Thiel-embalming method[1–2] is a soft-fix embalming method developed by W. Thiel in 1990s, and since its first report in 1992, has produced cadavers that are both flexible and able to be long preserved, suitable for many training and research activities[4–6]. Contrary to conventional preservation techniques like freezing preservation and formalin embalming, the flexibility of the cadavers is excellent from the start and no additional measures are needed to achieve this.
Due to the unique advantage of Thiel embalmed cadavers, they can be employed in multiple research and training procedures such as arteries research, evaluation of mortuary cooling equipment, MRI research, ventilation research, ear motion, orthopedic studies, research on explanted organs[8,13–14], anesthesia[15–16], oral surgery and, neural modulus[18–19], spinal motion, and developments of new surgical devices or evaluation of a surgeon's skill[4,21–22].
Despite the outstanding properties of Thiel's embalmed cadavers for various applications in biomedical and biomechanical research, a number of studies have described the unsuitability of the Thiel-embalmed cadavers for studies into failure loads of implants due to significant discrepancies in biomechanical properties between embalmed cadavers and living tissues. It is recommended that such differences need to be taken into consideration when planning studies and interpreting the findings[8,23]. Thiel embalmed tendons for biomechanical investigation was also dissuaded from since the Thiel embalmed tendons did not "faithfully represent the biomechanical characteristics of fresh frozen tendons". Fessel et al, in 2011, by finding the unsuitability of Thiel embalmed for biomechanical studies, suspected that the diminished elastic modulus and failure strength of Thiel preserved tendons may be related to collagen denaturing associated with the high salt (boric acid) concentration in the Thiel embalming solution[24–25].
Hence, Thiel specimens cannot be recommended for biomechanical load to failure test because of a significantly altered failure strain and plastic energy absorption[15,26– 27]. The plastic energy absorption of human and bovine specimens was altered significantly by Thiel's method. Moreover, loss of signal and contrast when Thiel-embalmed human cadavers are imaged using clinical magnetic resonance imaging (MRI) sequences, especially those based on spin-echo MRI[29–30]. Thiel-embalmed organs cannot be used as reliable model for radiofrequency ablation evaluation due to the much increased electrical conductivity by the embalming process[32–33]. The aim of this study was to modify the original Thiel embalming technique and produce embalmed organ with electrical conductivity comparable to its normal organ in order for radiofrequency ablation evaluation.
To understand Thiel's fluid, Benkhadra et al conducted a histological comparison between fresh cadavers, formalin preserved cadavers and Thielembalmed cadavers. Microscopic images showed that the muscle fibers had a cut-up "minced" appearance, but remained aligned, with conjunctive collagen fibrils also undisturbed, leading them to give the conclusion that the exceptional suppleness of these cadavers was caused by boric acid for its very corrosive effects on proteins. However, the mechanism proposed by Benkhadra et al is questionable. If the pH of the Thiel's tank fluid is taken into consideration, one would easily found that the solution is nearly neutral or slightly alkaline (Table 1). The corrosive effect of acids is the consequence of high concentration of hydronium ion, but boric acid in this case is almost neutralized by the basic ionic salt such as sodium sulphite, and the concentration of H+ is not high enough, as is indicated by the pH value, thus not capable to corrode biological tissues as a protonic acid.
Chemical* Concentration in Thiel's tank fluid Electrical conductivity of individual solution pH Boric acid 3.6 g/100 mL H2O 1.384 × 10−4 Sm−1 4.38 Potassium nitrate 6.0 g/100 mL H2O 7.25 Sm−1 9.37 Sodium sulphite 8.4 g/100 mL H2O 6.83 Sm−1 9.24 Ammonium nitrate 12.0 g/mL H2O 13.33 Sm−1 5.86 Ethylene alcohol None in tank fluid 7.16 × 10−3 Sm−1 7.18 Propylene glycol 12.0 mL/100 mL H2O 7.31 × 10−3 Sm−1 7.46 8.9% Formalin 10.0 mL/100 mL H2O 9.41 × 10−3 Sm−1 6.54 Thiel's tank fluid – 11.09 Sm−1 7.27 Note: *Chemical used: potassium nitrite (Alfa Aesar, 500 g, 97% cas 7758-09-0, shore road, Heysham, la3 2xy, England), Boric acid (99 + %, Alfa Aesar, cas 10043-35-3, bond street, ward hill, MA 01835), sodium sulphite (anhydrous, 98%. 500 g, cas 7757-83-7, shore road, Heysham, la3 2xy, England), Ammonium nitrate (99%, cas 6484-52-2, VWR chemicals, Prolabor, Leuven Belgium), 1,2-propanediol (99 + %, cas 57-55-6, Merck KGaA, 64271 Darmstadt, Germany), Ethanol (99.8 + %, cas 64-17-5, Sigma-Aldrich, 3050 Spruce Street, St louis, MO 63103), formaldehyde, 37% in aq. Sln stab 10%-15% methanol liquid, cas 50-00-0, Alfa Aesar)
Table 1. Measured pH and electrical conductivity data from each component of the Thiel's embalming fluid
While boric acid is able to react with substances with two or more neighboring hydroxyl groups such as saccharides to form stable chelates. The formations of chelate by boric acid and glucose chain in the glycoprotein might be able to reduce intercellular adhesion by reducing hydrogen bonding and possibly covalent bond as well. However, boric acid alone seems not to be able to make such drastic change of Thiel-embalmed cadavers. Propylene glycol, with its high concentration in the embalming solution is suspected to soften the embalmed tissue by weakening hydrogen bond and damaging cellular membrane structure for its amphiphilic property (soluble both in water and lipid). Also, the high salinity, due to high concentration of inorganic solutes, might contribute to the extraordinary suppleness, since high salinity is usually associated with high ionic strength in the solution which leads to higher solubility to some substances. Our measurement results have demonstrated that electrolytic components, i.e., potassium nitrate, ammonium nitrate and sodium sulphite, are the main contributors to the high electrical conductivity of Thiel's embalming. These three electrolytes are all strong electrolytes and have good solubility in aqueous environment. By solving some substances that are originally insoluble, biological tissue might become softer with increased hydration. Nonetheless, boric acid is not the sole contributor to the suppleness, which is also confirmed by a personal communication with Miss Amanda Hunter, a PhD student at Centre for Anatomy & Human Identification (CAHID) in University of Dundee, who stated that "The mouse embalmed with embalming solution without boric acid seemed to be soft enough as well".
Moreover, the pinkish or reddish color of Thiel-embalmed cadavers can be attributed to the existence of nitrate. Nitrate itself is not directly engaged in the color preserving but its product nitrite is highly suspected to play a vital role. Formaldehyde and sulphite are both agents with considerable reducing capability and can reduce nitrate to yield nitrite. Nitrite and its gaseous products, nitrogen monoxide NO and nitrogen dioxide NO2 can all coordinate to the ferrous ion Fe2+ in myoglobin in myocytes (muscle cells) and hemoglobin in blood red cell. The formation of the coordinate compound of Fe2+ with nitrite, NO or NO2 leads to the pinkish appearance of embalmed cadavers. So is the same in meat industry. To keep meat products, especially red meat product, it is the usual practice to add small amount of sodium nitrite to keep the product in inviting color and from decaying. However, the reaction is not that quick, while the products are supposed to be slowly released into the solution since the solution is not acid enough to realize quick release. Though slow, the reaction can take long enough since the concentration of nitrate, sodium and formaldehyde is high enough.
To achieve lower concentrations of electrolytes, we substituted nitrite for nitrate. Eight candidate embalming fluids were designed, listed in Table 2 in comparison with Thiel's original tank fluid. Fluid 1 has no electrolytic component in it at all, which aimed to understand the role of the electrolytic components as a whole. The use of sodium sulphite in fluid 2 was aiming to know the role of nitrate/nitrite. Potassium nitrite is the only strong electrolyte in it, which is to understand the role of sulphite. Fluid 4 is a combination of fluids 2 and 4 in order to explore possible synergism of the two. Fluids 5 to 8 have more electrolyte than the first four, trying to meet the balance between the properties of embalmed samples and their conductivity. The electrical conductivity and pH of the eight solutions were measured (Table 3). Porcine kidneys were purchased from local butcher shop (Scott Brothers, Dundee, UK). The fresh porcine kidneys were harvested from pigs sacrificed within 48 hours and had been under refrigeration since then. The selected samples had similar size and appearance. Seven porcine kidneys were embalmed in the fluids for 14 days before we measured their electrical conductivity.
Electrical conductivity (DC) pH Note Thiel's tank fluid 11.09 Sm−1 7.27 Clear and transparent Fluid 1 7.91 × 10−3 Sm−1 4.26 Clear and transparent Fluid 2 4.79 × 10−1 Sm−1 6.81 Clear and transparent Fluid 3 4.78 × 10−1 Sm−1 5.13 Brownish, transparent Fluid 4 8.46 × 10−1 Sm−1 6.81 Clear and transparent Fluid 5 1.248 Sm−1 5.38 Brown but transparent and emitting plenty of achromatic gas, very unstable Fluid 6 1.095 Sm−1 6.61 Clear and transparent, slightly green Fluid 7 1.075 Sm−1 6.14 Clear and transparent, slightly yellow Fluid 8 2.19 Sm−1 7.72 Clear and transparent, almost colorless
Table 3. Measurement of modified fluids
Thiel's Fluid 1 Fluid 2 Fluid 3 Fluid 4 Fluid 5 Fluid 6 Fluid 7 Fluid 8 Hot tap water/mL 500 500 500 500 500 500 500 500 500 B(OH)3/g 18 18 18 18 18 18 18 18 18 NH4NO3/g 60 - - - - 6 3 3 3 KNO3/g 30 - - - - - - - - KNO2/g - - - 3 3 3 1.5 3 1.5 NaSO3/g 42 - 4.2 - 4.2 4.2 8.4 4.2 21 Propylene glycol/mL 60 60 60 60 60 60 75 75 75 Stock Ⅱ/mL 12 12 12 12 12 12 18 18 18 8.9% formalin/mL 51 51 51 51 51 51 51 25.5 25.5
Table 2. Formulae of candidate embalming solutions
In this experiment study, porcine kidneys were embalmed with Thiel's tank fluid for 28 days instead of just 18 days used in previous experiments. The purposes were to make sure the sample completely Thiel-embalmed and to also compare duration's influence upon the electrical conductivity. Based on Thiel's original tank fluid, dilution fluids were designed by removing the strong electrolytes while the only weak electrolyte boric acid was kept. Chlorocresol (pre-mixed with propylene glycol, mixture referred to as stock Ⅱ) concentration did not change since it is assumed that this antiseptic agent does not greatly affect the overall quality of the embalmed tissues but purely prevents them from decaying. There were some minor adjustments of the other components. Propylene glycol was increased in dilution fluid 3 to 90 g per every 500 mL water but in dilution fluids 1 and 2, the dosage remained unchanged. Since the drop in ionic strength would probably lead to increase of stiffness in the embalmed tissues and formalin can stiffen the tissue, formalin was reduced to its one tenth in dilution fluids 2 and 3. In addition, ethanol is added in all three dilution fluids to strength anti-bacterial activity. Detailed formulae were shown in Table 4.
Component Dilution fluid 1 Dilution fluid 2 Dilution fluid 3 Hot tap water/mL 500 500 500 Boric acid/g 20 20 20 Propylene glycol/mL 60 60 90 Stock Ⅱ/L 18 18 18 Formalin (8.9%)/mL 51 5.1 5.1 Ethanol/mL 30 60 30
Table 4. Formulae of dilution fluids
All three dilution solutions were odourless, achromatic and transparent. Electrical conductivity and pH of the dilution fluid were measured as shown in Table 5.
Dilution fluid 1 Dilution fluid 2 Dilution fluid 3 pH 4.205 4.742 4.195 Electrical conductivity (DC) 6.80 × 10−3 Sm−1 1.672 × 10−3 Sm−1 6.76 × 10−3 Sm−1 Appearance Achromatic and transparent Yellowish but transparent Achromatic and transparent
Table 5. Measurement of dilution fluids
Three porcine kidneys that had been previously embalmed in Thiel's tank fluid for 28 days were later transferred in to three dilution fluids, respectively. Before dilution process, conductivity measurement was conducted. Then 800 mL of the dilution fluid was used to embalm each kidney for one week, after the first week the dilution fluids was then renewed with the same dilute solution, 800 mL of each. Two weeks of dilution was then followed by conductivity measurement.