The authors acknowledge funding from National Natural Science Foundation of China (No. 81771971), Shanghai Pujiang Program (No. 14PJD008 and 17PJ1401500), &#34;Chen Guang&#34; Project Supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation (No. 14CG06), Natural Science Foundation of Shanghai (Nos. 17411962800 and 17ZR1432900), and Science and Technology Commission of Shanghai Municipality (17JC1400200). W.Z. acknowledges funding from the National Natural Science Foundation of China (31501555 and 81772007, and 21734003), the China&#39;s 1000 Young Talents Program, Education Commission of Shanghai Municipality (Young Eastern Professorship Award), and Science and Technology Commission of Shanghai Municipality (17JC1400200 and 16391903900).

The present study was approved by the ethics committee of Zhongshan Hospital Fudan University (B2016-142R) and all participants gave their informed consent.
1. Diagnosis of the Aortic Anomaly by Symptoms and Acquisition of Imaging Data
<ol>
	<li>Identify patients who have symptoms such as chest pain, dysphagia, or a blood pressure difference of the upper limbs in out-patient clinic. Exclude patients who may be intolerant of the operation.</li>
	<li>Perform CT angiography in these patients to diagnose Kommerell&#8217;s diverticulum8.</li>
</ol>
2. Segmentation of Regions of Interest
<ol>
	<li>Import all the CT angiography images into the software in a DICOM format. The resolution of these images was 512 &#215; 512 pixels, and the slice thickness was 1 mm.
	<ol>
		<li>Double click the patient case from case library and open it.</li>
		<li>Select the DICOM series and click Model Recon to open the model recon page.</li>
	</ol>
	</li>
	<li>Have an engineer and a team of cardiac surgeons review the DICOM formatted raw data to identify key anatomic features and region of interest (ROI).</li>
	<li>Use gray value-based thresholding to segment the ROI.
	<ol>
		<li>Click the Threshold Segmentation button and adjust the threshold range for the vascular mask. The default range is between 226 to 3071.</li>
		<li>Click the Confirm button for threshold segmentation and the vascular mask will show in the object list. Click the Recon button from the right of the mask and the 3-D vascular mask will be reconstructed and shown in 3-D viewer.</li>
		<li>Click the Threshold Segmentation button and adjust the threshold range for the trachea mask. Click the Marquee Segmentation button to limit the region of interest to the mediastinum and the lung. The default range is between -1024 to -520.</li>
		<li>Click the Mask Edit button and erase the connection between the trachea and the lung.</li>
		<li>Click the Region Grow button and select a seed by clicking any point/pixel at the mask in any one of the 2-D viewers. Check and confirm that the region grows as result, and that the trachea mask will show in the object list.</li>
		<li>Click the Recon button on the right of the mask, and the 3-D trachea will be reconstructed in the 3-D viewer.</li>
	</ol>
	</li>
	<li>Save the ROI as masks for 3-D reconstruction.</li>
</ol>
3. 3-D Reconstruction of the ROI
<ol>
	<li>Adopt the gray value interpolation algorithm to calculate the surface mesh of the 3-D model. Make the surface a triangle to match the outmost voxels of the mask.</li>
	<li>Click the Export button to export the 3-D model as an STL file.</li>
	<li>Place the model on the center of the building platform. Orient the model by aligning the tangent of the vessel centerline at its extremity to be parallel to the Z axis of the building platform. Supports were automatically generated to the overhangs using the default parameters.</li>
	<li>Click the Slice | Save to save as a file ready for 3-D printing.</li>
</ol>
4. 3-D Printing
<ol>
	<li>Perform stereolithographic printing with a 3-D printer. Use the following parameters: a slice distance of 1 mm, a resolution of 512 &#215; 512 pixels, a building layer thickness of 0.1 mm, and a laser spot diameter of 80 &#956;m.</li>
	<li>Use ultraviolet light at 405 nm to harden the photosensitive resin by scanning the contours sliced by the software. The ultraviolet light laser speed is 3 m/s. 
	NOTE: When one slice of the digital 3-D model was built, the building platform went up 0.1 mm for the next slice. The physical model was built layer by layer. The next layer was formed on top of the previous layer. The 3-D physical model was built layer by layer in this way.</li>
</ol>
5. Preoperative Planning and Intraoperative Review Using 3-D Printed Models
<ol>
	<li>Before the surgery, have surgeons make detailed and accurate surgical plans for each patient by learning the 3-D printed models.</li>
	<li>During the surgery, place the 3-D printed models in the operation room and have a nurse hold them. The anatomic details were reviewed by surgeons during the vascular location and separation. 
	NOTE: The surgical treatment included resection of diverticulum and reconstruction of aortic branches. The impregnated woven polyester tube graft was applied to replace the resected aorta1,2,3,9. All patients were sent to cardiac surgery intensive care unit after surgery.</li>
</ol>

<ol>
	<li>Tanaka, A., Milner, R., Ota, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kommerell's+diverticulum+in+the+current+era:+a+comprehensive+review.">Kommerell's diverticulum in the current era: a comprehensive review.</a> General Thoracic and Cardiovascular Surgery. 63 (5), 245-259 (2015).</li><li>Rosu, C., Dorval, J. F., Abraham, C. Z., Cartier, R., Demers, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-stage+hybrid+repair+of+right+aortic+arch+with+Kommerell's+Diverticulum.">Single-stage hybrid repair of right aortic arch with Kommerell's Diverticulum.</a> The Annals of Thoracic Surgery. 103 (4), e381-e384 (2017).</li><li>Idrees, J., 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=Hybrid+repair+of+Kommerell+diverticulum.">Hybrid repair of Kommerell diverticulum.</a> The Journal of Thoracic and Cardiovascular Surgery. 147 (3), 973-976 (2014).</li><li>Kankala, R. 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=Fabrication+of+arbitrary+3-D+components+in+cardiac+surgery:+from+macro-,+micro-+to+nanoscale.">Fabrication of arbitrary 3-D components in cardiac surgery: from macro-, micro- to nanoscale.</a> Biofabrication. 9 (3), 032002 (2017).</li><li>Vukicevic, M., Mosadegh, B., Min, J. K., Little, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+3-D+printing+and+its+future+directions.">Cardiac 3-D printing and its future directions.</a> JACC Cardiovascular Imaging. 10 (2), 171-184 (2017).</li><li>Yoo, S. J., Spray, T., Austin, E. H., Yun, T. J., van Arsdell, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hands-on+surgical+training+of+congenital+heart+surgery+using+3-dimensional+print+models.">Hands-on surgical training of congenital heart surgery using 3-dimensional print models.</a> The Journal of Thoracic and Cardiovascular Surgery. 153 (6), 1530-1540 (2017).</li><li>Hermsen, J. L., 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=Scan,+print,+practice,+perform:+Development+and+use+of+a+patient-specific+3-dimensionalprinted+model+in+adult+cardiac+surgery.">Scan, print, practice, perform: Development and use of a patient-specific 3-dimensionalprinted model in adult cardiac surgery.</a> The Journal of Thoracic and Cardiovascular Surgery. 153 (1), 132-140 (2017).</li><li>Sun, X., Zhang, H., Zhu, K., Wang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-specific+three-dimensional+printing+for+Kommerell's+diverticulum.">Patient-specific three-dimensional printing for Kommerell's diverticulum.</a> International Journal of Cardiology. 255, 184-187 (2018).</li><li>Ota, T., Okada, K., Takanashi, S., Yamamoto, S., Okita, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+treatment+for+Kommerell's+diverticulum.">Surgical treatment for Kommerell's diverticulum.</a> The Journal of Thoracic and Cardiovascular Surgery. 131 (3), 574-578 (2006).</li><li>Agematsu, K., Ueda, T., Hoshino, S., Nishiya, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rupture+of+Kommerell+diverticulum+after+total+arch+replacement.">Rupture of Kommerell diverticulum after total arch replacement.</a> Interactive Cardiovascular and Thoracic Surgery. 11 (6), 800-802 (2010).</li></ol>

The authors have nothing to disclose.

Congenital aortic anomalies comprise a rare spectrum of cardiovascular diseases, which often show complex aortic anomalies. Medical imaging, such as CT and MR, are required to elucidate complex aortic arch anomalies, the abnormal branching pattern, their relationship with the trachea and the esophagus, and other associated pathologies. Both CT and MR angiography can provide 2-D information of aortic vessel locations. With 3-D digital reconstruction of 2-D imaging, the anatomical relationship of the aortic vessels can be ...

Congenital aortic anomalies are extremely rare congenital malformations of the aortic arch system. They can be diagnosed either by imaging analysis or by evaluation of entities like dysphagia or subclavian steal1. In clinical scenarios, it is important to identify the anatomical anomaly in the confined surgical space that has limited visualization during the surgery2,3. Currently, conventional planar two-dimensional (2-D) imaging, such as computed tomography (CT) and magnetic resonance imaging (MRI), are usually presented to surgeons before the surgery. However, it is difficult for surgeons to image the anomaly based on the 2-D imaging. Consequently, they could encounter unpredictable difficulties while trying to separate the complex aortic vessels during surgery. Unpredictable injury to the vessel, the trachea and the esophagus could occur and result in disastrous outcomes.
In the last decade, 3-D imaging modeling has been used in cardiac surgery to help surgeons understand the complex anatomic anomaly4,5,6,7. Three-dimensional (3-D) printing technology can help convert the modeling data into a physical model. Compared with the digital reconstruction, 3-D printed physical models could present a better understanding of the anatomical details and provide an intuitional view of the malformation. For aortic anomaly surgery, the printed intuitional 3-D model is significant because poor understanding of aortic locations could be disastrous to patients. During the surgery, any mistake could lead to unpredictable bleeding and injury. Using the printed models, surgeons can fully understand the spatial relationships of aortic branches. During the surgery, the surgeons can also perform real-time review of the 3-D models to avoid confusion of the complex vascular locations.
Here, we present a protocol to apply 3-D printed models for pre-operative planning and intra-operative guidance while dealing with congenital aortic diseases. Kommerell&#39;s diverticulum, a type of complex congenital aortic anomaly, was selected as a case study. The steps include diagnosis based on computed tomography angiography (CTA) imaging, partitioning regions of interest, building 3-D models, preoperative surgical planning, and intra-operative reviewing of 3-D printed models8. This 3-D printing strategy could substantially reduce the risk of unpredictable tissue injury during the surgery.

<table border="0" cellpadding="0" cellspacing="0" width="911">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:247px;">3D printer</td>
			<td style="width:231px;">Meditool Enterprise Co., Ltd</td>
			<td style="width:136px;"></td>
			<td style="width:297px;">For 3D printing</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Chaos Version 2.0</td>
			<td>Meditool Enterprise Co., Ltd</td>
			<td style="width:136px;"></td>
			<td>For 3D segmentation and reconstruction</td>
		</tr>
	</tbody>
</table>

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.

Acquisition of CT angiography images, digital modeling and 3-D printing were all done in a hospital. Two hours were spent to get the 3-D model from the CT angiography image ready for the 3-D printing. Using the procedure and 3-D printer here, a patient-specific 3-D physical model can be sent to physicians quickly and the surgical decision can be made in time. The workflow from acquisition of CT angiography data to 3-D printing was shown in Figure 1. From the ...

Watch this Scientific Journal Video about Three-Dimensional Printing of a Complex Aortic Anomaly at JoVE.com

Three-Dimensional Printing of a Complex Aortic Anomaly

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-of-a-complex-aortic-anomaly

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

6.6K Views.  Fudan University. 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.

Video: Three-Dimensional Printing of a Complex Aortic Anomaly

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

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. ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

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 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 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 ...

Multicolor 3D Printing of Complex Intracranial Tumors in Neurosurgery

Three-dimensional (3D) printing technologies offer the possibility of visualizing patient-specific pathologies in a physical model of correct dimensions. The model can be used for planning and simulating critical steps of a surgical approach. Therefore, it is important that anatomical structures such as blood vessels inside a tumor can be printed to be colored not only on their surface, but throughout their whole volume. During simulation this allows for the removal of certain parts (e.g., with a high-speed drill) and revealing internally located structures of a different color. Thus, diagnostic information from various imaging modalities (e.g., CT, MRI) can be combined in a single compact and tangible object.
However, preparation and printing of such a fully colored anatomical model remains a difficult task. Therefore, a step-by-step guide is provided, demonstrating the fusion of different cross-sectional imaging data sets, segmentation of anatomical structures, and creation of a virtual model. In a second step the virtual model is printed with volumetrically colored anatomical structures using a plaster-based color 3D binder jetting technique. This method allows highly accurate reproduction of patient-specific anatomy as shown in a series of 3D-printed petrous apex chondrosarcomas. Furthermore, the models created can be cut and drilled, revealing internal structures that allow for simulation of surgical procedures.

The protocol describes the fabrication of fully colored three-dimensional prints of patient-specific, anatomical skull models to be used for surgical simulation. The crucial steps of combining different imaging modalities, image segmentation, three-dimensional model extraction, and production of the prints are explained.

Three-dimensional (3D) printing technologies offer the possibility of visualizing patient-specific pathologies in a physical model of correct dimensions. ...

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, ...

Three-Dimensional Printing of a Complex Aortic Anomaly

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