Name: vacuum-sealed hot water bath immersion for the preparation of anatomical and surgical cadaveric bone models
Uploaded: 2022-12-02T14:50:00
Description: 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

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 harvest...

<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>

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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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...

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 ...

This method produces accurate anatomical bone models that depicts the native anatomy that helps to guide researchers, clinicians, and surgeons on the surgical approach and implant placement, amongst other benefits. This method produces clean and hygienic anatomical bone models, which is important, considering having these models at the bedside or in the surgical theater, for example. Compared to 3D-printed anatomical bone models, our models are low cost, safe, and effective to produce, and also compared to those 3D models, the architecture of the bone and the native anatomy is better represented by these surgical bone models.The technique described is to produce accurate anatomical bone models for the spine, but the technique could be easily extrapolated to other osseous structures of the body. The method better informs surgeons, researchers, and clinicians on how to plan and execute surgical techniques, and also informs them of what normal osseous anatomy is that will then help them to recognize pathology. But the technique could be easily extrapolated to a range of other osseous structures from a range of different species.To begin, manually remove soft tissue, such as muscle, connective tissue, fat, et cetera, using a sharp dissection tool from the bone sample, and seal the sample in a heat-safe sealable plastic bag after removing the air. Completely submerged the sealed sample in a 70-degree Celsius water bath for 24 hours, and then remove the bag from the bath, open the bag, and allow the sample to cool until handleable. Remove as much soft tissue from the bone as possible using a sharp scalpel and running water as needed.To disarticulate any joints to expose the cartilaginous tissue, keep the disarticulated pieces in situ using materials such as orthopedic wire or cable ties to maintain the anatomical position. Seal the sample in a vacuum bag along with 10 milliliters of dishwashing detergent and 10 milliliters of tap water, and submerge the sealed sample in a 70-degree Celsius water bath for 24 hours. After removing the bag from the bath, repeat the steps shown earlier to remove as much soft tissue as possible until the bone is free of soft tissue material.Finally, allow the sample to dry for approximately 48 hours. A qualitative comparison between 3D-printed and cadaveric bone models is presented here. These images show that the 3D-printed model does not accurately detail fine osseous detail of the cadaveric specimens, with a loss of finer details, such as contours of the bone, especially on the transverse processes.Sheep lumbar spine models processed with and without detergent at the same step of the processing are shown here. From these two images, a clear difference in color and texture can be noted. To reproduce this method, the individual or research group does need access to anatomical specimens, a hot water bath, and vacuum-sealing equipment.The dissection of soft tissues pre and post-hot water bath is not technically demanding, but it does require some fine motor skills and some experience using sharp dissection equipment and other surgical equipment.

Research

JoVE Journal

Medicine

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Adjustable Stiffness, External Fixator for the Rat Femur Osteotomy and Segmental Bone Defect Models

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices ...

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies ...

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 ...