The authors have no acknowledgments.

The study was approved by the ethics committee of our institution (reference 2020-277-1). Patients signed the CT scan informed consent.
1. Downloading the CT images
<ol>
	<li>Gain access to a picture archiving and communication system (PACS). 
	NOTE: Each software package has a different way of accessing a PACS, but all of them have a way to download a study in the DICOM format. If there is a question regarding how this is done, ask the system administrator of the center or the radiologists of the center.</li>
	<li>Download the full CT scan in the DICOM format while maintaining patient anonymity.</li>
</ol>
2. Obtaining the 3D biomodel (Supplementary File 1-Figure S1)
<ol>
	<li>Download the software 3D Slicer (see the Table of Materials). Install the program on the computer.</li>
	<li>Import the CT images in the DICOM format.
	<ol>
		<li>Click on the DCM icon on the top-left corner of the screen.</li>
		<li>Click on Import DICOM Files on the left side of the screen, and wait for a window to open that makes it possible to choose the folder where the CT study is saved in the DICOM format.</li>
		<li>Click on &#34;DummyPatName!&#34; on the top right of the screen. If the folder has more than one CT study, click on the series &#34;DummySeriesDesc!&#34; with the most images at the bottom of the screen.</li>
		<li>Click on Load on the lower-right margin.</li>
	</ol>
	</li>
	<li>Create the 3D biomodel.
	<ol>
		<li>Click the menu bar at the top of the screen, and wait for the DICOM to appear.</li>
		<li>In the dropdown menu, choose the Legacy | Editor option. Press ok on the message that appears (Supplementary File 1-Figure S2).</li>
		<li>Wait for a new menu to appear on the left side of the screen. Click on the Threshold Effect icon.</li>
		<li>Move the bar in the lower box until only the bone is painted in the images on the right. In this way, select the value of the Hounsfield units to be included in the model.</li>
		<li>Once the desired paint level is achieved, click on Apply. The selection is marked with the color green (Supplementary File 1-Figure S3).</li>
		<li>Select the option Make Model Effect from the side menu (the same as the previous one). Select Apply (Supplementary File 1-Figure S4).</li>
		<li>In the upper-right window, the 3D model is generated. Click on the golden frame, Center the 3D view on the scene, to center the image in the center of the window (Supplementary File 1-Figure S4).</li>
	</ol>
	</li>
	<li>Save the biomodel (Supplementary File 1-Figure S5).
	<ol>
		<li>Click on Save in the upper-left margin.</li>
		<li>In the box that appears, only select the file &#34;tissue&#34; (Supplementary File 1-Figure S5).</li>
		<li>In the second column, in the dropdown menu, select STL.</li>
		<li>In the third column, in the dropdown menu, choose where to save ithe STL file. Click on Save. This is the file that will be used in the following steps.</li>
	</ol>
	</li>
</ol>
3. Preparation of the biomodel
<ol>
	<li>Download the MeshMixer software (see the Table of Materials). Install the program on the computer.
	<ol>
		<li>Import the STL image by selecting the Import option in the center of the screen (Supplementary File 1-Figure S6).</li>
	</ol>
	</li>
	<li>Select the biomodel.
	<ol>
		<li>Look for the Select option in the menu on the left side. Use any of the following main methods to select.</li>
		<li>Use the Select tool, select the thickness of the brush, and double-click on the femur (Supplementary File 1-Figure S7). If it is not possible to separate only the femur, it means that it has direct contact with other bone structures or soft parts; in that case, select Edit | Generate face Groups from the menu (Supplementary File 1-Figure S7). Use the Angle Threshold option and move the bar until the different structures have a different color, indicating that the pieces have been recognized as separate (Supplementary File 1-Figure S8).
		<ol>
			<li>With the Select tool, hold down the left mouse button while painting the part of the biomodel of interest.</li>
			<li>With the Select tool, click on a point outside the model, and hold down the left mouse button while painting a circle that includes the part that is of interest.</li>
		</ol>
		</li>
		<li>Use the Select tool to select the part of interest. Look for the option Select | Modify | Invert in the side menu, and press Delete&#160;(Supplementary File 1-Figure S9) to delete the unselected parts. At this juncture, the biomodel of the clean femur is obtained (Supplementary File 1-Figure S9).</li>
		<li>Make the model solid (Supplementary File 1-Figure S10).</li>
		<li>Navigate to Edit | Make Solid | Solid Type| Accurate.</li>
		<li>Maximize the Solid Accuracy and Mesh Density values.</li>
	</ol>
	</li>
	<li>Save the biomodel. Select the Export option from the side menu. Select the STL format and the folder to which the biomodel is exported.</li>
</ol>
4. Calculation of the proximal femoral anteversion
<ol>
	<li>Download the 3D Builder software (see the Table of Materials). Install the program on the computer. 
	NOTE: The program can only be downloaded if the operating system of the computer is Windows.</li>
	<li>Click on the Insert icon at the top of the screen (Supplementary File 1-Figure S11). Click on Add to import the biomodel to the scene (Supplementary File 1-Figure S12). 
	NOTE: The left mouse button makes it possible to rotate the object to see it in a 360&#176; view. With the right button, it is possible to scroll along the object. The central wheel of the mouse allows for zooming in.</li>
	<li>Click on Object | Settle to fix the object to the work plane so that it rests on the femoral condyles and the trochanter mayor. 
	NOTE: It is advisable to put the object vertically parallel to the y-axis and perpendicular to the x-axis marked on the working plane (Supplementary File 1-Figure S13).</li>
	<li>Perform the femoral osteotomy.
	<ol>
		<li>Click on Edit | Split from the top menu . When a rectangular cut plane appears, select Keep Both (Supplementary File 1-Figure S14).
		<ol>
			<li>Use the Move Mode button on the bar in the lower margin of the screen to move the cutting plane horizontally and vertically.</li>
		</ol>
		</li>
		<li>Use the Rotate Mode button on the bar in the lower margin of the screen to rotate the plane around the femur (Roll: 90&#176;, Pitch: 0&#176;, Yaw: 0&#176;) (Supplementary File 1-Figure S14).</li>
		<li>Put the cutting plane parallel to the x-axis and perpendicular to the y-axis. Click on Split. In this case, perform the osteotomy above the trochanter minor (intertrochanteric) (Supplementary File 1-Figure S15).</li>
	</ol>
	</li>
	<li>Calculate the femoral anteversion.
	<ol>
		<li>Insert the guides, which help to establish the reference points to measure the femoral anteversion in the image in the 3D environment of the program according to Murphy&#39;s method (Supplementary File 1-Figure S16A,B). To insert the guides, click on Insert | Add, and choose the 3mf Supplementary File 2. 
		NOTE: These guides were designed in-house, and the 3mf file Supplementary File 2 is accessible as a supplementary material provided in this article. The Murphy 3D method was implemented by establishing three measurement points in the same way as in the conventional method11 but in a 3D environment. The usual circumference at the level of the femoral head was replaced by a sphere, and the measurement was established by a circumference at the level of the trochanter minor. As a distal reference, the posterior intercondylar line was taken, as defined in the original Murphy method.</li>
		<li>Select only the proximal part of the femur on the right side of the screen and click on CTRL + X to cut the selection. This is how the femoral diaphysis appears (Supplementary File 1-Figure S17).</li>
		<li>Select the red circular guide and the purple circular guide (doing this means that they will be together) on the right side of the screen. Use the commands in the bottom margin panel to move the guides. 
		NOTE: The red guide represents the rotational axis of the osteotomy, while the violet guide represents the rotational axis of the femur.</li>
		<li>Put the guides in the center of the femoral diaphysis, and use the commands of the inferior margin panel to adjust the size. Ensure that all the edges touch the cortex of the bone (Supplementary File 1-Figure S18). 
		NOTE: When the two guides, the red circumference and the purple circumference, are used for the first time, they are selected together so that they move as a block, as if they were one guide, to measure the FAV, and they are placed in the femoral diaphysis just above the lesser trochanter at the osteotomy line.</li>
		<li>Click on CTRL + V to paste the proximal femur again (Supplementary File 1-Figure S19).</li>
		<li>Select only the sphere on the right side of the screen. Use the commands in the bottom margin panel to move the sphere and place it on top of the femoral head. Adjust the size, including all the edges touching the bone cortex (Supplementary File 1-Figure S20).</li>
		<li>Select the proximal femur on the right side, and cut it (CTRL + X).</li>
		<li>Select only the red plane on the right side of the screen (Supplementary File 1-Figure S21).</li>
		<li>Use the commands in the lower margin panel to move the red plane, and place it so that it passes through the center of the sphere and through the center of the circular guides. 
		NOTE: The grades marked by the panel in the lower margin correspond to the pathological femoral anteversion calculated on the CT using Murphy&#39;s method.</li>
		<li>Press CTRL + V to paste the proximal femur again (Supplementary File 1-Figure S22 A,B) .</li>
	</ol>
	</li>
	<li>Perform the rotational osteotomy of the proximal femur.
	<ol>
		<li>Select the proximal femur + the red circumference (only the red) + the sphere on the right side.</li>
		<li>Make an internal derotational proximal femoral osteotomy of 20&#176; (use the commands on the panel of the lower margin; add 20 on Pitch) (Supplementary File 1-Figure S23).</li>
		<li>Measure the new femoral anteversion (Supplementary File 1-Figure S24). 
		NOTE: The two guides are used again to perform the osteotomy. In this case, only the red guide is selected together with the proximal femur (so that when the proximal femur rotates, the red guide also rotates; step 4.6.1), while the violet guide is not selected (Supplementary File 1-Figure S25). In this way, the violet guide remains in the femoral diaphysis and does not participate in the rotation of the proximal femur.
		<ol>
			<li>Select the proximal femur + the red circumference, and press CTRL + X to cut these two elements.</li>
			<li>Select only the red plane, and place it so that it passes through the center of the sphere and through the center of the purple circular guide. 
			NOTE: A 1:1 relationship between the magnitude of the osteotomy and the correction of the deformity is not achieved because the derotation of the proximal femur does not follow the anatomical axis of the femur.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Perform the adjustment of the rotational osteotomy.
	<ol>
		<li>Select the femoral diaphysis + the red plane. Press CTRL + X to cut (Figure 27). (Supplementary File 1-Figure S25).</li>
		<li>Select the proximal femur + the sphere + the red circumference.</li>
		<li>Move the three elements en bloc so that the center of the red circumference matches the center of the purple circumference&#160;(Supplementary File 1-Figure S26).</li>
		<li>Recalculate the new femoral anteversion with the adjustment made (Supplementary File 1-Figure S27). 
		NOTE: Through this 3D method, it is shown that the rotation axis of the femur and the rotation axis of the osteotomy do not coincide. For this reason, it is necessary to make an adjustment that involves realigning the two guides so that the original femoral axis and the osteotomy axis coincide.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to disclose.

The most important finding of this study is that 3D technology allows the planning of proximal external derotational femoral osteotomy. This technology can simulate the surgery that is to be performed on a specific patient on the computer. It is a simple, reproducible, and free technique that uses software adaptable to most computers. The only technical problem may be that the 3D builder software works only with the Windows operating system. The major limitation is the learning curve. This protocol is still in the prelim...

Anterior knee pain (AKP) is a common clinical issue among adolescents and young adults. There is a growing body of evidence that increased femoral anteversion (FAV) plays an important role in the genesis of AKP1,2,3,4,5,6,7,8,9,10,11. In addition, this same evidence suggests that a derotational femoral osteotomy is beneficial for these patients, as good clinical results have been reported1,2,3,4,5,6,7,8,9,10,11. However, this type of surgery is not widely used in daily clinical practice among orthopedic surgeons, especially in the cases of adolescents and young active patients with anterior knee pain27, because the many controversial aspects generate uncertainty. For example, it has been observed that sometimes the correction obtained after the osteotomy is not what was previously planned. That is, there is not always a 1:1 ratio between the amount of rotation planned when performing the osteotomy and the amount of FAV corrected. This finding has not been studied to date. Therefore, it is the subject of the present paper. To explain the discrepancy between the magnitude of the rotation performed with the osteotomy and the magnitude of the correction of FAV, it was hypothesized that the axis of rotation of the osteotomy and the axis of rotation of the femur may not coincide.
One of the main problems to be addressed is accurately locating the femoral axis of rotation and the axis of rotation of the osteotomy. The first femoral axis is the femoral axis measured on the CT scan at the time of the patient&#39;s diagnosis, while the second femoral axis is the femoral axis measured after performing the osteotomy. Over the last decade, 3D technology has become increasingly important in preoperative planning, especially in orthopedic surgery and traumatology, for simplifying and optimizing surgical techniques15,16. The development of 3D technology has supported the creation of anatomical biomodels based on 3D imaging tests such as CT, in which customized prosthetic implants can be adapted17,18,19 and osteosynthesis plates can be molded in the case of fractures20,21,22. Additionally, 3D planning has already been used in previous studies to analyze the origin of the deformity in unilateral torsional alterations of the femur14. Currently, there are several software programs that are completely free and adaptable to most computers and 3D printers on the market, making this technology easily accessible to most surgeons in the world. This 3D planning allows for the accurate calculation of the initial axis of rotation of the femur and the axis of rotation of the femur after the intertrochanteric osteotomy has been performed. The main purpose of this study is to demonstrate that the axis of rotation of the femoral intertrochanteric osteotomy and the axis of rotation of the femur do not coincide. This 3D technology makes it possible to visualize this discrepancy between the axes and correct it through an adjustment of the osteotomy. The ultimate goal is to stimulate greater interest from orthopedic surgeons in this type of surgery.
This protocol with a 3D methodology is conducted in four fundamental steps. First, CT images are downloaded, and the 3D biomodel is created from the DICOM (Digital Imaging and Communication in Medicine) files of the CT scan. Higher-quality CT scans allow for better biomodels but mean the patient receives more ionizing radiation. For surgical planning with biomodels, the quality of conventional CT is sufficient. The DICOM image of a CT scan consists of a folder with many different files, with one file for each CT cut made. Each of these files contains not only the CT cut&#39;s graphical information but also the metadata (data associated with the image). To open the image, it is essential to have a folder with all the files of the series (the CT). The biomodel is extracted from the totality of the files.
Second, to obtain the 3D biomodel, it is necessary to download the 3D Slicer computer program, an open-source program with many utilities. Furthermore, this is the most widely used computer software in international 3D laboratories and has the advantage of being completely free of cost and downloadable from its main page. As this software is an X-ray image viewer, the DICOM image must be imported into the program.
Third, the first biomodel obtained with 3D Slicer will not match with the definitive one, because there will be regions such as the CT table or bones and soft parts nearby that are of no interest. The biomodel is &#34;cleaned&#34; almost automatically with the 3D design software, MeshMixer, which can also be downloaded directly from its official website for free. Finally, femoral anteversion is calculated, and the osteotomy is simulated using another free software from the Windows Store, 3D Builder.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D Builder</td>
			<td>Microsoft Corporation, Washington, USA</td>
			<td></td>
			<td>open-source program; https://apps.microsoft.com/store/detail/3d-builder/9WZDNCRFJ3T6?hl=en-us&#38;gl=us</td>
		</tr>
		<tr>
			<td>3D Slicer</td>
			<td>3D Slicer Harvard Medical School, Massachusetts, USA</td>
			<td></td>
			<td>open-source program; https://download.slicer.org</td>
		</tr>
		<tr>
			<td>MeshMixer&#160;</td>
			<td>Autodesk Inc&#160;</td>
			<td></td>
			<td>open-source program; https://meshmixer.com/download.html</td>
		</tr>
	</tbody>
</table>

three-dimensional preoperative virtual planning in derotational proximal femoral osteotomy

Anterior knee pain (AKP) is a common pathology among adolescents and adults. Increased femoral anteversion (FAV) has many clinical manifestations, including AKP. There is growing evidence that increased FAV plays a major role in the genesis of AKP. Furthermore, this same evidence suggests that derotational femoral osteotomy is beneficial for these patients, as good clinical results have been reported. However, this type of surgery is not widely used among orthopedic surgeons.
The first step in attracting orthopedic surgeons to the field of rotational osteotomy is to give them a methodology that simplifies preoperative surgical planning and allows for the previsualization of the results of surgical interventions on computers. To that end, our working group uses 3D technology. The imaging dataset used for surgical planning is based on a CT scan of the patient. This 3D method is open access (OA), meaning it is accessible to any orthopedic surgeon at no economic cost. Furthermore, it not only allows for the quantification of femoral torsion but also for carrying out virtual surgical planning. Interestingly, this 3D technology shows that the magnitude of the intertrochanteric rotational femoral osteotomy does not present a 1:1 relationship with the correction of the deformity. Additionally, this technology allows for the adjustment of the osteotomy so that the relationship between the magnitude of the osteotomy and the correction of the deformity is 1:1. This paper outlines this 3D protocol.

Femoral anteversion can be measured by different methods. Some of them focus on the femoral neck, using the line passing through the center of the neck and one passing through the femoral condyles as references. Others add a third reference point at the lesser trochanter23. Murphy&#39;s method, which is the most reliable in clinical practice because it has the best clinical-radiological relationship, is one such method using a third reference point25,<sup class="...

Watch this Scientific Journal Video about Three-Dimensional Preoperative Virtual Planning in Derotational Proximal Femoral Osteotomy at JoVE.com

Three-Dimensional Preoperative Virtual Planning in Derotational Proximal Femoral Osteotomy

This work presents a detailed surgical planning protocol using 3D technology with free open-source software. This protocol can be used to correctly quantify femoral anteversion and simulate derotational proximal femoral osteotomy for the treatment of anterior knee pain.

Anterior knee pain (AKP) is a common pathology among adolescents and adults. Increased femoral anteversion (FAV) has many clinical manifestations, ...

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872 Views.  Hospital Arnau de Vilanova. This work presents a detailed surgical planning protocol using 3D technology with free open-source software. This protocol can be used to correctly quantify femoral anteversion and simulate derotational proximal femoral osteotomy for the treatment of anterior knee pain.

Video: Three-Dimensional Preoperative Virtual Planning in Derotational Proximal Femoral Osteotomy

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. At present, it is common practice to collect 3 mm skin biopsies from the distal leg (DL) and the proximal thigh (PT) for the evaluation of length-dependent polyneuropathies 3. However, due to the multidirectional nature of IENFs, it is challenging to examine overlapping nerve structures through the analysis of two-dimensional (2D) imaging. Alternatively, three-dimensional (3D) imaging could provide a better solution for this dilemma.
In the current report, we present methods for applying 3D imaging to study painful neuropathy (PN). In order to identify IENFs, skin samples are processed for immunofluorescent analysis of protein gene product 9.5 (PGP), a pan neuronal marker. At present, it is standard practice to diagnose small fiber neuropathies using IENFD determined by PGP immunohistochemistry using brightfield microscopy 4. In the current study, we applied double immunofluorescent analysis to identify total IENFD, using PGP, and nociceptive IENF, through the use of antibodies that recognize tropomyosin-receptor-kinase A (Trk A), the high affinity receptor for nerve growth factor 5. The advantages of co-staining IENF with PGP and Trk A antibodies benefits the study of PN by clearly staining PGP-positive, nociceptive fibers. These fluorescent signals can be quantified to determine nociceptive IENFD and morphological changes of IENF associated with PN. The fluorescent images are acquired by confocal microscopy and processed for 3D analysis. 3D-imaging provides rotational abilities to further analyze morphological changes associated with PN. Taken together, fluorescent co-staining, confocal imaging, and 3D analysis clearly benefit the study of PN.

In order to study the changes of nociceptive intraepidermal nerve fibers (IENFs) in painful neuropathies (PN), we developed protocols that could directly examine three-dimensional morphological changes observed in nociceptive IENFs. Three-dimensional analysis of IENFs has the potential to evaluate the morphological changes of IENF in PN.

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Surgical Fixation of Sternal Fractures: Preoperative Planning and a Safe Surgical Technique Using Locked Titanium Plates and Depth Limited Drilling

Different ways to stabilize a sternal fracture are described in literature. Respecting different mechanisms of trauma such as the direct impact to the anterior chest wall or the flexion-compression injury of the trunk, there is a need to retain each sternal fragment in the correct position while neutralizing shearing forces to the sternum. Anterior sternal plating provides the best stability and is therefore increasingly used in most cases. However, many surgeons are reluctant to perform sternal osteosynthesis due to possible complications such as difficulties in preoperative planning, severe injuries to mediastinal organs, or failure of the performed method.
This manuscript describes one possible safe way to stabilize different types of sternal fractures in a step by step guidance for anterior sternal plating using low profile locking titanium plates. Before surgical treatment, a detailed survey of the patient and a three dimensional reconstructed computed tomography is taken out to get detailed information of the fracture&#8217;s morphology. The surgical approach is usually a midline incision. Its position can be described by measuring the distance from upper sternal edge to the fracture and its length can be approximated by the summation of 60 mm for the basis incision, the thickness of presternal soft tissue and the greatest distance between the fragments in case of multiple fractures.
Performing subperiosteal dissection along the sternum while reducing the fracture, using depth limited drilling, and fixing the plates prevents injuries to mediastinal organs and vessels.
Transverse fractures and oblique fractures at the corpus sterni are plated longitudinally, whereas oblique fractures of manubrium, sternocostal separation and any longitudinally fracture needs to be stabilized by a transverse plate from rib to sternum to rib. Usually the high convenience of a patient is seen during follow up as well as a precise reconstruction of the sternal morphology.

Here we present a method to stabilize sternal fractures by using locked titanium plates in a low profile design. Performing subperiosteal dissection along the sternum while reducing the fracture, using depth limited drilling, and fixing the plates provides a safe surgical way.

Different ways to stabilize a sternal fracture are described in literature. Respecting different mechanisms of trauma such as the direct impact to the ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

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 ...

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 ...

Digital Hybrid Model Preparation for Virtual Planning of Reconstructive Dentoalveolar Surgical Procedures

Virtual, hybrid three-dimensional (3D) model acquisition is presented in this article, utilizing the sequence of radiographic image segmentation, spatial registration, and free-form surface modeling. Firstly cone-beam computed tomography datasets were reconstructed with a semi-automatic segmentation method. Alveolar bone and teeth are separated into different segments, allowing 3D morphology, and localization of periodontal intrabony defects to be assessed. The severity, extent, and morphology of acute and chronic alveolar ridge defects are validated concerning adjacent teeth. On virtual complex tissue models, positions of dental implants can be planned in 3D. Utilizing spatial registration of IOS and CBCT data and subsequent free-form surface modeling, realistic 3D hybrid models can be acquired, visualizing alveolar bone, teeth, and soft tissues. With the superimposition of IOS and CBCT soft tissue, thickness above the edentulous ridge can be assessed about the underlying bone dimensions; therefore, flap design and surgical flap management can be determined, and occasional complications may be avoided.

A workflow for creating three-dimensional (3D) virtual hybrid models has been designed based on cone-beam computed tomography dataset and intraoral optical scans utilizing radiographic image segmentation methods and free-form surface modeling. Digital models are used for the virtual planning of reconstructive dentoalveolar surgical procedures.

Virtual, hybrid three-dimensional (3D) model acquisition is presented in this article, utilizing the sequence of radiographic image segmentation, spatial ...

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.

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, ...