Approval or consent to perform this study was not required since the use of patients&#39; data is not applicable. In this study, DICOM data from a maxillary model consisting of extracted, de-identified human teeth are used. Teeth were extracted due to reasons not related to this study.
1. Virtual access cavity planning
<ol>
	<li>Start the digital planning program.</li>
	<li>Right-click on Expert to choose the advanced mode.</li>
	<li>Right-click on New to open a new case.</li>
	<li>Select the folder with DICOM image data to import the image data to the software.</li>
	<li>Adjust the Hounsfield Units (HU) thresholds if necessary for optimal visualization (check in the small window in the lower left).</li>
	<li>Click on Create Dataset to complete the data import. 
	NOTE: Here, DICOM data from a maxillary model consisting of extracted, de-identified human teeth are used.</li>
	<li>Choose the type of planning by left-clicking on Maxilla or Mandible and name the planning.</li>
	<li>Click on Edit Segmentations to start the image segmentation process. 
	NOTE: A new window opens automatically for the segmentation process.</li>
	<li>Choose the axial view by left-clicking on Axial in the upper left box.</li>
	<li>Click on Density Measurement to measure the high radiopaque tooth surface and the surrounding non-radiopaque states (air). Calculate the mean values between both densities. (Figure 1). 
	NOTE: The mean value needs to be calculated manually; there is no integrated tool in the software.</li>
	<li>Click on 3D Reconstruction.</li>
	<li>Set the lower threshold to the determined mean value (Figure 2A).</li>
	<li>Segment the dentition with the Flood Fill Tool and name the segmentation as desired (Figure 2B). 
	NOTE: When the flood fill tool is selected and active, the desired area can be segmented by a left click in the 3D-view.</li>
	<li>Complete the segmentation by clicking on Close Module.</li>
	<li>Add a model scan by selecting Add &#62; Object &#62; Model Scan. 
	NOTE: A surface scan needs to be generated beforehand (e.g., utilizing an intra-oral scanner, which provides the data as an stl-file).</li>
	<li>Import the stl File from the digital surface scan.</li>
	<li>Choose Align to Other Object.</li>
	<li>Select the performed segmentation (Figure 2C).</li>
	<li>Choose three different matching points for landmark registration in the 3D view in both datasets, the segmentation, and the surface scan. 
	NOTE: Spatial distribution of the points will facilitate the semi-automatic matching of the data.</li>
	<li>Verify correct registration in all planes and complete registration. 
	NOTE: Manual corrections can be required if deviations between CBCT and surface scan are apparent. If required, left-click and drag to adjust the alignment spatially, and right-click and drag to adjust angular deviation in the displayed planes (Figure 3)</li>
	<li>Add an implant (the utilized endodontic bur must be imported to the software&#39;s implant database beforehand) to plan the access to the root canal.</li>
	<li>Position the bur in the desired angulation and to the required depth, and verify in all planes (Figure 4A).</li>
	<li>Add the corresponding sleeve to the bur (the utilized sleeve system must be added to the database beforehand via Extras &#62; Edit Custom Sleeve System). 
	NOTE: The sleeve must not be in contact with the crown of the tooth. If the sleeve is in contact, a longer bur needs to be selected to provide space between the sleeve and tooth structure (Figure 4B).</li>
	<li>Select Object &#62; Add &#62; Surgical Guide to design the template as preferred (Figure 5A).</li>
	<li>Export the template as an stl file and manufacture it with a 3D printer (Figure 5B, Supplementary File 1). 
	&#8203;NOTE: After completing the 3D print, rework the template according to the manufacturer&#39;s instructions for the printer and printing material used. The accurate removal of support material is crucial for the fit of the template on the dental arch, and thus also for the accuracy of the preparation of the access cavity.</li>
</ol>
2. Access cavity preparation
<ol>
	<li>Check the fit of the template on the dentition (Figure 5C). 
	NOTE: Inspection windows can be added during the design process to enhance visual control of the fit and seat.</li>
	<li>Check the fit of the sleeve in the template.</li>
	<li>Mark the enamel at the access cavity site. Dye (e.g., caries detector) may be used at the bur&#39;s tip (Figure 6A, B).</li>
	<li>Remove the enamel at the access cavity site without using the template or endodontic bur. Use a diamond bur instead until dentin is exposed (Figure 6C).</li>
	<li>Place the sleeve containing the template on the dental arch.</li>
	<li>Insert the bur into the handpiece that was used for the planning.</li>
	<li>Perform the access cavity preparation with template guidance (Figure 6D). 
	&#8203;NOTE: The access cavity should be prepared intermittently. The drill and the cavity should be cleaned of debris to counteract heat generation. Hand files can be used to check if the root canal orifice can be entered before the apical position is reached. The apical position will be defined by the bur stop. Hand files can be used to search or enter the canal orifice. Once the canal orifice is located, conventional root canal treatment utilizing hand files and/or rotary instruments can be performed.</li>
</ol>
3. Treatment evaluation
<ol>
	<li>Use the preoperative CBCT settings to create postoperative image data.</li>
	<li>Start a new case planning.</li>
	<li>Import the image data analog to the preoperative planning.</li>
	<li>Click on Edit Segmentations.</li>
	<li>Set the lower threshold to the determined mean value, which was calculated for the preoperative data.</li>
	<li>Use the Flood Fill Tool to segment the dentition.</li>
	<li>Complete the segmentation by clicking on Close Module.</li>
	<li>Open the preoperative planning.</li>
	<li>Select Plan &#62; Treatment Evaluation.</li>
	<li>Select Postoperative Volume Dataset (Figure 7A).</li>
	<li>Load the correct postoperative dataset and choose the generated segmentation.</li>
	<li>Align pre- and postoperative CBCT data by choosing three different regions for landmark registration. 
	NOTE: Spatial distribution of the points will facilitate the semi-automatic matching of the data (Figure 7B).</li>
	<li>Verify correct registration in all planes and complete registration. 
	NOTE: Manual corrections can be required if deviations between CBCT and surface scan are apparent (Figure 8).</li>
	<li>Place the virtual endodontic bur in the direction of performed access cavity preparation, and check in all the planes (Figure 9). 
	NOTE: If the diameter of the calcified canal is larger than the diameter of the utilized endodontic bur, adjustment in the apical-coronal direction is not feasible. Thus, treatment evaluation can be determined for angular and lateral deviation only, not for apical or three-dimensional deviation.</li>
	<li>Select Finish, and the software will calculate the deviation automatically, showing the results in a table. Moreover, the deviation between planned and performed access cavity preparation can be visualized in a 3D-rendered view.</li>
</ol>

<ol>
	<li>Andreasen, F. M., Zhijie, Y., Thomsen, B. L., Andersen, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occurrence+of+pulp+canal+obliteration+after+luxation+injuries+in+the+permanent+dentition.">Occurrence of pulp canal obliteration after luxation injuries in the permanent dentition.</a> Endodontics &amp; Dental Traumatology. 3 (3), 103-115 (1987).</li><li>Fleig, S., Attin, T., Jungbluth, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Narrowing+of+the+radicular+pulp+space+in+coronally+restored+teeth.">Narrowing of the radicular pulp space in coronally restored teeth.</a> Clinical Oral Investigation. 21 (4), 1251-1257 (2016).</li><li>Linu, S., Lekshmi, M. S., Varunkumar, V. S., Sam Joseph, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+outcome+following+direct+pulp+capping+using+bioceramic+materials+in+mature+permanent+teeth+with+carious+exposure:+A+pilot+retrospective+study.">Treatment outcome following direct pulp capping using bioceramic materials in mature permanent teeth with carious exposure: A pilot retrospective study.</a> Journal of Endodontics. 43 (10), 1635-1639 (2017).</li><li>Robertson, A., Andreasen, F. M., Bergenholtz, G., Andreasen, J. O., Noren, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+pulp+necrosis+subsequent+to+pulp+canal+obliteration+from+trauma+of+permanent+incisors.">Incidence of pulp necrosis subsequent to pulp canal obliteration from trauma of permanent incisors.</a> Journal of Endodontics. 22 (10), 557-560 (1996).</li><li>Kiefner, P., Connert, T., ElAyouti, A., Weiger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+calcified+root+canals+in+elderly+people:+a+clinical+study+about+the+accessibility,+the+time+needed+and+the+outcome+with+a+three-year+follow-up.">Treatment of calcified root canals in elderly people: a clinical study about the accessibility, the time needed and the outcome with a three-year follow-up.</a> Gerodontology. 34 (2), 164-170 (2017).</li><li>Cvek, M., Granath, L., Lundberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Failures+and+healing+in+endodontically+treated+non-vital+anterior+teeth+with+posttraumatically+reduced+pulpal+lumen.">Failures and healing in endodontically treated non-vital anterior teeth with posttraumatically reduced pulpal lumen.</a> Acta Odontologica Scandinavica. 40 (4), 223-228 (1982).</li><li>Zehnder, M. S., Connert, T., Weiger, R., Krastl, G., Kuhl, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guided+endodontics:+accuracy+of+a+novel+method+for+guided+access+cavity+preparation+and+root+canal+location.">Guided endodontics: accuracy of a novel method for guided access cavity preparation and root canal location.</a> International Endodontic Journal. 49 (10), 966-972 (2016).</li><li>Moreno-Rabi&#233;, C., Torres, A., Lambrechts, P., Jacobs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+applications,+accuracy+and+limitations+of+guided+endodontics:+a+systematic+review.">Clinical applications, accuracy and limitations of guided endodontics: a systematic review.</a> International Endodontic Journal. 53 (2), 214-231 (2020).</li><li>Buchgreitz, J., Buchgreitz, M., Bj&#248;rndal, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guided+root+canal+preparation+using+cone+beam+computed+tomography+and+optical+surface+scans+-+an+observational+study+of+pulp+space+obliteration+and+drill+path+depth+in+50+patients.">Guided root canal preparation using cone beam computed tomography and optical surface scans - an observational study of pulp space obliteration and drill path depth in 50 patients.</a> International Endodontic Journal. 52 (5), 559-568 (2019).</li><li>Dula, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SADMFR+guidelines+for+the+use+of+cone-beam+computed+tomography/+digital+volume+tomography.">SADMFR guidelines for the use of cone-beam computed tomography/ digital volume tomography.</a> Swiss Dental Journal. 124 (11), 1169-1183 (2014).</li><li>Ender, A., Zimmermann, M., Mehl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+complete-+and+partial-arch+impressions+of+actual+intraoral+scanning+systems+in+vitro.">Accuracy of complete- and partial-arch impressions of actual intraoral scanning systems in vitro.</a> International Journal of Computerized Dentistry. 22 (1), 11-19 (2019).</li><li>Krug, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guided+endodontics:+a+comparative+in+vitro+study+on+the+accuracy+and+effort+of+two+different+planning+workflows.">Guided endodontics: a comparative in vitro study on the accuracy and effort of two different planning workflows.</a> International Journal of Computerized Dentistry. 23 (2), 119-128 (2020).</li><li>Chen, L., Lin, W. S., Polido, W. D., Eckert, G. J., Morton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy,+reproducibility,+and+dimensional+stability+of+additively+manufactured+surgical+templates.">Accuracy, reproducibility, and dimensional stability of additively manufactured surgical templates.</a> The Journal of Prosthetic Dentistry. 122 (3), (2019).</li><li>Tahir, N., Abduo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+evaluation+of+the+effect+of+3D+printing+orientation+on+the+accuracy+of+implant+surgical+templates+fabricated+by+desktop+printer.">An in vitro evaluation of the effect of 3D printing orientation on the accuracy of implant surgical templates fabricated by desktop printer.</a> Journal of Prosthodontics. , (2022).</li><li>Torres, A., Lerut, K., Lambrechts, P., Jacobs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guided+endodontics:+Use+of+a+sleeveless+guide+system+on+an+upper+premolar+with+pulp+canal+obliteration+and+apical+periodontitis.">Guided endodontics: Use of a sleeveless guide system on an upper premolar with pulp canal obliteration and apical periodontitis.</a> Journal of Endodontics. 47 (1), 133-139 (2021).</li><li>Dong, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+in+vitro+mandibular+volumetric+measurements+from+CBCT+of+different+voxel+sizes+with+different+segmentation+threshold+settings.">Accuracy of in vitro mandibular volumetric measurements from CBCT of different voxel sizes with different segmentation threshold settings.</a> BMC Oral Health. 19 (1), 206 (2019).</li><li>Oh, S. -. M., Lee, D. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+the+accuracy+of+postoperative+analysis+methods+for+locating+the+actual+position+of+implants:+An+in+vitro+study.">Validation of the accuracy of postoperative analysis methods for locating the actual position of implants: An in vitro study.</a> Applied Sciences. 10 (20), 7266 (2020).</li><li>Connert, T., Weiger, R., Krastl, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Present+status+and+future+directions+-+Guided+endodontics.">Present status and future directions - Guided endodontics.</a> International Endodontic Journal. , (2022).</li></ol>

All authors declare that they have no conflict of interest.

The introduction of template-aided access cavity preparations in endodontics has brought immense progress to non-surgical endodontic treatment in teeth with PCO. Conventional access cavity preparation can be very time consuming5 and is prone to error in cases with severe PCO. In vitro studies and clinical case reports demonstrate the feasibility of the guided endodontics approach, generating satisfying results in terms of root canal detection and an overall low deviation between the plann...

Pulp canal obliterations (PCO) are signs of vital pulp, and are often observed after dental trauma1 or as a response to stimuli like caries, restorative procedures2, or vital pulp therapy3. When no clinical or radiographic signs of pathology are present, root canal treatment is not indicated. In the long term, however, the remaining pulp tissue can develop a pathosis4. In cases where clinical or radiographic signs of pulpal or apical pathology are present, non-surgical root canal treatment would be the treatment of choice for tooth preservation. 
For a successful outcome of the root canal treatment, the preparation of an adequate access cavity is crucial. Teeth with PCO needing a root canal treatment are difficult to treat, even for dentists that are specialized in the field of endodontics5. Attempting to locate a calcified root canal may result in a high loss of tooth structure and thus weakening or even perforation of the root. This reduces the prognosis of the tooth, and extraction may be indicated6.
As template-based (static) navigation is already successfully used in oral implantology, its application in endodontics was first described in the literature a few years ago7. Since then, numerous case reports and studies have demonstrated the benefits of template-aided endodontic access cavity preparation in cases with PCO8,9. 
The aim of this work is to present the technique of guided access cavity preparation using guided endodontics. For research purposes, a treatment evaluation (determination of angular and spatial deviation between planned and performed access cavity) is possible after a postoperative CBCT scan, which is also presented in this article.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accuitomo 170</td>
			<td>Morita Manufacturing</td>
			<td>NA</td>
			<td>CBCT machine</td>
		</tr>
		<tr>
			<td>coDiagnostiX</td>
			<td>Dental Wings Inc</td>
			<td>Version 10.4</td>
			<td>Planning software, which is mainly intended for implant surgery. Endodontic access cavities can be planned by adding the utlized bur to the implant database</td>
		</tr>
		<tr>
			<td>Endoseal drill</td>
			<td>Atec Dental GmbH</td>
			<td>NA</td>
			<td>Carbide bur, which is used for the guided access cavity preparation</td>
		</tr>
		<tr>
			<td>StecoGuide Endo-Sleeve</td>
			<td>steco-system-technik</td>
			<td>REF M.27.28.D100L5</td>
			<td>Sleeves, which are inserted into the fabricated template</td>
		</tr>
		<tr>
			<td>TRIOS 3</td>
			<td>3Shape A/S</td>
			<td>NA</td>
			<td>Surface scanner</td>
		</tr>
		<tr>
			<td>P30</td>
			<td>Straumann</td>
			<td>NA</td>
			<td>3D Printer</td>
		</tr>
		<tr>
			<td>P pro Surgical Guide Clear</td>
			<td>Straumann</td>
			<td>NA</td>
			<td>Light-curing resin for the additive manufacturing</td>
		</tr>
	</tbody>
</table>

guided endodontics: three-dimensional planning and template-aided preparation of endodontic access cavities

Pulp canal obliterations (PCO) are often a consequence of dental trauma, such as luxation injuries. Even though dentin apposition is a sign of vital pulp, pulpitis or apical periodontitis may develop in the long term. Root canal treatment of teeth with severe PCO and pulpal or periapical pathosis is challenging for general practitioners and even for well-equipped endodontic specialists. To ensure detection of the calcified root canal and avoid excessive loss of tooth structure or root perforation, static navigation using templates ("Guided Endodontics") was introduced a few years ago. The general workflow includes three-dimensional imaging using cone-beam computed tomography (CBCT), a digital surface scan, and superimposition of both in a planning software. This is followed by virtual planning of the access cavity and the design of a template that will guide the drill to the desired target point. To do this, a true-to-scale virtual image of the drill must be placed in a way that the tip of the drill reaches the orifice of the calcified root canal. Once the template has been fabricated using computer-aided design and computer-aided manufacturing (CAD/CAM) or a 3D printer, guided preparation of the access cavity can be performed clinically. For research purposes, a postoperative CBCT image can be used to quantify the accuracy of the access cavity performed. This work aims to present the technique of static guided endodontics from imaging to clinical implementation.

Figure 10A shows the occlusal view of a prepared endodontic access cavity in a first maxillary molar after template-aided access cavity preparation of the mesio-buccal canal. Figure 10B shows the insertion of three endodontic handfiles to confirm successful root canal detection after preparation of the palatal and disto-buccal access cavities. After matching the postoperative CBCT data to the preoperative planning data, virtual bur placement generates informatio...

Watch this Scientific Journal Video about Guided Endodontics: Three-Dimensional Planning and Template-Aided Preparation of Endodontic Access Cavities at JoVE.com

Guided Endodontics: Three-Dimensional Planning and Template-Aided Preparation of Endodontic Access Cavities

Guided endodontics describes a template-aided approach for access cavity preparation. The procedure requires cone-beam computed tomography and a surface scan to produce a template. An incorporated sleeve guides the drill to the target point. This allows the preparation of minimally invasive endodontic access cavities in calcified teeth.

Pulp canal obliterations (PCO) are often a consequence of dental trauma, such as luxation injuries. Even though dentin apposition is a sign of vital pulp, ...

guided-endodontics-three-dimensional-planning-template-aided

Research

JoVE Journal

Medicine

4.1K Views.  University of Basel. Guided endodontics describes a template-aided approach for access cavity preparation. The procedure requires cone-beam computed tomography and a surface scan to produce a template. An incorporated sleeve guides the drill to the target point. This allows the preparation of minimally invasive endodontic access cavities in calcified teeth. 

Video: Guided Endodontics: Three-Dimensional Planning and Template-Aided Preparation of Endodontic Access Cavities

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Radiation Planning Assistant - A Streamlined, Fully Automated Radiotherapy Treatment Planning System

The Radiation Planning Assistant (RPA) is a system developed for the fully automated creation of radiotherapy treatment plans, including volume-modulated arc therapy (VMAT) plans for patients with head/neck cancer and 4-field box plans for patients with cervical cancer. It is a combination of specially developed in-house software that uses an application programming interface to communicate with a commercial radiotherapy treatment planning system. It also interfaces with a commercial secondary dose verification software. The necessary inputs to the system are a Treatment Plan Order, approved by the radiation oncologist, and a simulation computed tomography (CT) image, approved by the radiographer. The RPA then generates a complete radiotherapy treatment plan. For the cervical cancer treatment plans, no additional user intervention is necessary until the plan is complete. For head/neck treatment plans, after the normal tissue and some of the target structures are automatically delineated on the CT image, the radiation oncologist must review the contours, making edits if necessary. They also delineate the gross tumor volume. The RPA then completes the treatment planning process, creating a VMAT plan. Finally, the completed plan must be reviewed by qualified clinical staff.

Radiation therapy is a highly complex cancer treatment that requires multiple specialists to create a treatment plan and provide quality assurance (QA) prior to delivery to a patient. This protocol describes the use of a fully automated system, the Radiation Planning Assistant (RPA), to create high-quality radiation treatment plans.

The Radiation Planning Assistant (RPA) is a system developed for the fully automated creation of radiotherapy treatment plans, including volume-modulated ...

Three-Dimensional Printing of a Complex Aortic Anomaly

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal symptoms. These anomalies may be associated with other congenital heart diseases. It is hard to identify the accurate anatomic vessel location from two-dimensional imaging data, such as computed tomography. As an additive manufacturing method, three-dimensional (3-D) printing can covert the acquired imaging data into 3-D physical models. This protocol describes the procedure for modeling the volumetric DICOM imaging into 3-D data and printing it as an anatomically realistic 3-D model. Using this model, surgeons can identify the vessel location of complex aortic anomalies, which is helpful for pre-operative planning and intra-operative guidance.

Here, we present a protocol to use three dimensional printed models for pre-operative planning and intra-operative reorganization of complicated vascular locations when handling a congenital aortic anomaly.

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal ...

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty (PVP)

Percutaneous vertebroplasty (PVP) is considered an effective treatment for the back pain caused by osteoporotic vertebral compression fracture. The accuracy of PVP mainly depends on the surgeons&#39; experience and multiple fluoroscopes during a traditional procedure. Puncture related complications were reported all over the world. To make the surgical procedure more precise and decrease the rate of puncture-related complications, our team applied a three-dimensional printing guide template to PVP to modify the traditional procedure. This protocol introduces how to model target vertebrae DICOM imaging data into three-dimensions in the software, how to simulate operation in this 3-D model, and how to use all of the surgical data to reconstruct a patient specific template for application. Using this template, surgeons can identify suitable puncture points accurately to improve the accuracy of the operation. The whole protocol includes: 1) diagnosis of the osteoporotic vertebral compression fracture; 2) acquisition of CT imaging of the target vertebra; 3) simulation of the operation in the software; 4) design and fabrication of the 3-D printing guide template; and 5) application of the template into an operation procedure.

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty PVP

Herein, we present a three-dimensional printing guide template for percutaneous vertebraplasty. A patient with a T11 vertebral compression fracture was selected as a case study.

Percutaneous vertebroplasty (PVP) is considered an effective treatment for the back pain caused by osteoporotic vertebral compression fracture. The ...

Three-Dimensional Collagen Matrix Scaffold Implantation as a Liver Regeneration Strategy

Liver diseases are the leading cause of death worldwide. Excessive alcohol consumption, a high-fat diet, and hepatitis C virus infection promote fibrosis, cirrhosis, and/or hepatocellular carcinoma. Liver transplantation is the clinically recommended procedure to improve and extend the life span of patients in advanced disease stages. However, only 10% of transplants are successful, with organ availability, presurgical and postsurgical procedures, and elevated costs directly correlated with that result. Extracellular matrix (ECM) scaffolds have emerged as an alternative for tissue restoration. Biocompatibility and graft acceptance are the main beneficial characteristics of those biomaterials. Although the capacity to restore the size and correct function of the liver has been evaluated in liver hepatectomy models, the use of scaffolds or some kind of support to replace the volume of the extirpated liver mass has not been assessed.
Partial hepatectomy was performed in a rat liver with the xenoimplantation of a collagen matrix scaffold (CMS) from a bovine condyle. Left liver lobe tissue was removed (approximately 40%), and an equal proportion of CMS was surgically implanted. Liver function tests were evaluated before and after the surgical procedure. After days 3, 14, and 21, the animals were euthanized, and macroscopic and histologic evaluations were performed. On days 3 and 14, adipose tissue was observed surrounding the CMS, with no clinical evidence of rejection or infection, as was vessel neoformation and CMS reabsorption at day 21. There was histologic evidence of an insignificant inflammation process and migration of adjacent cells to the CMS, observed with the hematoxylin and eosin (H&#38;E) and Masson&#39;s trichrome staining. The CMS was shown to perform well in liver tissue and could be a useful alternative for studying tissue regeneration and repair in chronic liver diseases.

Liver diseases are induced by many causes that promote fibrosis or cirrhosis. Transplantation is the only option for recovering health. However, given the scarcity of transplantable organs, alternatives must be explored. Our research proposes the implantation of collagen scaffolds in liver tissue from an animal model.

Liver diseases are the leading cause of death worldwide. Excessive alcohol consumption, a high-fat diet, and hepatitis C virus infection promote fibrosis, ...

Sample Preparation for Computed Tomography-based Three-dimensional Visualization of Murine Hind-limb Vessels

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ function. Clarifying the mechanism of blood vessel formation is therefore extremely useful for elucidating developmental processes and pathological mechanisms. Murine hind-limb vessels are often used as a model for physiological and pathological angiogenesis. Evaluation is mainly performed via a two-dimensional method using tissue sections. However, methods for evaluating three-dimensional (3D) vascular morphology are particularly limited. This paper introduces a method for visualizing murine hind-limbs using computed tomography (CT). Radiation-opaque resin is injected through the descending aorta, and whole vessels are filled with dye. By adjusting the time of dye injection, arterial-specific filling is also possible, and samples can be obtained with any micro-X-ray CT device. This contrast method provides a basic technique for the 3D evaluation of murine blood vessels in the lower extremities. Furthermore, this method can be used to visualize all blood vessels below the diaphragm and evaluate blood vessels in the abdominal organs.

Here, we describe a visualization and quantification method for murine hind-limb vessels using micro-X-ray computed tomography.

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ ...

Dynamic Navigation in Endodontics: Guided Access Cavity Preparation by Means of a Miniaturized Navigation System

In the case of teeth with pulp canal calcification (PCC) and apical pathology or pulpitis, root canal treatment can be very challenging. PCC are common sequelae of dental trauma but can also occur with stimuli such as caries, bruxism, or after placing a restoration. In order to access the root canal as minimally invasive as possible in case of a necessary root canal treatment, dynamic navigation has recently been introduced in endodontics in addition to static navigation. The use of a dynamic navigation system (DNS) requires pre-operative cone-beam computed tomography (CBCT) imaging and a digital surface scan. If necessary, reference markers must be placed on the teeth before the CBCT scan; with some systems, these can also be planned and created digitally afterward. By means of a stereo camera connected to the planning software, the drill can now be coordinated with the help of reference markers and virtual planning. As a result, the position of the drill can be displayed on the monitor in real-time during preparation in different planes. In addition, the spatial displacement, the angular deviation, and the depth position are also displayed separately. The few commercially available DNS mostly consist of relatively large camera-marker-systems. Here, the DNS contains miniaturized components: a low-weight camera (97 g) mounted on the micromotor of the electric handpiece utilizing a manufacturer-specific connecting mechanism and a small marker (10 mm x 15 mm), which can be easily attached to an individually manufactured intraoral tray. For research purposes, a post-operative CBCT scan can be matched with the pre-operative one, and the volume of tooth structure removed can be calculated by the software. This work aims to present the technique of guided access cavity preparation by means of a miniaturized navigation system from imaging to clinical implementation.

Dynamic navigation systems (DNS) provide real-time visualization and guidance to the operator during endodontic access cavities preparation. The planning of the procedure requires three-dimensional imaging utilizing cone beam computed tomography and surface scans. After the export of the planning data to the DNS, access cavities can be prepared with minimal invasion.

In the case of teeth with pulp canal calcification (PCC) and apical pathology or pulpitis, root canal treatment can be very challenging. PCC are common ...

Whole-Kidney Three-Dimensional Staining with CUBIC

Although conventional pathology provided numerous information about kidney microstructure, it was difficult to know the precise structure of blood vessels, proximal tubules, collecting ducts, glomeruli, and sympathetic nerves in the kidney due to the lack of three-dimensional (3D) information. Optical clearing is a good strategy to overcome this big hurdle. Multiple cells in a whole organ can be analyzed at single-cell resolution by combining tissue clearing and 3D imaging technique. However, cell labeling methods for whole-organ imaging remain underdeveloped. In particular, whole-mount organ staining is challenging because of the difficulty in antibody penetration. The present protocol developed a whole-mount mouse kidney staining for 3D imaging with the CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) tissue clearing method. The protocol has enabled visualizing renal sympathetic denervation after ischemia-reperfusion injury and glomerulomegaly in the early stage of diabetic kidney disease from a comprehensive viewpoint. Thus, this technique can lead to new discoveries in kidney research by providing a macroscopic perspective.

The present protocol describes a tissue clearing method and whole-mount immunofluorescent staining for three-dimensional (3D) kidney imaging. This technique can offer macroscopic perspectives in kidney pathology, leading to new biological discoveries.