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

Summary

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.

Abstract

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 For clinical research and medical use, these methods have been superseded by three-dimensional (3D) printed models, which require substantial equipment and expertise, and are costly.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.

Introduction

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 complications^1,2,3. 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 software^1,4. 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 maceration^5,6. The success of these methods is generally based on the cost, time, labor, equipment, safety, and quality of the final product^5,7. 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 user^5,6. 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 infrequently^6,7. Chemical maceration usually involves the use of enzymes such as trypsin, pepsin, and papain, or commercial detergents containing substances such as surfactants and enzymes^5,8. 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 hood^5,7,8,9. 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.

Protocol

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

1. 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.
2. 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).

2. Procedure for the initial bath

1. Completely submerge the sealed sample in a 70 °C water bath for 24 h.
2. After 24 h, remove the bag from the bath, open the bag, and allow the sample to cool until handleable.

3. Preparation for subsequent baths

1. Remove as much soft tissue from the bone as possible using a sharp scalpel and running water as needed.
2. Disarticulate any joints using a sharp scalpel to expose the cartilaginous tissue.
   1. Keep the disarticulated pieces in situ using material such as orthopedic wire or cable ties (see Table of Materials) to maintain the anatomical position.
3. Seal the sample in a vacuum bag along with 10 mL of dishwashing detergent (see Table of Materials) and 10 mL of tap water.
   ​NOTE: The volume of detergent depends on the strength, concentration, and size of the sample.

4. Procedure for subsequent baths

1. Completely submerge the sealed sample in a 70 °C water bath for 24 h.
2. After 24 h, remove the bag from the bath, open the bag, and allow the sample to cool until handleable.
   1. Avoid letting the sample cool completely, as this allows softened cartilage to harden and adhere to bone, becoming more difficult to remove.
      ​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.
3. Remove as much soft tissue as possible using a sharp dissection tool (number 22 scalpel blade on a dedicated scalpel handle) and running water.
4. 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.

5. Completion of the procedure

1. Wash the sample with liquid detergent and rinse thoroughly with water.
   NOTE: Alcohol may be used to speed drying.
2. Allow the sample to dry for approximately 48 h.

Representative Results

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.

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). Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

Discussion

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 °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 use^6,10. While enzymatic maceration poses a higher health risk, it has the potential to reduce the processing time from days to hours^5,6. 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 processing^11,12. 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.

Materials

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