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 For clinical research and medical use, these methods have been superseded by three-dimensional (3D) printed models, which require substantial equipment and expertise, and are costly.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

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...

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...

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