Comparison of chemical and enzymatic maceration processes for soft tissue removal from arterial silicone casts on skull scaffolds

The various models used in gross anatomical studies to improve the visualization of blood vessels differ in the amount of manual labour, cost, equipment and time involved. This study aimed to compare chemical and enzymatic maceration processes for soft‐tissue removal from arterial silicone casts on skull scaffolds using ringed seal (Pusa hispida) skull specimens. Both processes produced specimens that covered all anatomical aspects required to visualize the intracranial arterial arrangement on a bone scaffold. Overall, the enzyme maceration process was better for production of such specimens, as this process is easy and safe to perform, is less harmful to the bony parts of the specimen, and the resulting specimens are visually more appealing for display and teaching. Compared with previously published models, the end result varied in the amount of dissolved bone tissue and the visual presentation of the model.


| INTRODUC TI ON
Various corrosion cast and other models are routinely used in gross anatomical studies and teaching to improve the visualization of blood vessels (Anderson et al., 1989;Cirak et al., 2021;Kiełtykaka-Kurz et al., 2015;Meyer et al., 2007;Sanan et al., 1999;Zdun et al., 2019).Depending on the purpose of the model, the specimen may be fixed and soft tissues removed only to a degree to allow visualization of the routes of blood vessels among the soft tissue (Cirak et al., 2021).If the goal is to remove all soft tissue from the finished model, an important step in the preparation process is the removal of soft tissue without damaging the cast or bone structures, which serve as orientation and topographical marks for the finished cast in the head, backbone, and limb regions.Besides boiling, microbial decomposition, and flesh-eating invertebrates (e.g.Banta, 1961;Brown & Twigg, 1967;Hall & Russell, 1933;Hurlin, 1918;Sealander & Leonard, 1954), various chemical and enzymatic maceration processes are the main methods for preparation of bones from carcasses (e.g.Hangay & Dingley, 1985;Kiełtykaka-Kurz et al., 2015;McDonald & Vaughan, 1999;Niederklopfer & Troxler, 2001;Onwuama et al., 2012;Simonsen et al., 2011;Solov'ev et al., 2013).These methods differ in the amount of manual labour, cost, equipment and time involved.Depending on the method, the end results also vary in the amount of dissolved bone tissue and the visual presentation of the model (Hangay & Dingley, 1985;Kiełtykaka-Kurz et al., 2015;Onwuama et al., 2012).
We have previously employed a silicone compound used in dentistry to produce casts for the visualization of organ systems (Laakkonen et al., 2014;Laakkonen & Kivalo, 2013;Vesterinen et al., 2014) and coronary arteries (Kareinen et al., 2020) for research and teaching purposes.The compound was chosen for its qualities of low viscosity and high hydrophilicity.The models can be produced relatively quickly using chemical maceration with sodium hypochlorite solution.Other than a slight colour change, the silicone does not dissolve or otherwise change in shape or form during the process or later.This is demonstrated by our oldest model, which has remained unchanged for 10 years (Laakkonen & Kivalo, 2013).As hypochlorite solution is a modifying agent that alters the physiological properties of bone, it is not recommended for preparation of bone specimens (Bromage, 1984;de la Torre, 1951;Kerbl et al., 2012).Biological museum preparators have been developing and describing various methods for cleaning osteological specimens at least since the late 1800s (Zander, 1886).We feel it is advantageous to adopt some of the techniques in use and optimized for them for osteological museum work for the preparation of injected anatomical specimens.Papain is a proteolytic enzyme widely used in enzymatic maceration.The advantages of papain include excellent price/ performance ratio, the ability to break down a wide range of soft tissues, and the general ease of use, since the solution can be reused several times and stored at room temperature.
The aim of this article was to compare chemical and enzymatic maceration processes for soft tissue removal from skulls of two ringed seal subspecies injected with silicone for the examination of intracranial arterial vasculature.Besides the inherent need to limit damage to the bone structures, this study had the additional challenge of working with skulls of the endangered Saimaa ringed seal.This limits the amount of skulls available, as all recovered skulls are stored preferably intact for museum collections.

| MATERIAL S AND ME THODS
All Saimaa ringed seal carcasses found dead are collected by the Due to the scarcity of endangered Saimaa ringed seal material, both maceration methods were tested first with injected dog (Canis familiars) specimens that were donated by the owners to the Faculty of Veterinary Medicine of the University of Helsinki for research and teaching purposes (Figure 1a).The dogs were euthanized for reasons unrelated to this study.Both dog specimens are now part of the anatomical collection of the faculty and used as teaching aids.

| Maceration with sodium hypochlorite
The heads of specimens 2698 and 458 were boiled separately in tap water for 2-3 h such that some of the soft tissue parts could be removed by hand without damaging the cast.For the removal of the remaining soft tissue parts in difficult-to-reach cavities and around the smallest vessels of the silicone model (which are difficult to remove without breaking some branches of the model), each head was placed separately in a fume hood in a 5-L plastic bucket containing 2-3 L of sodium hypochlorite solution (14%, Sigma-Aldrich, Switzerland) in a horizontal position.To prevent possible pressure build-up, the buckets were covered with a lid that was not completely airtight.During the maceration, the specimens were rotated daily.Any cleaned bone areas were placed above the liquid level as much as possible to limit the damage to the bone structures.Approximately 500 mL of sodium hypoclorite solution was then added to replace the inactive and evaporated liquid.The removal of soft tissues lasted for 7-10 days depending on the size of the specimen.After the chemical maceration process, the skulls were washed thoroughly under running water for 15 min and then placed separately in clean tap water in a 5-L bucket for 3 days, during which the water was changed twice a day.

| Maceration with papain
During routine bone preparation, the macerated specimens are manually cleaned from most of the soft tissue and treated with prolonged soaking to remove blood and water-soluble proteins.No soft tissue was cleaned and specimens were not soaked to prevent damage to the injected arteries.
Unless stated otherwise, the enzymatic maceration process was followed as outlined for osteological preparation by Niederklopfer and Troxler (2001).Medis Bone Maceration Unit MU 1360-2, a purpose-made machine at Finnish Museum of Natural History LUOMUS, was used to perform the enzymatic maceration.
The maceration solution was prepared in tap water, in which papain (5 g/L, Bauer Handels, Switzerland) was added.NaCl (15 g/L) was added both to inhibit the formation of lime soaps (calcium salts of animal fatty acids) and to increase the enzymatic activity of papain.The maceration process was further enhanced by adding SUPRALAN UF (0.5 g/L) as an emulsifier of fats and SUPRALAN 80 (0.5 g/L) as a wetting agent (to increase permeability of soft tissues) and as a disinfectant (to inhibit unwanted bacterial activity).Na 2 CO 3 was added during the process to maintain the pH between 8.0-8.5, as acidic pH is detrimental to bone material.The solution was maintained at 42°C (±2°C).The maceration solution was agitated regularly with circulation pumps (80-s run/5-min break).The maceration was complete within 24-36 h.

| RE SULTS
The specimens produced by the chemical and enzymatic maceration processes were relatively similar from a research perspective, although the enzymatic maceration method produced cleaner skulls (Figures 1 and 2).All teeth became loose in the enzymatic maceration process but remained attached both in the upper and lower jaw in the chemically macerated skulls.Due to the substantial amount of soft tissue and no soaking, the enzymatic maceration produced specimens with slight discoloration in the bone (Figure 1b).Damage to the bone was minimal in all structures except for the damage resulting from the chemical maceration to the very thin bones (Figure 2a,b).Enzymatic maceration was considerable faster and cheaper than the chemical maceration; the enzyme maceration solution can be reused several times, thus further lowering the costs.Professional maceration machinery was used in this study.If this is not feasible, the specimens can be processed in a heating cabinet.The enzyme maceration was user friendly compared with the chemical treatment, which involves hazardous materials (Table 1).
The silicone models remained unchanged except for a slight colour change in the chemical maceration process.With the chemical maceration process, a few hardened soft tissue residue spots remained in the skull in all specimens (Figure 2b,c) unless the process was continued for more than 10 days, which resulted in bone breakage.In both the chemical and enzymatic maceration processes, some soft tissue residues remained tightly attached around the smallest arteries even after through rinsing.These were difficult to remove without breaking some branches of the model.

| DISCUSS ION
Compared with the previous models prepared using similar procedures or fixed tissues, the methods used in this study yielded very similar bone scaffolds and the silicone casts (Ashwini et al., 2008;Brudnicki, 2011;Frąckowiak et al., 2015;Zdun et al., 2019).All prepared skulls have been stored for at least 2 years and have remained unchanged, although tiny bone pieces tend to come off from chemically macerated skulls during handling.Similar dissolving and cracking of bones was reported from bone preparations with sodium hydroxide (Onwuama et al., 2012).
Most of the Saimaa ringed seals caught as bycatch from gillnets are young individuals.This is a challenge for processing specimens for anatomical studies, as the skull bones of young individuals tend to come apart more easily than those of adult individuals regardless of the maceration and cleaning process used (Figure 2).Therefore, the maceration should be monitored regularly, especially towards the end of the maceration process.The enzymatic activity of papain is negligible below 38°C.If the maceration cannot be monitored, temperature regulation can be used to slow down or halt the maceration before the skulls begin to fall apart.
Papain only breaks down coagulated proteins in the pH range suitable for bone material.Therefore, a short pre-boiling before the maceration is necessary; after maceration, the specimens should be treated with hot water to denature the remaining enzyme inside the bone.Skipping the reheating at the end of the maceration process is advised for skulls of immature animals (Niederklopfer & Troxler, 2001).Since papain is only active in aqueous solution, the denaturation step at the end of the enzymatic maceration is not strictly necessary (however, see Shelton & Buckley, 1990).
Although the LUOMUS machinery was used to clean the specimens in this study, enzymatic maceration can be executed without special equipment.If no maceration machinery is available, the specimens can be processed in a heating cabinet equipped with a thermostat regulating temperature.The specimens can be housed in non-corrosive vessels, such as plastic containers.Small-scale processing of specimens enables one to increase the amount of enzyme and thus expedite the process without excess cost.Theoretically, it should be possible to produce a specimen in a matter of hours (Niederklopfer & Troxler, 2001, see also Simonsen et al., 2011).
However, the ratio of macerated specimen to maceration solution (in weight) should be no more than 1:20 (Niederklopfer & Troxler, 2001).
While it is possible to exceed this, the results may be suboptimal.
The enzymatic maceration process is less hazardous for the user and environment.Sodium hypochlorite is toxic if inhaled and harmful to aquatic life.Sodium hypochlorite becomes ineffective in removing soft tissue in a relatively short time and must be replenished or changed during the maceration process.Enzymatic maceration is highly versatile and adjustable with temperature control and enzyme concentration.The maceration solution can be reused several times and allowed to cool between processing specimens.
Once the solution is buffered with sodium carbonate, no additional buffering is needed.The major drawback of maceration with papain is the strong, characteristic smell.Eventually the solution becomes too odoriferous and saturated with soft-tissue residue.Good ventilation or an activated carbon air filter can reduce the smell, although a separate room within a building or even a separate building as a workspace is the optimal solution (cf. Brown & Twigg, 1967).
Disposing the spent solution is straightforward, as the small amount of biological waste can be (depending on local regulations) drained with excess water.Of other enzyme-based methods, maceration of injected skulls with an enzymatic household washing powder produced intact osseous material, although the process took 1 month or more (Frąckowiak et al., 2015;Kiełtykaka-Kurz et al., 2015).
In conclusion, both chemical and enzymatic maceration processes produced specimens that covered all anatomical aspects needed to visualize the intracranial arterial arrangement on a bone scaffold.In our opinion, the enzymatic maceration process was better for production of such specimens, as this process is easy and safe TA B L E 1 Comparison of factors involved in the chemical and enzymatic maceration process.

Quality of the finished specimen
Good to satisfactory, depending on the time needed for maceration.Bone separation in some specimens.
Good but teeth became loose.Bone separation in some specimens.

Finnish
state agency Metsähallitus and preserved frozen for conservation and research purposes.The Saimaa ringed seals (Pusa hispida saimensis) examined were bycaught in gillnets.Metsähallitus and the University of Helsinki share a research agreement that permits the use of this animal material in research.Two Saimaa ringed seal cadavers were prepared for this study.One was a subadult male (body weight 38 kg; Metsähallitus specimen number 2698) and the other was a subadult female (35 kg; Metsähallitus specimen number 2704).Baltic ringed seals (Pusa hispida botnica) are legally hunted in Finland, and the studied cadavers used in this study were hunted for reasons unrelated to this research.One adult female (53 kg; specimen number 668; the Natural Resources Institute Finland designates a specimen number for all Baltic ringed seals used in research) and one pup female (23 kg; specimen number 458) Baltic ringed seal were also prepared for this study.All specimens were frozen before being transported to the Faculty of Veterinary Medicine of the University of Helsinki for anatomical dissection.The specimens were prepared in 2019 and 2020.Once the cadavers were thawed, silicone casts were created of the head region of both ringed seal subspecies by injecting silicone (3 M Express™ 2 Light Body Standard Quick or 3 M Express™ 2, Light Body Flow Quick) as described in detail byLaakkonen and Kivalo (2013) into the right (specimen 458) or both common carotid arteries (specimens 2698, 2704 and 668).The silicone was left to dry for 10 min (drying time is usually 2-4 min).The skin and blubber were removed with a scalpel and the heads were removed from the body at the atlanto-occipital joint.The specimens 2704 and 668 were transported to the Finnish Museum of Natural History for the enzyme maceration process (see below).
After 1-2 days, depending on the mass of the specimen, the sodium hypochlorite was no longer effectively macerating the soft tissue.F I G U R E 1 Dog specimens pictured during the chemical maceration process (a) and at the end of the enzyme maceration process (b).Scale = 1 cm.

F
Finished specimens produced by the chemical (a; specimen 458 and b; specimen 2698) and enzymemaceration processes (c; specimens 668 and 2704).Note the damage (arrows) to the bones in the chemically macerated specimens (a and b) and the separation of bones (arrows) in the enzyme-macerated specimens (c).Scale (a and b) = 1 cm; scale bar (c) in centimetres.
Visible in thin bone structures, such as maxilloturbines Minimal of the biological waste liquid containing residue sodium hypochlorite can be added to water and drained with excessive water.Any unused sodium hypochlorite solution is disposed as a hazardous waste.