral characteristics assessed include intraoral and extraoral structures, soft and hard tissues, natural and artificial teeth, plaque, oral functions such as swallowing, and the impact this oral health status has on the patient&#39;s quality of life. Advantages of this method include its inclusion of measures and perceptions of both the observer and patient, and its ability to track changes in oral health over time. Results acquired are quantitative totals of questionnaire and oral screening items, which can be summed for an oral health status score. The scores of successive oral screenings can be used to track the progression of oral health across time and guide recommendations for both oral and overall health care.

The goal of this protocol is to enable non-dental professionals to assess oral health status for research or health-screening purposes. Aspects assessed include lips, tongue, soft and hard tissues, natural and artificial teeth, oral cleanliness, plaque, swallowing, and impact of oral health on quality of life.

Oral health is an often-undervalued contributor to overall health. The literature, however, underscores the myriad of systemic diseases influenced by oral ...

3D Planning and Printing of Patient Specific Implants for Reconstruction of Bony Defects

We are in the midst of the 3D era in most aspects of life, and especially in medicine. The surgical discipline is one of the major players in the medical field using the constantly developing 3D planning and printing capabilities. Computer-assisted design (CAD) and computer assisted manufacturing (CAM) are used to describe the 3D planning and manufacturing of the product. The planning and manufacturing of 3D surgical guides and reconstruction implants is performed almost exclusively by engineers. As technology advances and software interfaces become more user-friendly, it raises a question regarding the possibility of transferring the planning and manufacturing to the clinician. The reasons for such a shift are clear: the surgeon has the idea of what he wants to design, and he also knows what is feasible and could be used in the operating room. It allows him to be prepared for any scenario/unexpected results during the operation and allows the surgeon to be creative and express his new ideas using the CAD software. The purpose of this method is to provide clinicians with the ability to create their own surgical guides and reconstruction implants. In this manuscript, a detailed protocol will provide a simple method for segmentation using segmentation software and implant planning using a 3D design software. Following the segmentation and stl file production using segmentation software, the clinician could create a simple patient specific reconstruction plate or a more complex plate with a cradle for bone graft positioning. Surgical guides can be created for accurate resection, hole preparation for proper reconstruction plate positioning or for bone graft harvesting and re-contouring. A case of lower jaw reconstruction following plate fracture and nonunion healing of a trauma sustained injury is detailed.

This protocol describes the use of 3D planning and printing for reconstruction of bony defects. We use segmentation tools to create 3D models followed by 3D design software to create patient specific implants for reconstruction purposes concomitant to ablative surgery or as a second stage.

We are in the midst of the 3D era in most aspects of life, and especially in medicine. The surgical discipline is one of the major players in the medical ...

Treatment of Facial Deformities using 3D Planning and Printing of Patient-Specific Implants

Technological advancements in surgical planning and patient-specific implants are constantly evolving. One can either adopt the technology to achieve better results, even in the less experienced hand, or continue without it. As technology develops and becomes more user-friendly, we believe it is time to allow the surgeon the option to plan his/her operations and create his/her own patient-specific surgical guides and fixation plates allowing him full control over the process. We present here a protocol for 3D planning of the operation followed by 3D planning and printing of surgical guides and patient-specific fixation implants. During this process we use two commercial computer-assisted design (CAD) software. We also use a fused deposition&#160;modeling printer for the surgical guides and a selective laser sintering printer for the titanium patient-specific fixation implants. The process includes computed tomography (CT) imaging acquisition, 3D segmentation of the skull and facial bones from the CT, 3D planning of the operations, 3D planning of patient-specific fixation implant according to the final position of the bones, 3D planning of surgical guides for performing an accurate osteotomy and preparing the bone for the fixation plates, and 3D printing of the surgical guides and the patient-specific fixation plates. The advantages of the method include full control over the surgery, planned osteotomies and fixation plates, significant reduction in price, reduction in operation duration, superior performance and highly accurate results. Limitations include the need to master the CAD programs.

As technology develops and becomes more user-friendly, planning of operations and patient-specific surgical guides and fixation plates should be performed by the surgeon. We present a protocol for 3D planning of orthognathic skeletal movements and 3D planning and printing of patient-specific fixation plates and surgical guides.

Technological advancements in surgical planning and patient-specific implants are constantly evolving. One can either adopt the technology to achieve ...

Development and Evaluation of 3D-Printed Cardiovascular Phantoms for Interventional Planning and Training

Catheter-based interventions are standard treatment options for cardiovascular pathologies. Therefore, patient-specific models could help training physicians&#39; wire-skills as well as improving planning of interventional procedures. The aim of this study was to develop a manufacturing process of patient-specific 3D-printed models for cardiovascular interventions.
To create a 3D-printed elastic phantom, different 3D-printing materials were compared to porcine biological tissues (i.e., aortic tissue) in terms of mechanical characteristics. A fitting material was selected based on comparative tensile tests and specific material thicknesses were defined. Anonymized contrast-enhanced CT-datasets were collected retrospectively. Patient-specific volumetric models were extracted from these datasets and subsequently 3D-printed. A pulsatile flow loop was constructed to simulate the intraluminal blood flow during interventions. Models&#39; suitability for clinical imaging was assessed by x-ray imaging, CT, 4D-MRI and (Doppler) ultrasonography. Contrast medium was used to enhance visibility in x-ray-based imaging. Different catheterization techniques were applied to evaluate the 3D-printed phantoms in physicians&#39; training as well as for pre-interventional therapy planning.
Printed models showed a high printing resolution (~30 &#181;m) and mechanical properties of the chosen material were comparable to physiological biomechanics. Physical and digital models showed high anatomical accuracy when compared to the underlying radiological dataset. Printed models were suitable for ultrasonic imaging as well as standard x-rays. Doppler ultrasonography and 4D-MRI displayed flow patterns and landmark characteristics (i.e., turbulence, wall shear stress) matching native data. In a catheter-based laboratory setting, patient-specific phantoms were easy to catheterize. Therapy planning and training of interventional procedures on challenging anatomies (e.g., congenital heart disease (CHD)) was possible.
Flexible patient-specific cardiovascular phantoms were 3D-printed, and the application of common clinical imaging techniques was possible. This new process is ideal as a training tool for catheter-based (electrophysiological) interventions and can be used in patient-specific therapy planning.

Here we present development of a mock circulation setup for multimodal therapy evaluation, pre-interventional planning, and physician-training on cardiovascular anatomies. With the application of patient-specific tomographic scans, this setup is ideal for therapeutic approaches, training, and education in individualized medicine.

Catheter-based interventions are standard treatment options for cardiovascular pathologies. Therefore, patient-specific models could help training ...

3D Imaging of PDL Collagen Fibers during Orthodontic Tooth Movement in Mandibular Murine Model

Orthodontic tooth movement is a complex biological process of altered soft and hard tissue remodeling as a result of external forces. In order to understand these complex remodeling processes, it is critical to study the tooth and periodontal tissues within their 3D context and therefore minimize any sectioning and tissue artefacts. Mouse models are often utilized in developmental and structural biology, as well as in biomechanics due to their small size, high metabolic rate, genetics and ease of handling. In principle this also makes them excellent models for dental related studies. However, a major impediment is their small tooth size, the molars in particular. This paper is aimed at providing a step by step protocol for generating orthodontic tooth movement and two methods for 3D imaging of the periodontal ligament fibrous component of a mouse mandibular molar. The first method presented is based on a micro-CT setup enabling phase enhancement&#160;imaging of fresh collagen tissues. The second method is a bone clearing method using ethyl cinnamate that enables imaging through the bone without sectioning and preserves endogenous fluorescence. Combining this clearing method with reporter mice like Flk1-Cre;TdTomato provided a first of its kind opportunity to image the 3D vasculature in the PDL and alveolar bone.

We present a protocol for generating orthodontic tooth movement in mice and methods for 3D visualization of the collagen fibers and blood vessels of periodontal ligament without sectioning.

Orthodontic tooth movement is a complex biological process of altered soft and hard tissue remodeling as a result of external forces. In order to ...

Real-Time Dynamic Navigation System for the Precise Quad-Zygomatic Implant Placement in a Patient with a Severely Atrophic Maxilla

Zygomatic implants (ZIs) are an ideal way to address cases of a severely atrophic edentulous maxilla and maxilla defects because they replace extensive bone augmentation and shorten the treatment cycle. However, there are risks associated with the placement of ZIs, such as penetration of the orbital cavity or infra-temporal fossa. Furthermore, the placement of multiple ZIs makes this surgery risky and more difficult to perform. Potential intraoperative complications are extremely dangerous and may cause irreparable losses. Here, we describe a practical, feasible, and reproducible protocol for a real-time surgical navigation system for precisely placing quad-zygomatic implants in the severely atrophic maxilla of patients with residual bone that does not meet the requirements of conventional implants. Hundreds of patients have received ZIs at our department based on this protocol. The clinical outcomes have been satisfactory, the intraoperative and postoperative complications have been low, and the accuracy indicated by infusion of the designed image and postoperative three-dimensional image has been high. This method should be utilized during the entire surgical procedure to ensure ZI placement safety.

Here, we present a protocol to achieve precise quad-zygomatic implant placement in patients with severely atrophic maxilla using a real-time dynamic navigation system.

Zygomatic implants (ZIs) are an ideal way to address cases of a severely atrophic edentulous maxilla and maxilla defects because they replace extensive ...