Preservation of Porcine Biomodels for the Study of Comparative Human Anatomy

Summary

This article presents a protocol for preserving porcine biomodels. The proposed method involves utilizing a modified Chilean solution with a reduced formalin concentration. The preservation process consists of administering the solution through both repletion and immersion of the specimen, followed by deformolization and tissue softening using glycerin.

Abstract

The teaching of human anatomy in medical education faces challenges, necessitating effective alternatives for students to practically explore anatomical complexity. Porcine biomodels, with their anatomical similarities to humans, offer a promising solution. This article presents a detailed protocol for preserving porcine biomodels, addressing the need for accessible and efficient methods in comparative anatomy studies. This protocol proposes the use of a modified Chilean solution for biomodel preservation, employing formalinization techniques through repletion and immersion. Subsequently, deformalization is achieved using the modified Chilean solution without formaldehyde, followed by a glycerin softening process. Given the scarcity of literature on preservation techniques and the absence of a standardized procedure or test to evaluate tissue conservation, we suggest assessing tissue quality based on measures of resistance and elasticity. Our findings indicate a qualitatively high level of tissue preservation in our specimens. Furthermore, these biomodels are currently utilized successfully for morphology studies and in teaching human comparative anatomy to medical students.

Introduction

The teaching of anatomy in medical schools often encounters obstacles, such as difficulties in accessing human bodies for dissection and the lack of suitable facilities. These limitations underscore the need for effective alternatives that enable students to explore anatomical complexity practically and realistically. In this context, porcine biomodels have emerged as a promising alternative due to their anatomical similarities to humans, offering an accessible and efficient means of learning and teaching anatomy¹.

Anatomical preservation techniques aim to maintain the integrity of biological tissues while minimizing damage. These techniques have been employed for educational, academic, and research purposes in the study of both human and veterinary anatomy. Numerous substances have been tested to preserve bodies, organs, and tissues in their natural state for as long as possible². However, preserving and conserving anatomical specimens remains challenging, particularly for those intended for morphological studies, where high tissue preservation is required³.

Even though traditionally used solutions are widely available, limitations in day-to-day practice are evident. Formalin, one of the most common substances used, has several documented disadvantages for tissue preservation. These include its irritating odor, high toxicity, associated risks of cancer and mutagenicity for handlers, and the organoleptic changes it induces in tissues, such as stiffness and discoloration. These changes can alter the perception of the tissues' physiological properties when studied after fixation, potentially hindering precise and accurate morphological studies⁴. Studies have shown that fixation with high concentrations of formalin results in a greater degree of tissue stiffness⁵. Alternatives, such as the Thiel embalming technique, have demonstrated promising results with better conservation of original coloration and pliability of tissues⁶. However, this technique is more costly compared to other solutions⁷. These challenges present an opportunity to design and test new, affordable preservation techniques that still enable high-quality anatomy teaching.

The aim of this protocol is to describe the methodology employed at the Anatomy Laboratory of Universidad Icesi in Cali, Colombia, for the preservation of porcine biomodels used as educational tools for the comparative study of human anatomy.

Protocol

The technique described was developed in full compliance with the guidelines of the Institutional Ethics Committee for the Care and Use of Animals in Experimentation (CIECUAE) of Universidad Icesi, in accordance with Law 84 of 1989 and Rector's Resolution No. 847 (July 9, 2012). This ensures both scientific integrity and the welfare of the animals used, minimizing their suffering. Male Landrace pigs, 3 months old and weighing 15-20 kg, were utilized for this study. The reagents and equipment required are listed in the Table of Materials.

1. Porcine biomodel

1. Sacrifice of the animal
   1. Conduct a general veterinary evaluation and quarantine the selected pig specimen for 24 h. If the animal does not present any symptoms during this period, proceed with euthanasia.
2. Anesthesia
   1. Administer anesthesia intramuscularly using Ketamine + Xylazine + Atropine at doses of 10 mg/kg, 0.5 mg/kg, and 0.04 mg/kg, respectively (following institutionally approved protocols).
      NOTE: Ensure deep anesthesia. Assess the depth of anesthesia through physical examination. Deep anesthesia is indicated by a dilated and centered pupil, absence of the palpebral reflex, depression of the corneal reflex, and decreased respiratory rate, heart rate, and blood pressure.
3. Euthanasia
   1. Administer 5 mL of Sodium Pentobarbital/Diphenylhydantoin (390/50 mg/mL) intravenously. After administering the euthanasia agent, auscultate the animal's physiological parameters for the Suis Scrofa species: HR, 60-90; FR, 8-18; and T° 37-39°C⁸.
   2. Monitor the heart rate until it gradually decreases and stops completely. Observe immediate changes such as sphincter relaxation, cyanotic membranes, and absence of response to body stimuli. Consider euthanasia complete after 5 min of cardiac auscultation without detecting a heartbeat.
4. Storage and transportation
   1. Transport the porcine biomodel from the procedure room to the anatomy laboratory within half an hour of euthanasia to prevent decomposition and microbial proliferation, thereby optimizing the conservation of the biomodel.

2. Preservative solution based on Chilean solution

1. Preparation of the modified Chilean solution
   1. Use a modified Chilean solution containing Ethanol, Glycerin, Formaldehyde, Benzalkonium Chloride, coffee aromatic essence, and distilled water. Ensure the solution has a pH of 7.0.
   2. Prepare the preservative solution in a 50 L tank. First, add 10.6 L of distilled water to the tank. Next, add 7.6 L of 96% ethanol, 5 L of glycerin, 0.5 L of 50% benzalkonium chloride, and 0.1 L of coffee aromatic essence.
      NOTE: Refer to Table 1 for the required concentrations and volumes of each component needed to prepare 25 L of the modified Chilean solution.

3. Administration of the modified Chilean preservative solution

NOTE: Once the preservative solution is prepared, administer it through repletion and immersion.

1. Administration by repletion of the modified Chilean preservative solution
   NOTE: Use biosafety clothing for this procedure: a reusable half mask with A1P2 filters, biosafety goggles, a long-sleeved disposable surgical gown, a surgical cap, leggings, and nitrile gloves.
   1. Surgical approach
      1. Make an incision along the linea alba (using a scalpel handle 4, blade 22) from the xiphoid process to the pubic region.
      2. Identify the subcutaneous cellular tissue and the muscle fibers of the rectus abdominis muscle.
         NOTE: At the moment of the incision, identify the scant fatty tissue adhered to the skin, known as subcutaneous cellular tissue, and the muscle fibers of the rectus abdominis muscle located deeper⁹.
      3. Carefully cut to access the peritoneum, ensuring its opening to the abdominal cavity without injuring the liver and intestines, which will be exposed immediately.
      4. Retract the intestinal loops to the right side of the abdominal cavity. Identify the parietal peritoneum overlying the left kidney and renal vessels. Use these vascular structures to locate the abdominal aorta.
      5. Find the emergence of the left renal pedicle. Remove the peritoneum covering the aorta 5 cm above the renal artery.
      6. Upon exposing the left renal pedicle, mobilize the intestinal loops and incise the peritoneum covering the hilum. Dissect anteroposteriorly to identify first the renal vein, which is larger and located anteriorly.
      7. Locate the renal artery posteriorly, following its trajectory to its origin in the abdominal aorta. Finally, identify the renal pelvis, the most posterior structure of the hilum⁹.
      8. Carefully dissect the posterior part of the aorta. Use 0/0 silk to ligate the aorta with a knot above the level of the renal artery and another knot 5 cm above.
      9. Clamp the aorta immediately with two Kelly clamps at both ends of the isolated aortic segment.
      10. Incise the aortic wall with the tip of Metzembaum scissors, making a cut of approximately 0.3 cm in the anterior wall of the aorta, taking care not to perforate it.
      11. Knot the distal end while removing the Kelly clamp to insert a 3.2 mm blunt L-shaped needle into the arterial lumen. Remove the proximal Kelly clamp and start the perfusion of the modified Chilean preserving solution.
   2. Aortic perfusion of the modified Chilean preserving solution
   3. Proceed with the perfusion of the modified Chilean preserving solution.
      NOTE: Administer 0.25 L/kg with a pressure of 10 pounds using a perfusion pump.
   4. End of perfusion
      1. Once the perfusion of the preserving solution is complete, remove the perfusion cannula and knot the insertion site with 0/0 silk to prevent reflux of the preserving solution. Close the abdomen by suturing the incision at the linea alba with 0/0 silk.
         NOTE: To evaluate if adequate perfusion was achieved, check the acute indicators of saturation: extension of the upper and lower extremities, cervical and thoracic plethora, and runoff of the solution through the nostrils. The approximate perfusion time is 2 h.
2. Administration by immersion
   NOTE: Use biosafety clothing for this procedure: a reusable half mask with A1P2 filters, biosafety goggles, a long-sleeved disposable surgical gown, a surgical cap, leggings, and nitrile gloves.
   1. Administration by immersion of the modified Chilean preserving solution
      1. Store the porcine specimen in a 300 L tank filled with the same preservative solution described in step 2.
         NOTE: Ensure the biomodel is completely immersed using approximately 150 L of the solution. After immersion, store the biomodel at 20 °C for 6 months. Indicators of adequate preservation include the absence of decomposition signs (e.g., body edema, swelling, foul odor, lividity, and friability of the tissues).

4. Deformolization solution based on Chilean solution

1. Preparation of the deformolization solution
   1. Use a modified Chilean solution containing Ethanol, Glycerin, Benzalkonium Chloride, coffee aromatic essence, and distilled water. Ensure the solution has a pH of 7.0.
2. Prepare the deformolization solution in a 50 L tank. First, add 11.1 L of distilled water, then 7.8 L of 96% ethanol, 5 L of glycerin, 1 L of 50% benzalkonium chloride, and 0.1 L of coffee aromatic essence.
   NOTE: Refer to Table 2 for the required concentrations and volumes of each component needed to prepare 25 L of the formaldehyde-free modified Chilean solution.

5. Deformolization

NOTE: Use biosafety clothing for this procedure: a reusable half mask with A1P2 filters, biosafety goggles, a long-sleeved disposable surgical gown, a surgical cap, leggings, and nitrile gloves.

1. Administration by immersion of the formaldehyde-free modified Chilean preserving solution
   1. At the end of the 6-month immersion preservation period, transfer the biomodel to a 300 L tank filled with formaldehyde-free modified Chilean preserving solution. Store the biomodel for 4 weeks.
      NOTE: Ensure the biomodel is completely immersed using approximately 150 L of the solution.

6. Glycerin softening

1. Immediately after the deformolization process, immerse the biomodel in solutions with increasing concentrations of glycerin diluted in water: 50%, 70%, and 90% glycerin. Maintain the biomodel in each concentration for one week.

Results

The goal of this protocol is to present an effective and viable technique that allows for the preservation of porcine biomodels for comparative human anatomy teaching. There are currently no standardized methods or tests to evaluate model preservation. Therefore, the overall preservation of the model was assessed using indicators of solution repletion and signs of decomposition after the protocol was completed on the specimens. Furthermore, to objectively assess the biomodels' viability for teaching and research, a c...

Discussion

Traditionally, anatomical studies have relied on human cadavers; however, challenges in acquiring these specimens have led to the exploration of alternative methods. Porcine biomodels have proven to be valuable tools for studying human anatomy, offering anatomical similarities that facilitate learning and its extrapolation to medical applications in humans^12,13,14,15. Although cadaveric preserv...

Materials

Name	Company	Catalog Number	Comments
Benzalkonium Chloride	Protécnica Ingeniería	PROQUAT BC 50/80	CAS Number. 68424-85-1
Ethanol	Not applicable	Not applicable	Ethanol 96%
Formaldehyde	Albor químicos	Not applicable	Formaldehyde
Glycerin	Not applicable	Not applicable	Glycerin
Injection pump	Disánchez	Special Injector YA-02	Injection pump
Kelly forceps	Not applicable	Not applicable	Kelly forceps
Metzembaum scissors	Not applicable	Not applicable	Metzembaum scissors
Needle	Disánchez	L canule	L canule
Scalpel	Not applicable	Not applicable	Scalpel handle 4/ Scalpel blade 22
Suture Silk	Not applicable	Not applicable	Suture Silk 0/0