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In vivo reactions in mice and in vitro reactions in feline cells to implantable microchip transponders with different surface materials
  1. M. Linder, DrMedVet,
  2. S. Hüther, DrMedVet1 and
  3. M. Reinacher, DrMedVet, DECVP1
  1. 1 Institut für Veterinär-Pathologie, Justus-Liebig-Universität Giessen, Frankfurter Strasse 96, 35392 Giessen, Germany
  1. manfred.reinacher{at}vetmed.uni-giessen.de

Abstract

Tissues of mice that had had microchip transponders with surfaces made of bioglass, bioglass with a polypropylene cap, parylene C, titanium or aluminium oxide inserted were examined histologically, and the growth of two lines of feline fibroblastoid cells around these transponders was examined in vitro. The results for bioglass and aluminium oxide were similar. In vitro, there were almost no cells around or on the transponders; in vivo, there was often granulomatous inflammation in the surrounding tissue. In the case of the bioglass, this reaction seemed to be induced by petrolatum, which was added by the manufacturer for technical reasons, rather than by the bioglass itself. In some of the mice, polypropylene caused a proliferation of granulation tissue. In vitro, the cellularity around the transponders was high, but only a moderate number of cells were found on the material. In vivo, around the parylene C transponders, there were occasionally small fragments of foreign material, surrounded by a foreign body reaction; in vitro, the results for parylene C resembled those for polypropylene. In vivo, particles of titanium were sometimes visible in the connective tissue adjacent to the titanium transponders, and sometimes accompanied by a foreign body reaction; in vitro, a confluent layer of cells developed on the transponders, with a high cellularity around them.

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SINCE approximately 1990, microchip transponders have been established worldwide as a system of identification for domesticated and zoo animals, and for laboratory animals (Rao and Edmondson 1990). They have many advantages, but they may be lost shortly after injection (Pirkelmann and Kern 1994), they may cause inflammation at the implantation site, and they may migrate within the subcutaneous tissues. There have been two case reports of microchip-associated tumours (liposarcoma and fibrosarcoma) in dogs (Vascellari and others 2004, 2006).

Since 1997, 'microchip adverse reactions reporting forms' have been used to report such problems (British Small Animal Veterinary Association 2009). Migration has been the most frequently reported problem (Swift 2000, 2002, 2004). If a transponder has migrated from its original implantation site, an animal might be chipped twice or declared ownerless. Furthermore, if it migrates to a site with high mechanical stress, for example, the elbow, it may be damaged more easily. In cattle, microchips must be retrieved quickly after the animal is slaughtered, and any migration is therefore undesirable.

The migration of transponders is generally considered to be influenced by the animal's age and movement while the transponder is being implanted (Pirkelmann and Kern 1994, Lammers and others 1995), the choice of implantation site, the injection technique, whether the transponder is palpated after injection (Gruys and others 1993, Butcher 1998, Jansen and others 1999) and the surface material of the device (Pirkelmann and Kern 1994).

The surface materials in use are mainly either untreated bioglass or bioglass coated with different polymers. Bioglass consists mainly of silicon dioxide, but additional small amounts of alkaline oxides result in a surface activity thought to increase the microchip's interaction with the surrounding tissue (Williams 1999). The experiences with polyethylene and polytetrafluoroethylene have been rather negative. Both materials were referred to as antimigratory coatings (Mader and others 2002) but distinct inflammatory reactions have often occurred (Geisel and others 1998, Ober 1998).

One company (Digital Angel Corporation) added a polypropylene cap (a so-called antimigratory cap) to their bioglass transponders. The cap covers half of the device and has two holes intended to provide better tissue anchorage (Arndt and Wiedemann 1991). Good results have been obtained with these microchips in terms of tissue compatibility and lack of migration (Arndt and Wiedemann 1991). Sometimes connective tissue was observed to have grown into the gap between the polypropylene cap and the bioglass surface (Jansen and others 1999), and it is thought that this helps to fix the device in position and prevent it from migrating (Rao and Edmondson 1990, Murasugi and others 2003).

Another company (Pet-ID UK) uses a special polymer (parylene C) as a coating material to induce better tissue contact and inhibit migration of the transponder (Fry and Green 1999, Pet-ID 2009).

Aluminium oxide and titanium have also been used in making microchip transponders. Aluminium oxide belongs to the group of metal oxide ceramics. Since 1984, aluminium oxide has been ISO-standardised as a biocompatible and biostable bone substitute material, and it is often used in total hip replacement (Hannouche and others 2005). Titanium is a metal of the transition metal group. It is used as an implant material, for example, for extramedullary plates or intramedullary pins in human orthopaedic surgery (Leiting and others 2001), and forms a tight connection with bone tissue. Titanium is considered to have excellent biocompatibility (Pohler 2000).

The aim of this study was to analyse and compare the reactions of the tissues of mice to microchip transponders with surfaces made of bioglass, polypropylene, parylene C, titanium or aluminium oxide, and the reactions in vitro of two lines of feline cells to the same devices. The transponders either had a coating (parylene C, aluminium oxide or titanium) or were made of untreated bioglass. The polypropylene devices consisted of bioglass with a polypropylene cap covering half the surface.

Materials and methods

In vivo study

The transponders were sterilised with ethylene oxide, and implanted with either single- or multiple-use devices.

For stock surveillance in a commercial laboratory breeder facility, groups of female, three-week-old NMRI mice were chipped with transponders with the different surface materials. Seven weeks later, the animals were euthanased with carbon dioxide, and fixed in phosphatebuffered 4 per cent formalin.

Each mouse was scanned with a radio frequency identification reader, and on the basis of the code, the transponder's manufacturer and its surface material was determined.

The tissues containing the transponders were excised as a block. The transponders were then carefully retrieved after cutting the tissue ventrally along the longitudinal axis with a razor blade. The remaining tissue block was cut transversely into several slices and prepared for histological examination. Sections 3 to 5 μm thick were cut and stained with haematoxylin and eosin. The tissue around the polypropylene transponders was divided into the part around the polypropylene cap and the part round the bioglass before cutting each part into several pieces.

The histological findings were evaluated semi-quantitatively, mainly with respect to the presence of inflammatory reactions.

A piece of skin that had not been in contact with the transponder was taken from each mouse and examined histologically as a control, for comparison with the tissues around the microchip.

In vitro study

As a result of the differences observed between the different types of transponders in vivo, an in vitro investigation of the same devices was started. Two permanent feline fibroblastoid cell lines (FS I and FS VI) that had been established from feline fibrosarcomas (Löhberg-Grüne and others 2004) were used. Two transponders of each type were placed together in one cell culture flask. Then, 150 μl of each cell line (3 to 4 million cells/ml) and 6 ml cell culture medium (DMEM high glucose [4·5 g/l] with glutamine containing 1 per cent penicillin/streptomycin, 0.1 per cent gentamicin and 10 per cent fetal bovine serum; PAA Laboratories) were added. The transponders lay at the end of the flask (the angle between the bottom and the rear wall), and owing to capillary forces they did not move when the flasks were treated carefully. For each cell line one culture flask without a transponder served as a growth control. After incubation at 37°C in a humidified atmosphere containing 5 per cent CO2, for three days for cell line FSI and (owing to its slower growth) four days for cell line FSVI, cell growth and cell morphology around the transponders were examined with a tissue culture microscope (TCM). The density of the cell layer was evaluated semi-quantitatively (from 'minimal density' to 'very high density'). The results were compared with those of the controls.

For harvesting, the flasks were cut open and the transponders were removed with forceps, touching only the lower part of the microchip, and they were then put into a plastic block. They were then prepared for scanning electron microscopy (SEM) according to the protocol of Burkhardt (1979), with two slight modifications: the transponders were fixed in 2.5 per cent glutaraldehyde for two hours and a graded series of 1-propanol was used for dehydration. It was made sure that it was known which area of the transponder had faced the bottom and the wall of the culture flask and which part had been touched by the forceps when it was put into the plastic block. The bottoms of the control flasks were cut into several pieces and were prepared like the transponders for SEM.

The samples were examined for cells with a DSM 940 (Carl Zeiss), but only the parts of the transponders that had not been touched by forceps or been in contact with the walls or bottom of the culture flask were examined. The number of cells was estimated semi-quantitatively, from 'no cells' to 'large number of cells', and photographs were taken of representative locations. In the bioglass transponders with the polypropylene cap, only the polypropylene cap was examined by SEM because the bioglass part had been touched by the forceps during handling.

A viscous, wax-like, white to transparent mass was visible on the bioglass transponders and in the cannulae of their injection system when they were placed into the culture flasks in the in vitro experiment. According to their distributor, petrolatum is routinely added to the cannulae of the injection system to maintain sterility, and to lubricate the cannula.

Results

In two of the 48 mice, no transponder could be found. One other had developed an abscess around the transponder and this specimen was not evaluated. Two other specimens could not be evaluated owing to the poor histological quality of the tissues. Altogether, 43 specimens were available for evaluation.

In the in vitro studies, two transponders were not considered because their surface had been accidentally damaged mechanically.

The surfaces of all the aluminium oxide-coated transponders were eroded when they were removed from the mice or from the cell culture flasks; the coat of aluminium oxide was no longer intact so that the underlying bioglass was visible (Fig 1). A similar observation was made in two of the 10 titanium transponders.

Fig 1

Aluminium oxide-coated microchip transponder (a) before use and (b) after being removed from tissue

All the histological specimens had a capsule surrounding the transponders that consisted of more or less parallel concentric collagen fibres and fibrocytes (Fig 2). In most specimens a layer of macrophages or multinucleated giant cells was present at the interface between the fibrous capsule and the transponder (Fig 2). Hair granulomas sometimes occurred in the tissue surrounding all the types of transponders.

Fig 2

Fibrous capsule (arrows) around where a transponder (T) with a polypropylene cap had been, showing (a) epidermis, dermis and cutaneous muscle and (b) subcutis with brown fat and mammary gland tissues. Haematoxylin and eosin. Objective x 2.5. Inset: Fibrous capsule with collagen fibres, fibrocytes and a layer of multinucleated giant cells to where the transponder had been (arrows). Haematoxylin and eosin. Objective x 100

In the tissue around seven of the eight bioglass transponders there were optically empty spaces resembling lipid vacuoles, surrounded by granulomatous inflammation (Fig 3). In two of the eight specimens the fibrous capsule contained many macrophages. In all eight specimens there was a minimal mixed cellular infiltration in the tissue adjacent to the fibrous capsule (Fig 4).

Fig 3

Granulomatous inflammation around an optically empty space where a bioglass-coated transponder (T) had been. Haematoxylin and eosin. Objective x 10. Inset: x 40

Fig 4

Mixed cellular infiltration (arrows) and a multinucleated giant cell (arrowhead) in the tissue adjacent to where a bioglasscoated microchip had been. Haematoxylin and eosin. Objective x 40

In vitro, the density of the cell layer decreased distinctly from the middle of the culture flask towards the transponders. In their vicinity only a few round cells were visible, whereas in the control flasks there was a dense layer of spindle-shaped cells throughout the whole flask (Fig 5). Cells could be detected only rarely by SEM on the bioglass transponders.

Fig 5

(a) Highdensity layer of cells around a titaniumcoated transponder (T) exposed to a culture of feline fibroblastoid cells in vitro. (b) Lowdensity layer of round cells around an aluminium oxidecoated transponder (T) similarly cultured in vitro. (c) Control: dense layer of spindle-shaped cells in vitro. E Edge of the back wall of the culture flask. Objective x 20

The transponders with the polypropylene cap induced a focal proliferation of granulation tissue in four of the eight mice that received this type of transponder (Fig 6), but the others had no or only a few signs of inflammation. The hair granulomas were larger around the polypropylene transponders than around the other types and often contained multiple fragments of hair. The tissue around the bioglass part of the transponders had few signs of inflammation, and only one of them had granulation tissue in this area. In vitro, the density of the cell layer around the transponders with a polypropylene cap was high, but only moderate numbers of cells were detected on them (Fig 7). The surface of the polypropylene cap often had an uneven and squamous texture.

Fig 6

Proliferation of granulation tissue (arrows) around where a transponder (T) with a polypropylene cap had been. Haematoxylin and eosin. Objective x 40

Fig 7

Scanning electron micrograph showing moderate numbers of cells (arrows) on the polypropylene cap of a transponder exposed to a culture of feline fibroblastoid cells in vitro. x 100

The tissues around four of the 10 transponders coated with parylene C contained particles of foreign material surrounded by mild granulomatous inflammation (Fig 8); these particles were birefringent. No other signs of inflammation were detected. The results of the in vitro studies were similar to those with the polypropylene transponders, and the surface of the devices appeared to be quite uneven.

Fig 8

Foreign material (arrows) in and adjacent to a multinucleated giant cell (arrowhead) close to where a parylene C-coated transponder (T) had been. Haematoxylin and eosin. Objective x 40

Four of the 10 mice with titanium-coated transponders had small pieces of grey foreign material in the fibrous tissue capsule, accompanied by either a mild to moderate granulomatous inflammation (Fig 9) or with no reaction. In vitro, the titanium-coated devices developed a dense, confluent layer of cells around them (Fig 5a). By SEM, there was a dense layer of the cells of the FSI cell line (Fig 10), and a moderate to large number of the cells of the FS VI cell line on the surface of the transponders.

Fig 9

Fibrous capsule with granulomatous inflammation around grey foreign body particles (arrows) close to where a titanium-coated transponder (T) had been. Haematoxylin and eosin. Objective x 40

Fig 10

Scanning electron micrograph showing a dense layer of cells on a titaniumcoated transponder exposed to a culture of feline fibroblastoid cells in vitro. x 200

With five of the seven aluminium oxide-coated transponders there were foci of granulomatous inflammation in the fibrous tissue capsule and the surrounding tissue (Fig 11), and moderate numbers of eosinophils and mast cells. In vitro, the density of the cell layer around them was low, and there were only a few round cells in their vicinity, as with the bioglass transponders (Fig 5b). By SEM there were very few cells on the surface of the transponders, which regularly showed craterlike indentations.

Fig 11

Irregular fibrous capsule with inflammatory cells (arrows) close to where an aluminium oxide-coated transponder had been. Haematoxylin and eosin. Objective x 40

Discussion

The implantation of a microchip transponder leads initially to small tissue lesions that heal (Gruys and others 1993, Lammers and others 1995, Mader and others 2002, Murasugi and others 2003). A second reaction is the fibrous encapsulation of the device (Rao and Edmondson 1990, Gruys and others 1993, Geisel and others 1998, Mader and others 2002, Murasugi and others 2003). Jansen and others (1999) suggested that the relationship between the tissue reaction and the migration of the transponder could be explained by myofibroblasts inducing wound contraction during healing.

Good biocompatibility is a basic requirement for transponders (Gruys and others 1993, Pirkelmann and Kern 1994). An implant should evoke as little inflammation as possible because inflammatory cells can produce and secrete proteolytic enzymes (Kumar and others 2005). If its surface material causes permanent irritation and thus chronic inflammation, it is probable that proteolytic enzymes will be produced and released continuously, resulting in continuous collagenolysis and/or tissue reconstruction, reducing tissue stability. As a result, the transponder may be able to migrate more easily, for example, in combination with skin movements. Leiting and others (2001) suggested that the release of chemical mediators such as interleukin (IL)-1, IL-6, tumour necrosis factor-?? and prostaglandin E2 by activated macrophages might explain the aseptic loosening of implants. It is therefore likely that the fewer the signs of inflammation in the tissue around a microchip, the less likely it would be to migrate.

In vitro, cell growth is influenced by the chemical and physical properties of the materials added to the culture. Cytotoxic material, for example, polyvinyl chloride, may inhibit cell growth. Surface texture may also play a role. Neumann and others (2004) found that cell growth was significantly better on polished surfaces than on unpolished ones. Another factor is the hydrophilicity of the material. Kammer and others (2002) showed that cells grew better on a material that was made more hydrophilic by RIE than on the same material left untreated.

Unimpaired cell growth in the vicinity of and on transponders in vitro may indicate that the material may provide a favourable environment for cells - especially fibroblasts - in vivo. In contrast, weak cell growth in vitro may indicate that fibroblast proliferation and adhesion would be less likely in vivo, thus delaying wound healing and the encapsulation of the device. A cytotoxic material could even induce necrosis and inflammation.

It is therefore hypothesised that unaffected cell growth on the transponder and in its environment in vitro might indicate that it would be locally stable in vivo.

A fibrous tissue capsule was visible in all the histological specimens. It is considered that once a transponder is encapsulated it is unlikely to migrate any further (Behlert and Willms 1992, Gruys and others 1993). According to Ratner (2002), both the fibrous encapsulation and the macrophages at the interface of an implant are normal components of a foreign body reaction. This raises the question of why transponders can migrate, if they always become encapsulated. One possible explanation is that the fibrous capsule should not be considered as a stable and final product, but as a dynamic structure. Mechanical stresses, for example, as a result of the animal's movement, are likely to cause the reorganisation or even rupture of the capsule, and the stability of the capsule may be reduced by collagenolysis due to chronic inflammation in the vicinity of the transponder. An uncomplicated encapsulation and a capsule with few signs of inflammation are interpreted as signs of good tissue compatibility and local stability.

The in vivo data were obtained in mice and the in vitro data using feline fibrosarcoma cell lines. The results may be applicable to other species, but differences between different species and cell lines cannot be excluded.

The transponders with aluminium oxide and bioglass surfaces induced the least favourable reactions in both parts of the study. In contrast, other studies with bioglass transponders have observed little or no inflammation (Gruys and others 1993, Jansen and others 1999). No granulomatous inflammation or lipogranulomas have been reported in connection with bioglass transponders, and no such reactions were observed around the bioglass part of the transponders with the polypropylene cap. Bioglass is regarded as having high biocompatibility, in vivo and in vitro (Vrouwenvelder and others 1992, Jansen and others 1999, Mader and others 2002). The probable explanation for the lipogranulomatous inflammation and the highly affected cell growth in vitro was the petrolatum on the transponders and in their injection system; the distributor confirmed that petroleum was routinely applied for technical reasons, to facilitate handling of the transponders.

In both parts of the study, the results for aluminium oxide were unfavourable, in contrast with previous experiences with aluminium oxide; as an implant material in human medicine, aluminium oxide is considered to be biocompatible (Hannouche and others 2005), and it is used as a non-toxic reference material for in vitro cytotoxicity experiments in accordance with ISO 10993-12 (Neumann and others 2004). However, in the present study, the aluminium oxide-coated transponders had become corroded after seven weeks in tissue.

There was often a proliferation of granulation tissue around the polypropylene cap on the bioglass transponders. This finding cannot be explained by the healing of the small wound caused by the injection, because it should have been completed in much less than seven weeks. Furthermore, there was a similar reaction around the bioglass part of only one of these transponders. The polypropylene (or the special structure of the cap) clearly caused continuous irritation for the tissue, which reacted with a focal proliferation of granulation tissue.

In cell culture, polypropylene is an established material for culture flasks (Lindl 2000) because it is not cytotoxic. This was confirmed in the present study. The transponders were surrounded by a confluent layer of cells, and the fact that only a moderate number of cells were found on them can be explained by the unevenness of the surface.

Hair granulomas were found in specimens of tissue surrounding all the types of transponder, probably as a result of fragments of hair being carried from the epidermis to the subcutis as they were injected. The hair granulomas in the animals that received transponders with polypropylene caps were probably more pronounced because the diameter of the cannula was larger (3.0 mm) than in the other systems (2.6 mm).

The best results were obtained with titanium- and parylene C-coated transponders. The grey particles in the tissue close to some of the titanium-coated transponders were probably particles of titanium. In human medicine, there are reports of peri-implant metallosis in association with titanium implants (Leiting and others 2001) and, according to Pohler (2000), the particles are the result of abrasion. Such findings are quite rare and are associated with little or no inflammation. Similarly, in this study, the titanium particles were rarely accompanied by granulomatous inflammation. The undisturbed cell growth observed in vitro provides evidence that titanium is a convenient surface for the adhesion of fibroblasts and does not disturb their proliferation.

Fragments of foreign material were also observed in some of the mice with parylene C-coated transponders. These fragments were probably fragments of parylene C and caused a mild foreign body reaction. Parylene C is non-toxic in vitro (Institut für Technologie und Entwicklung von Medizinprodukten 2005). Kammer and others (2002) reported that parylene C that had been made more hydrophilic by a special etching process supported cell growth better than untreated parylene C. Those authors called untreated parylene C a biocompatible material that does not offer an appropriate surface for cell adhesion. This would explain why only moderate numbers of cells were observed on the transponders in spite of the high density of the cell layer around them. The fact that the surface of the parylene C was quite uneven also possibly affected cell adhesion (Neumann and others 2004).

The results of this study show that the surface material of a microchip transponder can influence the composition of the fibrous tissue capsule and the tissue reaction in vivo as well as cell growth in vitro. In vitro, cell growth in the presence of titanium microchips was much better than in the presence of any of the other devices; in vivo, mild to moderate granulomatous inflammation was observed around titanium particles in some of the mice. In vivo, parylene C elicited almost no inflammatory reactions in the surrounding tissue, whereas in vitro only a moderate number of cells could be detected on the parylene C transponders.

Titanium and parylene C therefore seem to be the most suitable of the materials tested for coating the surfaces of microchip transponders.

References

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