Biocompatibility screening in cardiovascular implants - Springer Link

Z Kardiol 94:383–391 (2005) DOI 10.1007/s00392-005-0231-4

M. Sigler T. Paul R. G. Grabitz

Biokompatibilitätsuntersuchungen an kardiovaskulären Implantaten n Zusammenfassung Hintergrund Zunehmendes Interesse gilt der Biokompatibilität kardiovaskulärer Implantate. Ziel dieser Arbeit ist die Vorstellung von Methoden und Ergebnissen der pathologischen Aufarbeitung von explantierten Implantaten. Methoden Die Standardeinbettung von Implantaten zur histologischen Beurteilung in Paraffin ist nur eingeschränkt geeignet, da metallische Anteile vor der Einbettung unter Beschädigung der Grenzfläche Gewebe/Implantat entfernt werden müssen. Alternativ kommt eine Einbettung in Kunst-

Received: 14 September 2004 Accepted: 14 January 2005

Dr. Matthias Sigler ()) Prof. Dr. Thomas Paul Herzzentrum Göttingen Pädiatrische Kardiologie und Intensivmedizin Georg-August-Universität Göttingen 37099 Göttingen, Germany E-Mail: [email protected]

Biocompatibility screening in cardiovascular implants

harze in Frage, wobei histologische Schnitte mittels Schneiden und Schleifen angefertigt werden. Dies ermöglicht die Untersuchung lokaler entzündlicher Vorgänge an der Oberfläche des Implantates. Zusätzlich interessieren bei der Aufarbeitung der Implantate die Reaktion und Struktur des umgebenden Gewebes sowie der benachbarten Grenzfläche zum Blutstrom. Neben der Histologie kommen immunhistochemische Verfahren sowie die Elektronenmikroskopie zum Einsatz. Unter Verwendung der genannten Methoden demonstrieren wir Befunde von Implantatpräparaten aus Tierversuchen und entsprechende Ergebnisse von Implantaten, die bei Patienten im Rahmen von Korrekturoperationen bei angeborenen Herzfehlern entfernt wurden. Ergebnisse Nach der Implantation kommt es unabhängig vom Implantattyp zu einer raschen Re-Endothelialisierung der Gefäßoberfläche. In das nach Okkluder-Implantation initial gebildete Thrombusgewebe sprossen fibromuskuläre Zellen ein, wie sie auch nach Stentimplantation in der Intimahyperplasie gesehen werden. Entzündliche Reaktionen sind in Qualität und zeitlichem Verlauf materialabhängig. Zusammenfassung Mit einer vollständigen pathologischen Aufarbeitung kardiovaskulärer Implantate nach

Explantation können Informationen über Einwachsen, Endothelialisierung und Entzündungsreaktionen gewonnen werden. n Schlüsselwörter Biokompatibilität – Implantate – Angeborener Herzfehler – Histologie – Immunhistochemie n Summary Background Interest in information on biocompatibility of implants is increasing. The purpose of this paper is to discuss methods and results of pathological biocompatibility screening of explanted cardiovascular implants. Methods Use of standard histology after embedding in paraffin is limited since metallic implants have to be removed during workup with disruption of the specimen. Alternatively, tissue blocks containing an implant can be embedded in methylmethacrylate or hydroxyethylmethacrylate and processed by sectioning with a diamond cutter and grinding, thus leaving the implant in situ and saving the tissue/implant interface for detection of local inflammatory reactions. Another important aspect of evaluation is the progress of thrombus organisation after initial fibrin clotting on the metal surface or in the inner part of occlusion devices. New methacrylate resins and embedding techniques allow for specific immunohisto-

ZFK 0231

Prof. Dr. Ralph G. Grabitz Universitätsklinik und Poliklinik für Kinderkardiologie Martin-Luther-Universität Halle-Wittenberg 06097 Halle, Germany

ORIGINAL PAPER

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Zeitschrift für Kardiologie, Band 94, Heft 6 (2005) © Steinkopff Verlag 2005

chemical staining of the specimen thus enabling characterisation of tissues surrounding the implant. Information on endothelialisation of the vascular surface of the implant can be obtained by means of immunohistochemistry or by scanning electron microscopy. Results Illustrating the use of these technologies, we demonstrate findings in tissue specimens from animal studies with different types of devices (i.e. stents, occlusion devices). We present corresponding findings in human specimens with implants that were removed

during corrective surgery for congenital heart defects. Early endothelialisation of the vascular surface was seen after implantation in all types of devices. Cells within occlusion devices could be characterised histologically and immunohistochemically as fibromuscular cells as seen in intimal hyperplasia after stent implantation. Inflammatory implant-host reactions ranged from mild to moderate (medical grade stainless steel, nitinol) to severe (polytetrafluoroethylene [PTFE]). Conclusions With an optimal work-up of

Introduction Studies on new implants for interventional therapy of congenital heart defects mainly focus on feasibility and safety as well as clinical results [16, 22, 27]. In contrast, data on biocompatibility are rare and often of a more general character. Local tissue reactions after implantation appear important for long-term success of interventional therapy. While interventional procedures become increasingly relevant for patients with congenital heart defects, more attention is paid to in vivo biocompatibility [25, 32, 35]. After implantation most biomaterials are left in the body for life. This is true for the adult patient who has received a stent in a coronary artery stenosis. But it is even more relevant to the paediatric patient who has undergone interventional therapy and who will live with the implant for possibly more than 80 years. The purpose of this overview is 1) to present a systematic approach to the work-up of surgically removed implants and 2) to demonstrate results that have been obtained using the presented protocol of biocompatibility screening.

Implant work-up n Tissue specimens Animal specimens presented in this review were obtained from animal experiments that were conducted according to the guidelines of the German Animal Protection Law and were approved by the State Agency supervising animal experiments. Human tis-

cardiovascular implants, ingrowth and endothelialisation as well as inflammatory reactions in the surrounding tissue can be assessed. This information allows evaluation of individual tissue reactions to the implant and may serve as valuable basis for optimisation of biocompatibility by implant modification. n Key words Biocompatibility – implants – congenital heart defect – histology – immunohistochemistry

sue specimens were collected during corrective surgery for congenital heart defects and were sent to us for histopathological work-up. So far, we have evaluated explants from 49 patients and from more than 200 animals.

n Tissue preparation Immediately after explantation, the tissue block containing the implant was dissected free with a minimum of surrounding tissue. After briefly flushing with normal saline, macroscopic evaluation and documentation were accomplished. Parts of the specimens arranged for histology and immunohistochemistry were fixed in ethanol/methanol (50/50) or formalin (buffered 4%). For scanning electron microscopy, usually one part of the specimen was placed in glutaraldehyde (2.5%).

n Embedding and sectioning After fixation, the main part of the tissue block with the device was embedded in the resin hydroxyethylmethacrylate (Technovit 7200 or Technovit 8100, Kulzer & Co, Wehrheim, Germany) or methylmethacrylate (Technovit 9100, Kulzer & Co, Wehrheim, Germany) (Fig. 1). After hardening, the resin blocks were subsequently sectioned into slices of 0.8 mm using a diamond cutter. These slices were ground down to 5–30 lm. Parts not containing metal (e.g. structures neighbouring the site of the implant, or parts only containing polymeric textile fibres or polyvinyl alcohol foam material) were dehydrated and placed in paraffin wax in the usual way.

M. Sigler et al. Implant biocompatibility

Fig. 1 Methylmethacrylate block containing the right atrial disc of an Amplatzer ASD occluder after slicing through half the specimen

n Histology Staining of resin-embedded specimens was performed with Toluidine blue or Richardson blue. Paraffin wax-embedded specimens were stained with haematoxylin and eosin (standard staining) or elastica van Gieson (staining of elastic fibres and fibrous tissue).

n Immunohistochemical staining Immunohistochemical staining was performed in paraffin-embedded specimens as well as in resin-embedded specimens (Technovit 8100 and Technovit 9100, Kulzer & Co, Wehrheim, Germany). For immunohistochemical staining, binding of primary antibodies was detected using horseradish peroxidase conjugated secondary antibodies. The sections were counterstained with hemalaun.

n Scanning electron microscopy Usually, one part of the specimen was submitted for scanning electron microscopy and placed in glutaraldehyde (2.5%). Images were obtained after critical point drying and gold coating of the specimen.

n Reactions on the vascular surface at the site of device application Endothelial damage during implantation of an intravascular device is a well described phenomenon [7, 9]. Endothelial damage again causes activation of

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the coagulation system with local fibrin formation and thrombocyte aggregation beside its effects on intimal growth which will be discussed later [26]. Thrombus formation is a feared complication at the site of implantation not only due to endothelial damage but also due to a foreign body surface directly exposed to the blood stream [17, 37]. Major events such as vessel occlusion or embolisation may occur as a consequence of thrombus formation. For prevention, anticoagulants are applied locally or systemically [8, 36]. Systemic anticoagulation using thrombocyte aggregation inhibitors is standard therapy following any interventional device application. In contrast, local administration of heparin, r-hirudin, or other anticoagulants by means of biologically active coating has not reached clinical use despite promising results in vitro [15]. Beside all efforts to reduce superficial thrombus formation using anticoagulants it is widely accepted, that early re-endothelialisation is desirable because of the antithrombotic nature of endothelial cells [28]. Up to now, no clinically relevant reduction of thrombogenicity has been achieved by pre-seeding interventionally applied implants with endothelial cells [13, 14]. Animal experiments have shown some acceleration of re-endothelialisation by local application of vascular endothelial growth factor (VEGF) or by fibronectin coating of the implant material [2, 15, 38]. In contrast, positive clinical results were achieved with surgically implanted vascular PTFE grafts pre-seeded with endothelial cells [20]. New insight in to the mechanism of re-endothelialisation after endothelial damage has been gained from experiments demonstrating that cells of the regenerated endothelium derive from hematopoetic stem cells (endothelial progenitor cells) [29, 34]. In an animal series of interventional closure of the patent ductus arteriosus (PDA) using three different types of implant, the time patterns of formation of a cellular monolayer on the aortic and pulmonary surface of the implants were not different [35]. As demonstrated by electron microscopy, the first step of coverage of the implants was shown to be formation of a fibrin net with inclusion of blood cells on the surface of the biomaterial exposed to blood. This was followed by spreading of endothelial cells over the fibrin net (Fig. 2 a). The earliest time point after implantation at which endothelial cells were observed adjacent to the surface of the fibrin net was 10 days in our series. A confluent monolayer of endothelial cells could be demonstrated in all specimens excised 4 weeks or more after implantation (Fig. 2 b). Corresponding findings were observed after stent implantation in the ductus arteriosus. Endothelialisation of the vascular surface was completed in all

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specimens at least 4 weeks after implantation. In accordance with this finding, all human specimens examined in our laboratory with implantation times of more than 4 weeks showed an intact monolayer of endothelial cells. Immunohistochemical staining of superficial cells in both paraffin- and resin-embedded specimens and in both human and animal samples were positive for von Willebrand factor (DAKO A/S, Glostrup, Denmark) which identifies these cells as endothelial cells (Fig. 3). Thrombus formation is of paramount importance if seen adjacent to the vessel wall at the site of implantation. In our series, however, no thrombi were found. a

n Intimal reactions and tissue formation after implantation

Fig. 2 a Single endothelial cells overgrow a fibrin network with blood cells on the vascular surface of a medical grade stainless steel coil (completely covered) 20 days after interventional closure of a PDA in a lamb model. Scanning electron microscopy. b A medical grade stainless steel coil is completely covered with endothelium (view of the aortic side) 112 days after interventional PDA closure in a lamb. Scanning electron microscopy

The main clinical problem after coronary stent implantation is intimal hyperplasia [33]. As described for coronary stents, we also see intimal hyperplasia in stents after implantation in the ductus arteriosus, pulmonary arteries, the aorta, or other locations (Fig. 4). The mechanisms leading to intimal proliferation are well described. Endothelial damage during implantation leads to activation of both the inflammatory as well as the coagulation system causing an activation of smooth muscle cells of the media by changing the state from contractile to synthetic. This is followed by migration of activated media cells into the intima with formation of intimal hyperplasia [24, 30]. Although most of the migrating cells origi-

Fig. 3 Positive immunohistochemical staining (brown) with von Willebrand factor (DAKO, 1:400) of superficial cells 117 days after interventional PDA closure by means of a polyvinyl alcohol foam plug in a lamb. Embedded in paraffin, counterstained with hemalaun. PVA polyvinyl alcohol foam material

Fig. 4 Intimal hyperplasia in a medical grade stainless steel stent 11 months after implantation in an infant with aortic coarctation. Embedded in methylmethacrylate, ground section, stained with Richardson blue. M media; NI neointima; SS stent strut

b


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a Fig. 5 c Cardioseal ASD occluder 24 months after implantation in a child surrounded by cells and connective tissue with no residual thrombus material. Embedded in methylmethacrylate, ground section, stained with Toluidine blue. T thrombus, VW vessel wall, D Dacron membrane; MS metal strut

b Fig. 5 a Central portion of a ductus arteriosus 48 days after interventional PDA closure by means of a medical grade stainless steel coil in a lamb showing the typically septated thrombus material within the windings of the coil (black). Embedded in methylmethacrylate, ground section, stained with Toluidine blue. b Central portion of a ductus arteriosus 32 days after interventional PDA closure by means of a nitinol coil in the lamb showing complete cellular organisation with little connective tissue within the windings of the coil (black). Embedded in methylmethacrylate, ground section, stained with Toluidine blue

nate in the media, adventitial myofibroblasts were also shown to contribute to neointima formation [40]. Intimal hyperplasia after stent implantation is unwanted and a variety of attempts have been undertaken to avoid or at least to minimise activation and proliferation of smooth muscle cells of the media [5, 10, 12, 21, 23]. In contrast to the situation after stent implantation, cell proliferation plays a different role after implantation of an occlusion device. The initial mechanism of occlusion of a PDA after implantation of a coil is thrombus formation within the implant (Fig. 5 a) [35]. Thus, thrombotic occlusion is part of the therapeutic concept after coil implantation, while

it represents a major complication after stent implantation. To enhance thrombogenicity of coils or other occlusion devices, often textile fabric is incorporated into the implant design (e. g. polyester fibres in Cook Coils or in Amplatzer occlusion devices). Cellular organisation of the thrombus material within the device proceeds in a material dependent time pattern and usually is completed 6 months after implantation (Fig. 5 b). The same pattern of early thrombus formation within the implant with gradual cellular organisation is seen after implantation of septal occluders (Fig. 5 c). Recanalisation of the interventionally occluded structure is possible as long as “unorganised” thrombus material is present [18, 31]. Thus, in contrast to attempts to avoid proliferation after stent implantation, cell proliferation is desirable after occluder implantation to avoid recanalisation. We have reason to believe that tissue organisation after implantation of an occlusion device occurs in a similar sequence as described for stents. It is a constant finding in specimens with incomplete cellular organisation that cells seem to grow into the implant originating from the implant-vessel interface (Fig. 6). This applies to the mechanism of migration of activated smooth muscle cells and the subsequent formation of intimal hyperplasia after stent implantation. In addition, cells within an implant after thrombus organisation and cells in intimal hyperplasia exhibit a corresponding pattern of antigen characteristics as demonstrated by immunohistochemical staining with positive reaction for smooth muscle actin (monoclonal antibody, clone 1A4, Sigma Immuno Chemicals, St. Louis, USA), desmin (monoclo-

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a Fig. 6 Amplatzer VSD occluder 71 days after implantation in a child surrounded by cells in the outer part (right) and by thrombus material in the inner part (left) of the device. Embedded in methylmethacrylate, ground section, stained with Richardson blue. C cells; D Dacron fibres; NS nitinol struts; T thrombus.

nal antibody, Laboserv GmbH, Giessen, Germany; both indicative for muscle cells), and vimentin (monoclonal antibody, clone V9, DAKO A/S, Glostrup, Denmark; indicative for mesenchymal origin) thus identifying these cells as fibromuscular cells (Fig. 7). It would be of great interest to examine molecular mechanisms of cell proliferation within the implant and in the surrounding tissue after occluder implantation to correlate these findings to cellular reactions after stent implantation.

n Implant related inflammatory reactions Foreign body surface in contact with blood serves as an attractive site for bacterial adhesion. But even without the presence of infective agents, inflammation occurs at the site of implantation. Three different types of local inflammatory reaction can be described associated with cardiovascular implants [1]. The first type is accumulation of granulocytes locally related to the implant surface [6]. This reaction is caused by neutrophil migration into the site of damage to the endothelium and underlying structures during implantation. It is seen as early as one day after implantation and appears to be transient since it is not seen in specimens from implants with implantation times of more than 4 weeks. The second type of inflammation is lymphocyte infiltration which is found adjacent to the implant. It is characterised by a rather loose accumulation of inflammatory cells with a large and dense cell nucleus. It develops within the first week after implan-

b Fig. 7 Positive immunohistochemical staining (brown) of neointima cells and media cells for smooth muscle actin (a 1:50, clone 1A4, Sigma Immuno Chemicals, St. Louis, USA) and vimentin (b 1:100, clone V9, DAKO A/S, Glostrup, Denmark) 8 months after implantation of a medical grade stainless steel stent in PDA (human). Embedded in methylmethacrylate, ground sections, counterstainined with hemalaun. M media; NI neointima; SS stent strut (in b the stent strut dropped away during the grinding procedure)

tation and may persist [1, 33]. In our series, we have observed lymphocytic infiltration even 50 months after implantation (Fig. 8 a). Presence of lymphocytes indicates a persistent inflammatory stimulus. The origin of this reaction is still unclear in the face of a rapid encapsulation of the foreign material by fibrin and the subsequent cellular ingrowth. An explanation may be the persistent metal ion elution described for metallic implant materials both in vitro and in vivo [11, 19, 32]. A positive correlation was shown for local nickel release by nickel-containing implants and tissue inflammation [39]. The third type of inflammation is foreign body reaction with monocyte infiltration. Monocytes


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a

b

Fig. 8 c Foreign body giant cell (arrow) adjacent to a nitinol coil (black) 216 days after interventional PDA closure in a lamb. Embedded in methylmethacrylate, grounded section, stained with Toluidine blue

Conclusions

Fig. 8 a Palmaz-Schatz coronary stent in a stenotic left pulmonary artery 50 months after implantation in a child showing a lymphocytic infiltrate (arrow) close to a metal strut. Embedded in hydroxyethylmethacrylate, ground section, stained with Richardson blue (M media; NI neointima; SS stent strut. b Fibres of the Dacron membrane (PTFE; brown) of a Cardioseal PFO occluder 3 years after implantation in an adult showing multiple foreign body giant cells (arrows). Embedded in methylmethacrylate, ground section, stained with Toluidine blue

eventually differentiate into macrophages and fuse with the formation of polynuclear foreign body giant cells [3, 4]. This type of inflammatory reaction is considered the normal wound healing response and is accompanied by formation of well vascularised granulation tissue. It is typically more pronounced in specimens containing non metal implant materials such as polytetrafluoroethylene (PTFE) or other polymeric textile fibres in Amplatzer or Cardioseal devices (Fig. 8 b). It may also be seen on metal surfaces (Fig. 8 c). Foreign body reaction occurs 1 to 4 weeks after implantation and persists as described for the lymphocytic inflammatory reaction. All types of inflammatory reaction demonstrated above are encountered in both animal and human tissue specimens in a corresponding pattern.

A valuable evaluation of in vivo biocompatibility can be achieved using the preparation protocol described above. Electron microscopy and immunohistochemistry allow for examination of the time course and completeness of endothelialisation as well as for identification of antigen characteristics of endothelial cells. In our series, rapid endothelialisation was demonstrated after initial endothelial damage during the implantation procedure. Cellular organisation of the initially accumulated thrombus material at the blood/implant interface or within occlusion devices was seen to proceed in a material-dependent time pattern. Immunohistochemistry allowed characterisation of newly formed tissues. Furthermore, inflammatory processes could be detected at the site of implantation and may be related to the type of implant material and implantation time of the device. So far, all information on biocompatibility of cardiovascular devices for interventional implantation has been derived from animal experiments. Implantation time in experimental series is often very short when compared to the possible implantation time of many decades in a young human being. This is due to limited resources in many respects and the desire to obtain prompt results from experiments that are highly demanding with regard to time and effort. In addition, animals in experimental series are healthy subjects in contrast to most of our young patients.

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Transfer of findings from animal experiments to humans may be less reliable than desired since no standardised information has been published yet with regard to this problem. To fill this gap and to learn more about the interaction between an implant and surrounding tissue, every explanted device should undergo careful pathological examination. Hope is expressed that a growing number of cardiologists, cardiovascular sur-

geons, and pathologists may become interested in the study of the important field of biocompatibility. The authors would like to offer the opportunity to work-up formalin fixed tissue specimens containing cardiovascular devices.

n Acknowledgements The authors thank Simone Freund, Andrea Poppe, and Karin Baer for technical assistance.

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