Lumbar spines were harvested from 4-year-old Merino cross adult ewes (Ovis aries) following the ethical guidelines of the Animal Care and Ethics Committee of the Surgical and Orthopaedic Research Laboratories. Following the institutionally approved method of humane euthanasia, the lumbar spines were harvested using a sharp dissection tool, first incising through the skin and subcutaneous tissues, followed by fascia and musculature prior to disarticulation at the thoracolumbar and lumbosacral junctions. A harvested sample is shown in Figure 1A.
1. Preparation for the initial bath
<ol>
	<li>Manually remove soft tissue (muscle, connective tissue, fat, etc.) using a sharp dissection tool (number 22 scalpel blade) from the bone sample before processing further. 
	NOTE: This step is optional; however, removing as much soft tissue as possible reduces the time the water bath takes to macerate the tissues. The specimen size (~20 cm x 10 cm x 8 cm) is also reduced; therefore, more samples can be fitted into the bath.</li>
	<li>Seal the sample in a heat-safe sealable plastic bag after removing the air. It is recommended to use a vacuum bag using a commercial vacuum-sealing device (see Table of Materials). 
	NOTE: No additives are required for the initial 24 h bath. If there is significant muscle covering all surfaces of the bone and if there is already minimal soft tissue and most of the bone surfaces of the sample are exposed, proceed to step 3.2 (Figure 1B).</li>
</ol>
2. Procedure for the initial bath
<ol>
	<li>Completely submerge the sealed sample in a 70 &#176;C water bath for 24 h.</li>
	<li>After 24 h, remove the bag from the bath, open the bag, and allow the sample to cool until handleable.</li>
</ol>
3. Preparation for subsequent baths
<ol>
	<li>Remove as much soft tissue from the bone as possible using a sharp scalpel and running water as needed.</li>
	<li>Disarticulate any joints using a sharp scalpel to expose the cartilaginous tissue.
	<ol>
		<li>Keep the disarticulated pieces in situ using material such as orthopedic wire or cable ties (see Table of Materials) to maintain the anatomical position.</li>
	</ol>
	</li>
	<li>Seal the sample in a vacuum bag along with 10 mL of dishwashing detergent (see Table of Materials) and 10 mL of tap water. 
	&#8203;NOTE: The volume of detergent depends on the strength, concentration, and size of the sample.</li>
</ol>
4. Procedure for subsequent baths
<ol>
	<li>Completely submerge the sealed sample in a 70 &#176;C water bath for 24 h.</li>
	<li>After 24 h, remove the bag from the bath, open the bag, and allow the sample to cool until handleable.
	<ol>
		<li>Avoid letting the sample cool completely, as this allows softened cartilage to harden and adhere to bone, becoming more difficult to remove. 
		&#8203;NOTE: The time required for sample processing may vary based on size and type, and repeated removal of subsequent baths may be unnecessary. Additionally, the sample can remain in the bath for extended periods, which may aid in the interim removal of tissues.</li>
	</ol>
	</li>
	<li>Remove as much soft tissue as possible using a sharp dissection tool (number 22 scalpel blade on a dedicated scalpel handle) and running water.</li>
	<li>Repeat step 4 as required until the bone is free of soft tissue material. In our experience, this was required to be repeated only once.</li>
</ol>
5. Completion of the procedure
<ol>
	<li>Wash the sample with liquid detergent and rinse thoroughly with water. 
	NOTE: Alcohol may be used to speed drying.</li>
	<li>Allow the sample to dry for approximately 48 h.</li>
</ol>

<ol>
	<li>Leary, O. P., 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=Three-dimensional+printed+anatomic+modeling+for+surgical+planning+and+real-time+operative+guidance+in+complex+primary+spinal+column+tumors:+single-center+experience+and+case+series.">Three-dimensional printed anatomic modeling for surgical planning and real-time operative guidance in complex primary spinal column tumors: single-center experience and case series.</a> World Neurosurgery. 145, 116-126 (2021).</li><li>Tack, P., Victor, J., Gemmel, P., Annemans, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-printing+techniques+in+a+medical+setting:+a+systematic+literature+review.">3D-printing techniques in a medical setting: a systematic literature review.</a> BioMedical Engineering OnLine. 15 (1), 115 (2016).</li><li>Ventola, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medical+applications+for+3D+printing:+current+and+projected+uses.">Medical applications for 3D printing: current and projected uses.</a> Pharmacy and Therapeutics. 39 (10), 704-711 (2014).</li><li>Wilcox, B., Mobbs, R. J., Wu, A. M., Phan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+review+of+3D+printing+in+spinal+surgery:+the+current+state+of+play.">Systematic review of 3D printing in spinal surgery: the current state of play.</a> Journal of Spinal Surgery. 3 (3), 433-443 (2017).</li><li>Mairs, S., Swift, B., Rutty, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detergent:+an+alternative+approach+to+traditional+bone+cleaning+methods+for+forensic+practice.">Detergent: an alternative approach to traditional bone cleaning methods for forensic practice.</a> The American Journal of Forensic Medicine and Pathology. 25 (4), 276-284 (2004).</li><li>Husch, C., Berner, M., Goldammer, H., Lichtscheidl-Schultz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+note:+A+novel+method+for+gentle+and+non-destructive+removal+of+flesh+from+bones.">Technical note: A novel method for gentle and non-destructive removal of flesh from bones.</a> Forensic Science International. 323, 110778 (2021).</li><li>Couse, T., Connor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+maceration+techniques+for+use+in+forensic+skeletal+preparations.">A comparison of maceration techniques for use in forensic skeletal preparations.</a> Journal of Forensic Investigation. 3, 1-6 (2015).</li><li>Mahon, T. J., Maboke, N., Myburgh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+different+detergents+in+skeletal+preparations.">The use of different detergents in skeletal preparations.</a> Forensic Science International. 327, 110967 (2021).</li><li>Hussain, M., Hussain, N., Zainab, H., Qaiser, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+preservation+techniques+to+enhance+veterinary+anatomy+teaching.">Skeletal preservation techniques to enhance veterinary anatomy teaching.</a> IJAVMS. 1, 21-23 (2007).</li><li>Simonsen, K. P., Rasmussen, A. R., Mathisen, P., Petersen, H., Borup, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fast+preparation+of+skeletal+materials+using+enzyme+maceration.">A fast preparation of skeletal materials using enzyme maceration.</a> Journal of Forensic Science. 56 (2), 480-484 (2011).</li><li>Burkhard, M., Furnstahl, P., Farshad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensionally+printed+vertebrae+with+different+bone+densities+for+surgical+training.">Three-dimensionally printed vertebrae with different bone densities for surgical training.</a> European Spine Journal. 28 (4), 798-806 (2019).</li><li>Rose, A. S., 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=Multi-material+3D+models+for+temporal+bone+surgical+simulation.">Multi-material 3D models for temporal bone surgical simulation.</a> The Annals of Otology, Rhinology, and Laryngology. 124 (7), 528-536 (2015).</li><li>Werz, S. M., Zeichner, S. J., Berg, B. I., Zeilhofer, H. F., Thieringer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+printed+surgical+simulation+models+as+educational+tool+by+maxillofacial+surgeons.">3D printed surgical simulation models as educational tool by maxillofacial surgeons.</a> European Journal of Dental Education. 22 (3), 500-505 (2018).</li></ol>

The authors have to disclose.

This technical note aims to describe a simple, safe, and low-cost method to produce an anatomical bone model for the benefit of veterinary and medical education and for use in anatomical education and surgical planning. 
Pilot testing found that a bath temperature of 70 &#176;C provided the fastest processing time without causing damage to the samples. Higher temperatures caused an extensive breakdown of collagen within the bone, resulting in brittle samples with a chalky texture. The hot bath in this experiment was specifically for processing 3D-printed samples and was used due to its convenience; however, other less expensive commercial options, such as slow cookers or sous vide devices, may be more accessible. 
The addition of detergent was a critical step in the protocol. Compared to samples without detergent, adding detergent reduced the completion time from 168 h to 96 h. Samples without detergent did not dry completely and appeared noticeably darker with a greasy feel, both presumably due to lipid accumulation within the bone surface. Completed samples that appear dark or greasy could indicate a need for additional detergent (Figure 3). During pilot testing, detergent alone failed to disperse evenly throughout the samples once sealed, necessitating the use of water in addition. When water and detergent were added to the bags before sealing, there was occasionally difficulty in creating a reliable vacuum seal, which can be avoided by freezing liquids beforehand. The detergent used in this protocol, a general dishwashing detergent, was chosen based on cost and availability. This method can be performed using any dishwashing detergent to obtain similar results. Other multi-purpose household cleaners containing 2-Butoxyethanol may aid in degreasing and soft tissue breakdown more effectively and, therefore, may be beneficial for samples with excessive or tough cartilage, such as intervertebral discs7. Enzymatic detergents that actively digest soft tissue could be used to reduce maceration time compared to regular cleaning detergents, but the effects can be unpredictable, particularly if the user is not familiar with their use6,10. While enzymatic maceration poses a higher health risk, it has the potential to reduce the processing time from days to hours5,6. This method also benefits significantly from stirring to promote an enzymatic reaction with soft tissue, which may be hindered by a lack of flow within the vacuum-sealed bag.
The disarticulation of vertebrae was notably easier when detergent was added; further, after subsequent detergent baths, the intervertebral discs were softer and easier to remove, presumably due to a higher surface area for detergent penetration once disarticulated. Therefore, the samples were completed in a shorter time when disarticulated beforehand. Similarly, if the initial samples contained thick areas of cartilage, manual removal may be required in between baths to promote detergent penetration. Additionally, stirring a typical maceration bath has been shown to reduce the time required significantly, but this is not possible with bagged samples10. Adding detergent with small amounts of water in the bag and disarticulating joints as early as possible may reduce the impact of these limitations. 
Another critical step was to avoid letting samples cool to room temperature after removal from the hot bath, As residual heat allowed for easier removal of the softened cartilage from the samples. When allowed to cool, this cartilage formed a tough, gelatinous coating on the bone that was difficult to remove without reheating the sample. For the same reason, if running water is used to aid soft tissue removal, it is recommended to use warm water.
As opposed to 3D printed models, this method produces bone samples to replicate real subjects for surgical planning. The use of gentle detergents in this method may avoid changes to the integrity of the bone surface, as seen when using alternate products such as bleach and hydrogen peroxide6. With further developments in 3D printing for medical research, models can be printed with customized parameters such as cortical thickness, which have been shown to provide a positive haptic simulation of pedicle screw placement in spinal surgery11. However, these samples can be expensive to produce and require specific software and equipment, as well as meticulous planning and processing11,12. Additionally, the quality of 3D printed models is limited by the equipment and can risk the loss of finer detail that is preserved within the bone sample13. Patient-specific and 3D-printed models have several practical applications, while anatomical models sourced from bone can provide unrivaled anatomical detail with minimal cost and equipment. 
Limitations of this method include the availability of cadaveric specimens specific to their application. The described method completely removes soft tissue from bone, creating models that can only be used for surgical planning related to bone, and is not as practical for visualizing articulation, biomechanics, or surrounding structures. Due to this loss of articulation, reconstruction of models may be required. The reconstruction method varies with the bone type, and can include silicone, wire, cable ties, or glue. While approximating the relative locations of the bones, these techniques cannot replicate in vivo mechanics and reduce translation to surgical practice. Other limitations include the time taken to remove excess soft tissues from large samples as well as cartilage and other soft tissues, if present, based on the anatomical region of the specimen. 
The ability to produce simple bone models can significantly impact surgical success. Models for surgical planning have proven benefits, including reduced surgical time, more accurate screw and implant placement, and fewer complications such as blood loss4. This is especially useful in the case of veterinary surgery, where anatomy varies greatly by species. This simple protocol has the potential to produce clean bone models from cadavers without hazardous chemicals and constant odors and requires minimal equipment and labor, especially when compared to modern 3D printing techniques.

Removing soft tissue and cleaning bones are required for forensics, medical and biological research, and veterinary and medical education. Most techniques have been developed for forensic purposes, minimizing damage to the bone to preserve as much detail as possible. This can provide an accurate, tangible bone model for preoperative surgical planning, as well as intraoperative decision-making to help minimize complications1,2,3. This is beneficial in surgery by reducing operation times and blood loss and improving communication between surgeons, compared to planning with 2D images4. The use of these models may also reduce the reliance on intraoperative imaging, such as fluoroscopy, which may reduce radiation exposure to personnel.
Skeletal bone from cadavers has historically been used for these models; however, technological advances have pushed toward the use of manufactured models and, more recently, three-dimensional (3D) printed models. Bone models rely on the availability of cadaveric samples and the efficiency of processing these samples into usable models. 3D printing has the advantage of creative freedom, allowing for anatomical and patient-specific models, especially when anatomical abnormalities or neoplasms are present, or if the hardware needs to be manufactured or augmented to fit the patient1. These samples are also able to be sterilized and manipulated by surgeons during a procedure. However, this freedom comes with a cost, as it requires computed tomography (CT) scans, the materials and equipment required can be expensive, and expertise is essential to create the models in the required software1,4. Additionally, these factors can limit the precision and quality of the model, and hence the surgical planning and success1. 3D printed models may not be the best choice for cases where there is no need for patient-specific anatomy and where there is an immediate requirement for the model.
Commonly applied methods for the removal of soft tissue from cadaveric bone include manual cleaning, bacterial maceration, chemical maceration, cooking, and insect maceration5,6. The success of these methods is generally based on the cost, time, labor, equipment, safety, and quality of the final product5,7. Manual cleaning requires the most labor and a significant amount of time, but involves minimal equipment5. Bacterial maceration consists of leaving the sample in a cold or warm water bath for long periods of time, often up to 3 weeks, allowing bacteria to decompose the tissue6. This creates unpleasant odors, requires additional equipment to treat the bacteria, and creates a biosecurity hazard for the user5,6. The use of dermestid beetles is very effective with minimal labor, but requires the acquisition of a colony and husbandry of the animals, and is not considered an economic investment if used infrequently6,7. Chemical maceration usually involves the use of enzymes such as trypsin, pepsin, and papain, or commercial detergents containing substances such as surfactants and enzymes5,8. Although this method provides faster results, the chemicals used, such as sodium hydroxide, ammonia, bleach, and gasoline, may represent a health and safety risk and produce noxious odors that require personal protective equipment (PPE) and a fume hood5,7,8,9. Finally, extended heating provides another minimally intensive method but may produce odors requiring ventilation10.
A simple, safe, and low-cost method for the preparation of anatomical bone models would provide a useful tool for surgeons, students, educators, and researchers. This article describes a novel method for preparing skeletal bone models that avoids unpleasant odors and noxious chemicals, and produces a detailed surgical model with minimal equipment and labor.

<table><tbody><tr><td>Dimension Elite 3D printer</td><td>Stratasys, Eden Prairie, MN, United States</td><td></td><td>3D printer for production of surgical bone models based on reconstructed CT scans</td></tr><tr><td>Mimics Innovation Suite</td><td>Materialise NV, Leuven, Belgium</td><td>Suite 24</td><td>Software to create 3D models from imaging scans</td></tr><tr><td>Nylon cable ties</td><td>4Cabling, Alexandria, NSW, Australia</td><td>011.060.1042/011.060.1039</td><td>Used to maintain connection between vertebral bodies</td></tr><tr><td>Orthopaedic wire</td><td>B Braun, Bella Vista, NSW, Australia</td><td></td><td>Used to maintain connection between vertebral bodies</td></tr><tr><td>Support Cleaning Apparatus</td><td>Phoenix Analysis and Design Technologies, Tempe, AZ, United States</td><td>SCA-1200</td><td>Hot water bath for immersion of the sealed sample.</td></tr><tr><td>Ultra Strength Original Dishwashing Liquid</td><td>Colgate-Palmolive, New York, NY, United States</td><td></td><td>Dishwashing liquid added to sealed bag with sample for cleaning of the bone model.</td></tr><tr><td>Vacuum bags</td><td>Pacfood PTY LTD</td><td></td><td>Heat safe, sealable plastic bags</td></tr><tr><td>Vacuum Food sealer</td><td>Tempoo (Aust) PTY LTD</td><td></td><td>Vacuum food sealer to seal vacuum bags prior to bath immersion</td></tr></tbody></table>

vacuum-sealed hot water bath immersion for the preparation of anatomical and surgical cadaveric bone models

Bone models serve many purposes, including improving anatomical understanding, preoperative surgical planning, and intraoperative referencing. Several techniques for the maceration of soft tissues have been described, mainly for forensic analysis. For clinical research and medical use, these methods have been superseded by three-dimensional (3D) printed models, which require substantial equipment and expertise, and are costly. Here, cadaveric sheep vertebral bone was cleaned by vacuum sealing the specimen with commercial dishwashing detergent, immersing in a hot water bath, and subsequently manually removing the soft tissue. This eliminated the disadvantages of the previously existing maceration methods, such as the existence of foul odors, usage of hazardous chemicals, substantial equipment, and high costs. The described technique produced clean, dry samples while maintaining anatomical detail and structure to accurately model the osseous structures that can be useful for preoperative planning and intraoperative referencing. The method is simple, low-cost, and effective for bone model preparation for education and surgical planning in veterinary and human medicine.

Following this protocol, clean and dry sheep lumbar vertebral column models were created for surgical planning and reference. Samples consisting of seven lumbar vertebrae were processed within 4 days using this method, with one initial bath to remove the bulk of the muscle and three subsequent baths. Completion of the baths was indicated by the ease at which cartilage and connective tissue were removed from the bone. This varied based on the type and location of cartilage; thin layers were easily removed after one or two baths, but thick material surrounded by other tissue, such as intervertebral discs, took three or four baths. After drying for 48 h, the bones were expected to be much lighter in color and weight and feel dry and non-greasy. The bone models produced provide an accurate representation of the anatomy, preserving the fine osseous structures and contours of the bone, specifically articular facets, the vertebral endplate, and transverse processes, compared to 3D printed models. For comparison, an ovine lumbar sheep spine was CT scanned at a 0.5 mm slice thickness, imported into 3D modeling software (see Table of Materials), and segmented to produce a model of an individual vertebra. This was then printed using acrylonitrile butadiene styrene (ABS) filament in a 3D printer. Figure 2 showcases the 3D printed model of a sheep lumbar spine compared to the anatomical cadaveric model produced from the vacuum-sealed hot water bath immersions. The comparative images show that the 3D printed model does not accurately detail fine osseous details of the cadaveric specimens, with a loss of finer details such as contours of the bone, especially on the transverse processes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64764/64764fig01.jpg" /> 
Figure 1: Sheep lumbar spine samples at different stages during processing. (A) The freshly harvested lumbar spine requires preparation and an initial bath (steps 1-2), while (B) the sample with minimal soft tissue can proceed to subsequent baths (step 3.2). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64764/64764fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64764/64764fig02.jpg" /> 
Figure 2: Qualitative comparison between 3D printed and cadaveric bone models. A comparison of (A,B) a 3D printed model and (C,D) a cadaveric bone model demonstrates loss of detail at finer points, such as the ends of transverse processes and facet details in the 3D printed model relative to the bone. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64764/64764fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64764/64764fig03.jpg" /> 
Figure 3: Sheep lumbar spines processed using hot water bath immersion. Processed sheep lumbar spine models at the same step of the processing; however, the sample on the (A) left was processed with detergent, and the sample on the (B) right was without. The difference in color and texture must be noted. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64764/64764fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Vacuum-Sealed Hot Water Bath Immersion for the Preparation of Anatomical and Surgical Cadaveric Bone Models at JoVE.com

Vacuum-Sealed Hot Water Bath Immersion for the Preparation of Anatomical and Surgical Cadaveric Bone Models

The present protocol describes the maceration and cleaning of cadaveric bone with a vacuum-sealed, hot water bath immersion technique. This is a low-cost, safe, and effective method to produce anatomical specimens for surgical planning and medical education as an alternative to three-dimensional (3D) printed models.

Bone models serve many purposes, including improving anatomical understanding, preoperative surgical planning, and intraoperative referencing. Several ...

vacuum-sealed-hot-water-bath-immersion-for-preparation-anatomical

Nanyang Technological University

Research

JoVE Journal

Medicine

1.2K Views.  University of New South Wales (UNSW) Sydney. Deze methode produceert nauwkeurige anatomische botmodellen die de inheemse anatomie weergeven die helpt om onderzoekers, clinici en chirurgen te begeleiden bij de chirurgische aanpak en de plaatsing van implantaten, naast andere voordelen. Deze methode produceert schone en hygiënische anatomische botmodellen, wat belangrijk is, gezien het feit dat deze modellen bijvoorbeeld aan het bed of in het operatietheater staan. In vergelijking met 3D-geprinte anatomische botmodellen zijn onze modelle...

Video: Vacuum-Sealed Hot Water Bath Immersion for the Preparation of Anatomical and Surgical Cadaveric Bone Models

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Developmental Biology

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Cancer Research

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides vital information on bone turnover or bone formation and resorption. This has tremendous importance in a variety of clinical and research applications. It yields beautiful images1 and allows for techniques such as fluorochrome assessment and histomorphometry2. Fluorochrome analysis is a technique where fluorescent dyes that bind to calcium are injected at a particular time point, which allows for quantification of the amount of mineralisation at that given time. Histomorphometry is a process of bone quantification at the microscopic level. 
Performing undecalcified bone histology is technically challenging, particularly with large size specimens. It requires variations in technique from those used in standard paraffin embedded histology. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them. Specimen preparation, fixation and processing are achieved with a manner similar to other soft tissues, however due to the density and lower permeability of bone considerably longer fixation and processing times are required, often taking several weeks. Embedding is achieved using a supporting medium with similar or equal hardness and density to the bone such as methacrylate- based resins, but unlike paraffin infiltration and embedding, this is an irreversible step. Sectioning can be achieved by grinding which produces a thicker section, which is optimal for studies such as fluorochrome analysis. This is best achieved using a diamond blade on a macrotome. Alternatively, thinner sections can be produced for light microscopy and this is achieved using a sledge microtome with a very sharp blade. The sledge microtome provides the additional strength and stability required for large, hard blocks. Resin embedded sections can be stained with a variety of stains, which are demonstrated.

Undecalcified bone histology provides important information for a variety of clinical and research applications. It is technically challenging, particularly with large size specimens. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them.

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides ...

Biology

Proximal Cadaveric Femur Preparation for Fracture Strength Testing and Quantitative CT-based Finite Element Analysis

Cadaveric fracture testing is routinely used to understand factors that affect proximal femur strength. Because ex vivo biological tissues are prone to lose their mechanical properties over time, specimen preparation for experimental testing must be performed carefully to obtain reliable results that represent in vivo conditions. For that reason, we designed a protocol and a set of fixtures to prepare the femoral specimens such that their mechanical properties experienced minimal changes. The femora were kept in a frozen state except during preparation steps and mechanical testing. The relevant clinical measures of total hip and femoral neck bone mineral density (BMD) were obtained with a clinical dual X-ray absorptiometry (DXA) bone densitometer, and the 3D geometry and distribution of bone mineral were obtained using CT with a calibration phantom for quantitative estimations based on the greyscale values. Any possible bone disease, fracture, or the presence of implants or artifacts affecting the bone structure, was ruled out with X-ray scans. For preparation, all bones were carefully cleaned of excess soft tissue, and were cut and potted at the internal rotation angle of interest. A cutting fixture allowed the distal end of the bone to be cut off leaving the proximal femur at a desired length. To allow positioning of the femoral neck at prescribed angles during later CT scanning and mechanical testing, the proximal femoral shafts were potted in polymethylmethacrylate (PMMA) using a fixture designed specifically for desired orientations. The data collected from our experiments were then used for validation of quantitative computed tomography (QCT)-based finite element analysis (FEA), as described in a different protocol. In this manuscript, we present the protocol for the precise bone preparation for mechanical testing and subsequent QCT/FEA modeling. The current protocol was successfully applied to prepare about 200 cadaveric femora over a 6-year time period.

We present a robust protocol on how to carefully preserve and prepare cadaveric femora for fracture testing and quantitative computed tomography imaging. The method provides precise control over input conditions for the purpose of determining relationships between bone mineral density, fracture strength, and defining finite element model geometry and properties.

Cadaveric fracture testing is routinely used to understand factors that affect proximal femur strength.  Because ex vivo biological tissues are prone to ...

The Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Mediated Drug Delivery Studies

The chicken embryo and the blood-vessel rich chorioallantoic membrane (CAM) is a valuable in vivo model to investigate biomedical processes, new ultrasound pulsing schemes, or novel transducers for contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery. The reasons for this are the accessibility of the embryo and vessel network of the CAM as well as the low costs of the model. An important step to get access to the embryo and CAM vessels is to take the egg content out of the eggshell. In this protocol, three methods for taking the content out of the eggshell between day 5 and&#160;8 of incubation are described thus allowing the embryos to develop inside the eggshell up to these days. The described methods only require simple tools and equipment and yield a higher survival success rate of 90% for 5-day, 75% for 6-day, 50% for 7-day, and 60% for 8-day old incubated eggs in comparison to ex ovo cultured embryos (~50%). The protocol also describes how to inject cavitation nuclei, such as microbubbles, into the CAM vascular system, how to separate the membrane containing the embryo and CAM from the rest of the egg content for optically transparent studies, and how to use the chicken embryo and CAM in a variety of short-term ultrasound experiments. The in vivo&#160;chicken embryo and CAM model is extremely relevant to investigate novel imaging protocols, ultrasound contrast agents, and ultrasound pulsing schemes for contrast-enhanced ultrasound imaging, and to unravel the mechanisms of ultrasound-mediated drug delivery.

This protocol describes three methods on how to obtain and use 5 to 8-day old chicken embryos and their chorioallantoic membrane (CAM) as an in vivo&#160;model to study contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery.

The chicken embryo and the blood-vessel rich chorioallantoic membrane (CAM) is a valuable in vivo model to investigate biomedical processes, new ...

Bioengineering

Surgical Training for the Implantation of Neocortical Microelectrode Arrays Using a Formaldehyde-fixed Human Cadaver Model

This protocol describes a procedure to assist surgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. Recent technological progress has enabled the fabrication of microelectrode arrays that allow recording the activity of multiple individual neurons in the neocortex of the human brain. These arrays have the potential to bring unique insight onto the neuronal correlates of cerebral function in health and disease. Furthermore, the identification and decoding of volitional neuronal activity opens the possibility to establish brain-computer interfaces, and thus might help restore lost neurological functions. The implantation of neocortical microelectrode arrays is an invasive procedure requiring a supra-centimetric craniotomy and the exposure of the cortical surface; thus, the procedure must be performed by an adequately trained neurosurgeon. In order to provide an opportunity for surgical training, we designed a procedure based on a human cadaver model. The use of a formaldehyde-fixed human cadaver bypasses the practical, ethical and financial difficulties of surgical practice on animals (especially non-human primates) while preserving the macroscopic structure of the head, skull, meninges and cerebral surface and allowing realistic, operating room-like positioning and instrumentation. Furthermore, the use of a human cadaver is closer to clinical daily practice than any non-human model. The major drawbacks of the cadaveric simulation are the absence of cerebral pulsation and of blood and cerebrospinal fluid circulation. We suggest that a formaldehyde-fixed human cadaver model is an adequate, practical and cost-effective approach to ensure proper surgical training before implanting microelectrode arrays in the living human neocortex.

We designed a procedure in which a formaldehyde-fixed human cadaver is used to assist neurosurgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain.

This protocol describes a procedure to assist surgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. ...

Creation of a High-Fidelity, Low-Cost, Intraosseous Line Placement Task Trainer via 3D Printing

The description of procedural task trainers includes their use as a training tool to hone technical skills through repetition and rehearsal of procedures in a safe environment before ultimately performing the procedure on a patient. Many procedural task trainers available to date suffer from several drawbacks, including unrealistic anatomy and the tendency to develop user-created &#39;landmarks&#39; after the trainer tissue undergoes repeated manipulations, potentially leading to inappropriate psychomotor skill development. To ameliorate these drawbacks, a process was created to produce a high-fidelity procedural task trainer, created from anatomy obtained from computed tomography (CT) scans, that utilize ubiquitous three-dimensional (3D) printing technology and off-the-shelf commodity supplies.
This method includes creating a 3D printed tissue mold capturing the tissue structure surrounding the skeletal element of interest to encase the bony skeletal structure suspended within the tissue, which is also 3D printed. A tissue medium mixture, which approximates tissue in both high-fidelity geometry and tissue density, is then poured into a mold and allowed to set. After a task trainer has been used to practice a procedure, such as intraosseous line placement, the tissue media, molds, and bones are reclaimable and may be reused to create a fresh task trainer, free of puncture sites and manipulation defects, for use in subsequent training sessions.

We describe a procedure to process computed tomography (CT) scans into high-fidelity, reclaimable, and low-cost procedural task trainers. The CT scan identification processes, export, segmentation, modeling, and 3D printing are all described, along with the issues and lessons learned in the process.

The description of procedural task trainers includes their use as a training tool to hone technical skills through repetition and rehearsal of procedures ...

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve causes aortic stenosis and contributes to heart failure and stroke. Disease pathogenesis is multifactorial, and stresses such as inflammation, extracellular matrix remodeling, turbulent flow, and mechanical stress and strain contribute to the osteogenic differentiation of valve endothelial and valve interstitial cells. However, the precise initiating factors that drive the osteogenic transition of a healthy cell into a calcifying cell are not fully defined. Further, the only current therapy for CAVD-induced aortic stenosis is aortic valve replacement, whereby the native valve is removed (surgical aortic valve replacement, SAVR) or a fully collapsible replacement valve is inserted via a catheter (transcatheter aortic valve replacement, TAVR). These surgical procedures come at a high cost and with serious risks; thus, identifying novel therapeutic targets for drug discovery is imperative. To that end, the present study develops a workflow where surgically removed tissues from patients and donor cadaver tissues are used to create patient-specific primary lines of valvular cells for in vitro disease modeling. This protocol introduces the utilization of a cold storage solution, commonly utilized in organ transplant, to reduce the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing with the benefit of greatly stabilizing cells of the excised tissue. The results of the present study demonstrate that isolated valve cells retain their proliferative capacity and endothelial and interstitial phenotypes in culture upwards of several days after valve removal from the donor. Using these materials allows for the collection of control and CAVD cells, from which both control and disease cell lines are established.

This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity.

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve ...

Surgical Bone Implantation Technique for Rat Tibia Models of Diabetes and Osteoporosis

The rat has long served as a valuable animal model in implant dentistry and orthopedics, particularly in studying the interactions between biomaterials and bone tissue. The rat's tibia is frequently chosen due to its easy surgical access through thin tissue layers (skin and muscle) and the flattened shape of its medial face, facilitating the surgical insertion of intraosseous devices. Additionally, this model enables the induction of specific diseases, mimicking various clinical conditions to assess biological responses to different implant conditions like geometry, surface texture, or biological cues. However, despite its robust cortical structure, certain intraosseous devices may require adaptations in design and size for successful implantation. Therefore, establishing standardized surgical methods for manipulating both soft and hard tissues in the implantation region is essential for ensuring proper implant or screw device placement, particularly in fields like implant dentistry and orthopedics. This study included eighty Sprague Dawley rats divided into two groups based on their respective diseases: Group 1 with osteoporosis and Group 2 with Type 2 Diabetes. Implantations were performed at 4 weeks and 12 weeks, with the same surgeon following a consistent surgical technique. A positive biological response was observed, indicating complete osseointegration of all implants placed. These results validate the success of the surgical protocol, which can be replicated for other studies and serve as a benchmark for the biomaterials community. Notably, osseointegration values remained stable at both 4 weeks and 12 weeks for both disease models, demonstrating a durable integration of the implant over time and emphasizing the establishment of an intimate bone connection as early as 4 weeks.

The placement of implants in a rat model is an essential experimental procedure for clinical research. This study presents a comprehensive surgical protocol for implanting titanium implants into the tibia of rat models with diabetes and osteoporosis.

The rat has long served as a valuable animal model in implant dentistry and orthopedics, particularly in studying the interactions between biomaterials ...

Vacuum-Sealed Hot Water Bath Immersion for the Preparation of Anatomical and Surgical Cadaveric Bone Models

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video