INTRODUCTION

The conservation of soft tissues is a complex task, because it is necessary to use chemical agents that cause some undesirable tissue alterations (loss of color or shrinkage) and are toxic. Among the different substances that can be used as preservative agents, formalin (i.e., aqueous formaldehyde solution) is preferred because it guarantees durability; however, it may cause human health issues (Godish, 1990; Noisel et al., 2007; Whitehead and Savoia, 2008; Dorairajan, 2010; Schwilk et al., 2010; Latorre et al., 2011; Raharyaningsih et al., 2018; Thetkathuek et al., 2020; Adamović et al., 2021). This situation has motivated the search for alternative techniques that help the process of conservation by minimizing the risk of toxicity. Within this framework, the plastination technique was developed (von Hagens, 1979), whose underlying principle is the replacement of organic fluids and tissue fats by harmless polymers (von Hagens et al., 1987). Thereby, through a four-stage procedure (fixation, dehydration, forced vacuum impregnation and curing), odorless, dry, durable, non-toxic materials are obtained, which can therefore be manipulated without the requirement of protective equipment. Because of its widely recognized benefits (Prasad et al., 2015), it represents an advanced solution, currently used by more than 300 scientific-educational institutions in the world (Sora, 2016), and already practised in several South American countries (Bula Calderón, 2012; Muñetón Gómez and Ortiz 2012; Miranda Solis, 2015; Ottone et al., 2015, 2016, 2018; Peralta Pineda, 2017, among others).

Although plastinated specimens are not dangerous, the type of chemicals used during the production process requires that biosecurity measures must be considered. The most critical procedure is dehydration and degreasing of the specimens, since the best solvent for this purpose is acetone, a flammable liquid which gives off a flammable vapor. So, a main concern in designing a plastination lab is to control the risk of fire or explosion. Therefore, to prevent the accumulation of flammable vapors, the space must have an adequate air extraction system, equipped with filters that capture organic vapors to avoid environmental pollution (Beltrán Guerra, 2010; Schill, 2018). During the plastination procedure, acetone must occasionally be handled in open containers; for this reason, and even if the air extraction system works properly, it is necessary to avoid all possible sources of ignition or explosion. This means that electrical circuits (plugs, outlets, switches, and motors) should be explosion-proof, or located outside the laboratory. All these requirements result in high construction costs, and therefore the adaptation of the existing facilities is an alternative that has been implemented elsewhere (Reina de la Torre et al., 2004; Ottone et al., 2014; Zerlotini et al., 2020). The objective of this work is to report our experience at the Universidad Nacional del Sur, starting from the modification of a pre-existing space to transform it into a laboratory suitable for the implementation of the plastination technique with silicones at room temperature.

MATERIALS AND METHODS

The Universidad Nacional del Sur (UNS) is located in the central-eastern part of Argentina, at the south of the province of Buenos Aires (38° 44' S; 62° 16' W). The facility, located away from the classroom complex, occupies a total area of ~15 m2, subdivided into two adjoining rooms (Fig. 1A). The largest room was used as a

Figure 1. Ceiling view of 3D rendering designs of original space (A) and of the planned modifications (B). AC: air conditioner; AD: access door; CD: communicating door; CTP: countertop; EF1: electric fan at 0.60 m from the floor; EF2: electric fan at 1.80 m from the floor; EP: electrical control panel; F: chest freezer; IC: impregnation chamber; R: refrigerator; SH: shelf; SI: sink; W: wall; WE: air vent with a wind air extractor

conventional laboratory and had an L-shaped tiled countertop with sink. Next to the entrance door, a low dividing wall delimited a corridor, which reduced the useable space and hindered movement. The other room was used for storage, but it also had a countertop and a sink. Both rooms had hot and cold running water and conventional electrical circuits. Considering the biosafety recommendations associated with the technique (Holladay et al., 2001; Schill, 2018, 2019), the largest room was destined for the plastination laboratory and other anatomical techniques, and the smaller used for storage of non-hazardous materials.

Modifications were planned, taking into account the fact that silicones would be used at room temperature. The building changes planned for the plastination room were: removal of the dividing wall, modification of the electrical circuit (rewired, removal of all existing plug in and light keys, and installation of LED lights), installation of air extractors with filtering system, change of the access door for a safety one, and making the countertop sanitary (Fig. 1B). Equipment to be acquired included: chest freezer, refrigerator, impregnation chamber, and furniture (such as shelves, side tables and desk chairs). The construction of an external booth for the motors was designed.

Both the planning and the entire process were supervised by the Occupational Hygiene and Safety Service of our institution (Servicio de Higiene y Seguridad en el Trabajo; Departamento de Biología, Bioquímica y Farmacia; UNS).

RESULTS

Modification of the facilities began in January 2020, with an estimated completion time of eight months. Given the strong and prolonged restrictions due to COVID, the total time was much longer than expected (25 months). Completion was February 2022. The total investment to set up the laboratory was US$7500 (Table 1).

Table 1. Plastination lab modifications and equipment costs

The modifications resulted in an area of 14.8 m2, between two rooms, one of 9 m2 for the laboratory, and the other 5.8 m2 intended for the storage room along with a refrigerator and necessary furniture (shelves, desk chair and side table) that were installed (Figs. 2 and 3).

Figure 2. Views of laboratory from A) access door, and B) communicating door

All the original electrical circuitry was removed from the entire site and new wiring, explosion-proof LED lights, and touch keys were installed. All the electrical outlets were installed outside the laboratory room. As an extra measure of protection, an electrical control panel with independent thermal keys and circuit breakers (one for each device and circuit), was installed in the storage unit (Fig. 3). In the laboratory room, the dividing wall was removed, and the entrance door was changed for a hermetic, aluminum door, with an anti-panic bar (Fig. 2). A hot and cold air conditioner with temperature control was installed, and the countertop was covered with stainless steel to facilitate cleaning and disinfection. To ensure proper removal of toxic vapors, generated by acetone and other chemical agents, two electric fans (flow: 200 m³/hour, at 1.8 m and 0.6 m above the floor respectively) and an air vent (flow: 100m³/hour, at 0.60 m above the floor) were installed. All air handling units were equipped with an organic vapor filtering system (3M™ Organic Vapor/Acid Gas/Filter).

For the dehydration phase, a 205 L chest freezer, and solvent containers (capacity: 20 liter, D.O.T. carboy) for the acetone, were acquired. A 0.30 m3 cylindrical vacuum chamber (30 cm diameter x 100 cm tall) was built of iron tubing and coated with epoxy paint. A 2 cm-thick toughened glass lid was selected. Both the chest freezer     motor and the vacuum pump motor were installed in a special cabinet outside the building, with underground electrical and vacuum line connections.

Figure 3. Storage room. Arrow: electrical control panel containing the thermal keys and circuit breakers

In addition to the described modifications, generic laboratory equipment (instruments, glassware, scales, and various containers) was acquired, as well as supplies for plastination (aquarium pump and two acetonometers).

All the construction and improvements carried out allowed us to start the plastination process with organs from Wistar rats (heart, kidneys, and testicles). For the final disposal of waste products, both liquid (such as acetone, alcohol, and formol) and solid (biological waste), were stored in appropriated containers (biological waste in red biohazard bags, and liquids in waste containers), and collected weekly by the personnel of the Occupational Hygiene and Safety Service of the Universidad Nacional del Sur (https://www.uns.edu.ar/contenidos/64/967#retiro-de-residuos).

DISCUSSION

The undergraduate and postgraduate courses of the UNS associated with the natural sciences (Biology, Pharmacy, and Biochemistry Medicine) depend on a large number of specimens preserved in formalin. In recent years, efforts have been made to replace this preservative with less harmful ones (isopropyl alcohol), but this has only been possible with the small-sized samples.

Among the existing methodologies to replace or reduce the use of formaldehyde, plastination emerged as a valuable option, since it allows creating quality specimens which are durable and safe, for manipulation by students and teachers (Latorre & López Albors, 2015). However, the construction of a plastination laboratory that meets biosecurity requirements, demanded by the technique, is linked with high costs and a long time for start-up. Our experience and others, demonstrates the possibility to reduce both factors by modifying a pre-existing space (Reina de la Torre et al., 2004; Ottone et al., 2014; Zerlotini et al., 2020). However, since acetone is used in large quantities in the plastination process, it is essential to bear in mind that the process does not only involve a re-adaptation of the space, but certain security requirements must be taken into consideration (Schill, 2018), such as:

• The laboratory must be isolated from other working areas
• The electric installations must be explosion safe/proof
• Proper ventilation of the space must be achieved, and fans must be equipped with organic vapor filtering systems to avoid environmental pollution as well as being explosion safe
• Refrigeration units and pump motors must be placed outside the enclosure, properly

The progress we have achieved with the installation of the Plastination Laboratory at the UNS correlates with the need for finding a method of preservation of organic tissues with minimal toxicity, as well as using a room-temperature impregnation technique. The samples generated in this way will be added to our collections to complement the traditional collection of hard tissues (bones, skins, teeth, horns, and shells), which are used both in research and teaching. The availability of these materials will reduce the number of animal cadavers needed for teaching and eliminate human exposure to hazardous substances. The manipulation of these products without the need for gloves is also presented as a promising tool in teaching blind students, who depend on touch as a fundamental sense of information.

Acknowledgments

Fundings: PIP-CONICET 11220200101668CO; PICT-2020-SERIEA-03298, PGI 24/B332, Programa Prombio IF-2016-00614844-APN-SECPU#ME. Additional financial support came from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) through a PhD fellowship to A.I.P.