No funding was received for this work.

This study followed the Declaration of Helsinki on medical protocol and ethics and the Institutional Ethical Review Board approved the study.
1. Segmentation using a segmentation software
NOTE: The import process of the DICOM files requires the orientation of the axial, coronal and sagittal planes in the pop-up window to finish the setup.
<ol>
	<li>In the Bone Segmentation menu, choose the General feature. Use the marker &#34;-&#34; for unwanted segments and &#34;+&#34; for the segment of interest. Add markers on the 3D reconstructed model or on the different cross sections when scrolling and moving throughout the scan.</li>
	<li>Choose the Set button that demonstrates the segmentation. At this point, correct the markings and add new ones for better accuracy. Press Apply to create the new segment. Multiple segments can be created this way.</li>
	<li>After segmentation is complete, export the files as stl 3D files for 3D printing or planning of 3D reconstruction implants in 3D design CAD programs.</li>
</ol>
2. Designing reconstruction implants using 3D design software
<ol>
	<li>After preforming the bone segmentation using the segmentation software, import the stl files into the 3D design software (see Table of Materials).</li>
	<li>If further separation is needed (e.g., if one part is intended to be moved separately), do so here. In the Sculpt Clay menu, use the shave tool to separate the bone into two parts. In the Select/Move Clay menu, select the clay and mark the part to work on. Then, copy this part and create a new identical object in the object list in order to manipulate its position as observed in the next stage.</li>
	<li>Perform segment movement at this stage. Make sure that the axis of rotation is set on the part of the bone to stay in the same position. In the Select/Move Clay menu, select Reposition and set the rotation axis as planned.</li>
	<li>As the human skull is mostly symmetrical, use the healthy side for guidance to obtain the right positioning/replacement of the missing/mal-positioned segment. Use a mirroring technique to create a mirroring image of the normal side. In the Construct Clay menu, use the Mirror Clay option and set the plane at the center of the skull.</li>
	<li>Based on the mirrored half, perform segment rotation if needed and reconstruct the avulsed bony part using the Add Clay tool in the Construct Clay menu. This reconstruction is performed in order to build a patient specific reconstruction implant in the next stages, which will reconstruct the correct facial contour.</li>
	<li>After reconstructing the bony segment, create the patient specific reconstruction implant. In the Curves menu, use the Draw Curve option and create a continuous outer shape of the desired implant.</li>
	<li>At this stage, duplicate the bony segment as it will be needed to perform a Boolean function to separate the constructed implant. This is performed in the object list window by right clicking the segment and pressing the Duplicate option.</li>
	<li>Work in the new duplicated segment. In the Detail Clay menu, use the Emboss With Curve option to create the volume of the reconstruction implant. Pick the outer form of the sketched implant, and then place the circle-shaped cursor inside the sketched implant, on the surface of the bone. Note that the emboss will work outwards or on the inside of the bone, depending on the placement of the cursor. Then, choose the desired parameters - most importantly, the distance option that controls the thickness of the implant.</li>
	<li>Separate the implant from the bony segment. In the object list, choose the previously duplicated object from step 2.7, right-click and select Boolean &#8594; Remove From. Then choose the object containing the created implant.</li>
	<li>In case holes for screw fixation or for allowing angiogenesis are required, select Planes Category &#8594; Create Plane to create a parallel plane in which the holes for the plate are designed. Using manual manipulation, place the planes in maximum parallelism to the implant. In the Sketch menu, choose a circle and create circles in the desired size and position. A second larger circle can be created, which will serve as the countersink for the head of the intendent screw.</li>
	<li>In the Curves menu, use the Project Sketch option and choose the sketches designated to be transferred from the plane to the implant.</li>
	<li>To generate the countersink for the screws, In the Detail Clay menu, use the Emboss With Curve option. Pick the outer circles of the sketch, place the circle-shaped cursor inside the marked circular area on the surface and enter the distance that controls the depths of the countersink (e.g., 0.3 mm). To complete the process, press Apply and Lower to make sure that the emboss is performed in a subtraction manner and not an additive one.</li>
	<li>To complete the holes, in the SubD Surfaces menu, use the Wire Cut SubD option to create rods perpendicular to the implant based on the small circles created in step 2.10.</li>
	<li>To create the holes using the rods, use Boolean &#62; Remove From as in step 2.9. Choose one rod after the other, right click in the object list &#8594; Boolean &#8594; Remove From &#8594; Created Implant. 
	NOTE: Alternatively, the desired screws can be created/scanned and the Boolean function can be used to create the desired holes.</li>
	<li>To create a mesh in the implant (allowing for angiogenesis for instance), first generate a sketch (using the curve option) of the planned mesh as in step 2.6.
	<ol>
		<li>In the Detail Clay menu, use the Emboss with Wrapped Image option. Choose an image according to which the mesh will be created (there are several templates which come with the program). The white parts of the image will be subtracted in the mesh, and the black parts will be spared.</li>
		<li>Using manual control, adjust the direction and size of the design. Set the distance that represents the thickness of the holes generated and press Apply. The patient specific implant is ready for production.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Goodsaid, F., Frueh, F., Burczynski, M. E., Hock, F., Gralinski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Personalized+Medicine.">Personalized Medicine.</a> Drug Discovery and Evaluation: Methods in Clinical Pharmacology. , (2019).</li><li>Hull, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apparatus+for+production+of+three-dimensional+objects+by+stereolithography.">Apparatus for production of three-dimensional objects by stereolithography.</a> Google Patents. , (1986).</li><li>Petzold, R., Zeilhofer, H. F., Kalender, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+prototyping+technology+in+medicine--basics+and+applications.">Rapid prototyping technology in medicine--basics and applications.</a> Computerized Medical Imaging and Graphics. 23 (5), 277-284 (1999).</li><li>Schmauss, D., Gerber, N., Sodian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+printing+of+models+for+surgical+planning+in+patients+with+primary+cardiac+tumors.">Three-dimensional printing of models for surgical planning in patients with primary cardiac tumors.</a> The Journal of Thoracic and Cardiovascular Surgery. 145 (5), 1407-1408 (2013).</li><li>Tam, M. D., Laycock, S. D., Bell, D., Chojnowski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-D+printout+of+a+DICOM+file+to+aid+surgical+planning+in+a+6+year+old+patient+with+a+large+scapular+osteochondroma+complicating+congenital+diaphyseal+aclasia.">3-D printout of a DICOM file to aid surgical planning in a 6 year old patient with a large scapular osteochondroma complicating congenital diaphyseal aclasia.</a> Journal of Radiology Case Reports. 6 (1), 31 (2012).</li><li>Emodi, O., Shilo, D., Israel, Y., Rachmiel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+planning+and+printing+of+guides+and+templates+for+reconstruction+of+the+mandibular+ramus+and+condyle+using+autogenous+costochondral+grafts.">Three-dimensional planning and printing of guides and templates for reconstruction of the mandibular ramus and condyle using autogenous costochondral grafts.</a> British Journal of Oral and Maxillofacial Surgery. 55 (1), 102-104 (2017).</li><li>Leiser, Y., Shilo, D., Wolff, A., Rachmiel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+reconstruction+in+mandibular+avulsion+injuries.">Functional reconstruction in mandibular avulsion injuries.</a> Journal of Craniofacial Surgery. 27 (8), 2113-2116 (2016).</li><li>Mazzoni, S., Bianchi, A., Schiariti, G., Badiali, G., Marchetti, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer-aided+design+and+computer-aided+manufacturing+cutting+guides+and+customized+titanium+plates+are+useful+in+upper+maxilla+waferless+repositioning.">Computer-aided design and computer-aided manufacturing cutting guides and customized titanium plates are useful in upper maxilla waferless repositioning.</a> Journal of Oral and Maxillofacial Surgery. 73 (4), 701-707 (2015).</li><li>Rachmiel, A., Shilo, D., Blanc, O., Emodi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+complex+mandibular+defects+using+integrated+dental+custom-made+titanium+implants.">Reconstruction of complex mandibular defects using integrated dental custom-made titanium implants.</a> British Journal of Oral and Maxillofacial Surgery. 55 (4), 425-427 (2017).</li><li>Xu, N., 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=Reconstruction+of+the+upper+cervical+spine+using+a+personalized+3D-printed+vertebral+body+in+an+adolescent+with+Ewing+sarcoma.">Reconstruction of the upper cervical spine using a personalized 3D-printed vertebral body in an adolescent with Ewing sarcoma.</a> Spine. 41 (1), E50-E54 (2016).</li></ol>

The authors have nothing to disclose.

With the constant developing use of computers for virtual planning of surgical procedures, the combination with another developing technology, 3D printing, led to a whole new era of surgical treatment. Accuracy is the goal of these technologies and patient specific care, as the future goal, is presented in the form of surgical guides and patient specific reconstruction implants. We discuss surgical guides as part of a different future protocol. In the current protocol, we discuss the segmentation of DICOM images into 3D ...

Personalized medicine is developing rapidly in many fields of medicine1. Oncologic personalized treatment is a subject of much discussion and thus is well known to the general population. 3D printing was first introduced by Charles Hull showing 3D printing of objects using stereolithography2. Since then, different technologies for 3D printing were developed. The method used is selected based on the purpose of the device.
The surgical field is rapidly embracing personalized medicine. Personalized treatment in the surgical field requires virtual planning using a computer-assisted design (CAD) software. The first stage always includes segmentation to create a 3D stl file. Computer assisted manufacturing (CAM) is referred as the manufacturing process of the 3D designed part. The first utilization of the technology was used in pre-operative model printing for surgical planning and mock surgery3,4,5. With the development of technology, virtual planning of the surgeries followed by the planning and manufacturing of surgical guides to assist in the surgery itself and patient specific reconstruction implants fitted perfectly on the bone of the patient became more popular6,7,8,9,10. The purpose of this protocol is to provide clinicians with the ability to create their own surgical guides and reconstruction patient specific implants. This method is more accurate than using stock plates because it fits perfectly and can be designed based on the characteristics of the specific defect. It also reduces the dependency on the surgeon&#39;s experience and reduces operation time.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>D2P (DICOM to Print)</td>
			<td>3D systems</td>
			<td></td>
			<td>Segmentation software to create 3D stl files</td>
		</tr>
		<tr>
			<td>Geomagic Freeform</td>
			<td>3D systems</td>
			<td></td>
			<td>Sculpted Engineering Design</td>
		</tr>
	</tbody>
</table>

3d planning and printing of patient specific implants for reconstruction of bony defects

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

A 40 year old female patient with a broken, stock supplied, reconstruction fixation plate from a previous injury and a non-union fracture in the left body of her lower jaw presented to the department. Imaging shows the broken fixation plate and the mal-positioned left segment of the lower jaw (Figure 1). Using segmentation software, segmentation of the lower jaw was performed separating the broken fixation plate (Supplemental Figure 1 and <st...

Watch this Scientific Journal Video about 3D Planning and Printing of Patient Specific Implants for Reconstruction of Bony Defects at JoVE.com

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

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

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

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6.3K Views.  Rambam Medical Care Center. This protocol describes the use of 3D planning and printing for reconstruction of bony defects. We use segmentation tools to create 3D models followed by 3D design software to create patient specific implants for reconstruction purposes concomitant to ablative surgery or as a second stage.

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

A Pipeline for 3D Multimodality Image Integration and Computer-assisted Planning in Epilepsy Surgery

Epilepsy surgery is challenging and the use of 3D multimodality image integration (3DMMI) to aid presurgical planning is well-established. Multimodality image integration can be technically demanding, and is underutilised in clinical practice. We have developed a single software platform for image integration, 3D visualization and surgical planning. Here, our pipeline is described in step-by-step fashion, starting with image acquisition, proceeding through image co-registration, manual segmentation, brain and vessel extraction, 3D visualization and manual planning of stereoEEG (SEEG) implantations. With dissemination of the software this pipeline can be reproduced in other centres, allowing other groups to benefit from 3DMMI. We also describe the use of an automated, multi-trajectory planner to generate stereoEEG implantation plans. Preliminary studies suggest this is a rapid, safe and efficacious adjunct for planning SEEG implantations. Finally, a simple solution for the export of plans and models to commercial neuronavigation systems for implementation of plans in the operating theater is described. This software is a valuable tool that can support clinical decision making throughout the epilepsy surgery pathway.

We describe the steps to use our custom designed software for image integration, visualization and planning in epilepsy surgery.

Epilepsy surgery is challenging and the use of 3D multimodality image integration (3DMMI) to aid presurgical planning is well-established. Multimodality ...

A Postoperative Evaluation Guideline for Computer-Assisted Reconstruction of the Mandible

Valid comparisons of postoperative accuracy results in computer-assisted reconstruction of the mandible are difficult due to heterogeneity in imaging modalities, mandibular defect classification, and evaluation methodologies between studies. This guideline uses a step-by-step approach guiding the process of imaging, classification of mandibular defects and volume assessment of three-dimensional (3D) models, after which a legitimized quantitative accuracy evaluation method can be performed between the postoperative clinical situation and the preoperative virtual plan. The condyles and the vertical and horizontal corners of the mandible are used as bony landmarks to define virtual lines in the computer-assisted surgery (CAS) software. Between these lines the axial, coronal, and both sagittal mandibular angles are calculated on both pre- and postoperative 3D models of the (neo)mandible and subsequently the deviations are calculated. By superimposing the postoperative 3D model to the preoperative virtually planned 3D model, which is fixed to the XYZ axis, the deviation between pre- and postoperative virtually planned dental implant positions can be calculated. This protocol continues and specifies an earlier publication of this evaluation guideline.

Here, we propose a practical, feasible and reproducible evaluation guideline for computer-assisted reconstruction of the mandible in order to create uniformity between studies regarding postoperative accuracy evaluation. This protocol continues and specifies an earlier publication of this evaluation guideline.

Valid comparisons of postoperative accuracy results in computer-assisted reconstruction of the mandible are difficult due to heterogeneity in imaging ...

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. ...

Combining Augmented Reality and 3D Printing to Display Patient Models on a Smartphone

Augmented reality (AR) has great potential in education, training, and surgical guidance in the medical field. Its combination with three-dimensional (3D) printing (3DP) opens new possibilities in clinical applications. Although these technologies have grown exponentially in recent years, their adoption by physicians is still limited, since they require extensive knowledge of engineering and software development. Therefore, the purpose of this protocol is to describe a step-by-step methodology enabling inexperienced users to create a smartphone app, which combines AR and 3DP for the visualization of anatomical 3D models of patients with a 3D-printed reference marker. The protocol describes how to create 3D virtual models of a patient&rsquo;s anatomy derived from 3D medical images. It then explains how to perform positioning of the 3D models with respect to marker references. Also provided are instructions for how to 3D print the required tools and models. Finally, steps to deploy the app are provided. The protocol is based on free and multi-platform software and can be applied to any medical imaging modality or patient. An alternative approach is described to provide automatic registration between a 3D-printed model created from a patient&rsquo;s anatomy and the projected holograms. As an example, a clinical case of a patient suffering from distal leg sarcoma is provided to illustrate the methodology. It is expected that this protocol will accelerate the adoption of AR and 3DP technologies by medical professionals.

Presented here is a method to design an augmented reality smartphone application for the visualization of anatomical three-dimensional models of patients using a 3D-printed reference marker.

Augmented reality (AR) has great potential in education, training, and surgical guidance in the medical field. Its combination with three-dimensional (3D) ...

Novel Percutaneous Approach for Deployment of 3D Printed Coronary Stenosis Implants in Swine Models of Ischemic Heart Disease

Minimally invasive methods for creating models of focal coronary narrowing in large animals are challenging. Rapid prototyping using three-dimensionally (3D) printed coronary implants can be employed to percutaneously create a focal coronary stenosis. However, reliable delivery of the implants can be difficult without the use of ancillary equipment. We describe the use of a mother-and-child coronary guide catheter for stabilization of the implant and for effective delivery of the 3D printed implant to any desired location along the length of the coronary vessel. The focal coronary narrowing was confirmed under coronary cineangiography and the functional significance of the coronary stenosis was assessed using gadolinium-enhanced first-pass cardiac perfusion MRI. We showed that reliable delivery of 3D printed coronary implants in swine models (n = 11) of ischemic heart disease can be achieved through repurposing mother-and-child coronary guide catheters. Our technique simplifies the percutaneous delivery of coronary implants to create closed-chest swine models of focal coronary artery stenosis and can be performed expeditiously, with a low procedural failure rate.

We describe a novel, cost-effective, and efficient technique for percutaneous delivery of three-dimensionally printed coronary implants to create closed-chest swine models of ischemic heart disease. The implants were fixed in place using a mother-and-child extension catheter with high success rate.

Minimally invasive methods for creating models of focal coronary narrowing in large animals are challenging. Rapid prototyping using three-dimensionally ...

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

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

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

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

Biotribological Testing and Analysis of Articular Cartilage Sliding against Metal for Implants

Osteochondral defects in middle-aged patients might be treated with focal metallic implants. First developed for defects in the knee joint, implants are now available for the shoulder, hip, ankle and the first metatarsalphalangeal joint. While providing pain reduction and clinical improvement, progressive degenerative changes of the opposing cartilage are observed in many patients. The mechanisms leading to this damage are not fully understood. This protocol describes a tribological experiment to simulate a metal-on-cartilage pairing and comprehensive analysis of the articular cartilage. Metal implant material is tested against bovine osteochondral cylinders as a model for human articular cartilage. By applying different loads and sliding speeds, physiological loading conditions can be imitated. To provide a comprehensive analysis of the effects on the articular cartilage, histology, metabolic activity and gene expression analysis are described in this protocol. The main advantage of tribological testing is that loading parameters can be adjusted freely to simulate in vivo conditions. Furthermore, different testing solutions might be used to investigate the influence of lubrication or pro-inflammatory agents. By using gene expression analysis for cartilage-specific genes and catabolic genes, early changes in the metabolism of articular chondrocytes in response to mechanical loading might be detected.

This protocol describes the preparation, biotribological testing, and analysis of osteochondral cylinders sliding against metal implant material. Outcome measures included in this protocol are metabolic activity, gene expression and histology.

Osteochondral defects in middle-aged patients might be treated with focal metallic implants. First developed for defects in the knee joint, implants are ...

Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes the atrioventricular node (AVN), to allow excitation and contraction of the whole heart. Any dysfunction of either SAN or AVN results in arrhythmias, indicating their fundamental role in electrophysiology and arrhythmogenesis. Mouse models are widely used in arrhythmia research, but the specific investigation of SAN and AVN remains challenging.
The SAN is located at the junction of the crista terminalis with the superior vena cava and AVN is located at the apex of the triangle of Koch, formed by the orifice of the coronary sinus, the tricuspid annulus, and the tendon of Todaro. However, due to the small size, visualization by conventional histology remains challenging and it does not allow the study of SAN and AVN within their 3D environment.
Here we describe a whole-mount immunofluorescence approach that allows the local visualization of labelled mouse SAN and AVN. Whole-mount immunofluorescence staining is intended for smaller sections of tissue without the need for manual sectioning. To this purpose, the mouse heart is dissected, with unwanted tissue removed, followed by fixation, permeabilization and blocking. Cells of the conduction system within SAN and AVN are then stained with an anti-HCN4 antibody. Confocal laser scanning microscopy and image processing allow differentiation between nodal cells and working cardiomyocytes, and to clearly localize SAN and AVN. Furthermore, additional antibodies can be combined to label other cell types as well, such as nerve fibers.
Compared to conventional immunohistology, whole-mount immunofluorescence staining preserves the anatomical integrity of the cardiac conduction system, thus allowing the investigation of AVN; especially so into their anatomy and interactions with the surrounding working myocardium and non-myocyte cells.

We provide a step-by-step protocol for whole-mount immunofluorescence staining of the sinoatrial node (SAN) and atrioventricular node (AVN) in murine hearts.

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes ...

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

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

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

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

Combining 3D-Printing and Electrospinning to Manufacture Biomimetic Heart Valve Leaflets

Electrospinning has become a widely used technique in cardiovascular tissue engineering as it offers the possibility to create (micro-)fibrous scaffolds with adjustable properties. The aim of this study was to create multilayered scaffolds mimicking the architectural fiber characteristics of human heart valve leaflets using conductive 3D-printed collectors.
Models of aortic valve cusps were created using commercial computer-aided design (CAD) software. Conductive polylactic acid was used to fabricate 3D-printed leaflet templates. These cusp negatives were integrated into a specifically designed, rotating electrospinning mandrel. Three layers of polyurethane were spun onto the collector, mimicking the fiber orientation of human heart valves. Surface and fiber structure was assessed with a scanning electron microscope (SEM). The application of fluorescent dye additionally permitted the microscopic visualization of the multilayered fiber structure. Tensile testing was performed to assess the biomechanical properties of the scaffolds.
3D-printing of essential parts for the electrospinning rig was possible in a short time for a low budget. The aortic valve cusps created following this protocol were three-layered, with a fiber diameter of 4.1 &#177; 1.6 &#181;m. SEM imaging revealed an even distribution of fibers. Fluorescence microscopy revealed individual layers with differently aligned fibers, with each layer precisely reaching the desired fiber configuration. The produced scaffolds showed high tensile strength, especially along the direction of alignment. The printing files for the different collectors are available as Supplemental File 1, Supplemental File 2, Supplemental File 3, Supplemental File 4, and Supplemental File 5.
With a highly specialized setup and workflow protocol, it is possible to mimic tissues with complex fiber structures over multiple layers. Spinning directly on 3D-printed collectors creates considerable flexibility in manufacturing 3D shapes at low production costs.

The presented method offers an innovative way for engineering biomimetic fiber structures in three-dimensional (3D) scaffolds (e.g., heart valve leaflets). 3D-printed, conductive geometries were used to determine shape and dimensions. Fiber orientation and characteristics were individually adjustable for each layer. Multiple samples could be manufactured in one setup.

Electrospinning has become a widely used technique in cardiovascular tissue engineering as it offers the possibility to create (micro-)fibrous scaffolds ...