In vitro evaluation of cytocompatibility of shellac as coating for intravascular devices.
Drug-eluting intravascular devices such as stents and balloon catheters carried the hope to prevent restenosis caused by smooth muscle cell (SMC) proliferation. However, in the recent years reports raise concerns about the long-term safety of drug-eluting stents (DES) regarding a delayed overgrowth of the prostheses by endothelial cells (EC). This delay of endothelial growth is induced by an inappropriate long-term drug release and can lead to late stent thrombosis (1). A suitable coating should not impair viability, proliferation and function of EC in the long run. In addition, the proliferative activity of SMC should not be activated.
Shellac is a purified resinous secretion of the lac insect Kerria lacca and used for decades as food additive E904 and in pharmaceutical industry for moisture protection, glossing, and enteric coating. Thus, the characteristics of shellac are well known, and the potential of shellac develops further due to technological progress (2).
Atherosclerosis is a progressive disease characterized by multifactorial injury to the vessel wall. Important cell types involved in the pathogenesis of atherosclerosis are EC and SMC. EC cover the inner surface of blood vessels and form the interface between the blood and the surrounding tissues. EC dysfunction denotes the beginning of atherosclerotic processes, and extensive damage of EC lining is supposed to trigger stent thrombosis (3). SMC constitute the middle layer of larger blood vessel walls (tunica media), and SMC proliferation accounts to the progression of atherosclerotic transformation leading to blood vessel stenosis as well as to restenosis often occurring in the medically treated blood vessel area (4). Thus, EC and SMC are important cell types in atherosclerotic progression and come into direct contact with intravascular devices during intervention and can thus be affected by their degradation products.
In this study, we characterised shellac coating regarding its in vitro cytocompatibility. We tested EC and SMC viability and inflammatory status after contact to shellac extraction products.
Materials and Methods
All chemicals, enzymes, antibiotics and biological factors were, if not indicated otherwise, supplied by Sigma Aldrich. Cell culture plastics were from Nunc and Greiner.
Shellac coating onto glass specimens was performed by application of 6 % ethanol-dissolved shellac by spraying. The thin coated layer was blow-dried at room temperature. This coating step was performed four times. The resulting shellac multilayer was approx. 0.7 mg shellac per 1 [cm.sub.2] (1.1 mg/specimen, as determined by weighing). Final drying and sterilization were performed at 135[degrees]C for 1 h in a heating chamber. Shellac extracts were produced by incubation of shellac-coated glass discs (see above) at 37[degrees]C for 24 h in cell culture medium. Tested extract concentrations are indicated in the results section.
Human dermal microvascular EC were isolated from juvenile foreskin as described previously (5). These experiments were conducted with the approval of the ethics committee (Medical Faculty, University of Rostock) and the patients consent. Cultivation of isolated EC was with Endothelial Basal Medium MV (PromoCell) containing 15 % fetal calf serum, basic fibroblast growth factor (bFGF, 2.5 ng/ml), sodium heparin (10 [micro]g/ml), 100 U/ml penicillin and 100 [micro]g/ml streptomycin (humidified atmosphere, 37[degrees]C, 5 % C[O.sub.2]). All experiments were performed with HDMEC in passage 3 or 4.
Human coronary artery SMC (PromoCell) were cultivated in SMC growth medium 2 (PromoCell) in passage 4 or 5 in a humidified atmosphere at 37[degrees]C and 5 % C[O.sub.2].
Assessment of cell viability and fluorescence staining
Cells were seeded on the sample discs with 32.000 EC or 8.500 SMC per [cm.sup.2], respectively. After 24 and 48 h, cell culture supernatants were discarded. MTS reagent (Promega) containing culture medium was added optionally.
Thereafter, cells were fixed and stained for quantification of relative cell number (by crystal violet staining) or for the interendothelial contact molecule CD31 and f-actin. Crystal violet staining and fluorescent staining for CD31, f-actin and nuclei were performed according to protocols described previously (6).
The proinflammatory cytokine IL8 was assayed by ELISA (R&D-Systems). Therefore, cell culture supernatants were collected after 24 h of incubation and frozen until execution of the experiment. The ELISA was performed according to the manufacturer's instructions.
To examine cytocompatibility, extraction products of shellac were made. EC and SMC were cultivated on standard tissue culture polystyrene surfaces until subconfluency and exposed to different concentrations of shellac extracts obtained following 24 h incubation at 37[degrees]C in cell culture medium. Cell number of both cell types, EC (Fig. 1a) and SMC (Fig. 1b), was unaffected by exposure to shellac extractions products, whereas exposure of EC with TNF as pro-inflammatory control showed a reduction of about 25 % after 24 h (data not shown). Further more, metabolic activity of SMC did not undergo articulate changes after 24 h of extract exposure compared with untreated cells (Fig. 1c). Thus, shellac extraction products do not impair viability or metabolic activity of EC and SMC.
EC are involved in barrier function, which is regulated by a specifically organized area located to the interendothelial contacts (IC). These IC are maintained by a number of different proteins, such as CD31 and f-actin (Fig. 2a). In non-inflammatory activated EC CD31 is nearly exclusively located within the IC, and the f-actin is organized as a peripheral actin ring. IC are affected during (patho-) physiological processes such as inflammation (Fig. 2b) (6). CD31, for example, can be activated by different soluble biological factors such as tumour necrosis factor a (TNF). When unaffected, EC show a continuous fringe of CD31 within their interendothelial contacts (Fig. 2c), and a peripheral actin ring is detectable (Fig. 2d). In case of inflammation (here induced by exposure to TNF as positive control), CD31 distribution is drastically changed: the CD31 distribution within the IC is discontinuous and also distributed on the cell surface (Fig. 2e). F-actin is reorganized as socalled stress fibres (Fig. 2f). Exposure of EC to shellac extraction products did not induce obvious changes from the unaffected controls: CD31 is continuously distributed within the IC (Fig. 2g), F-actin is localized within the peripheral actin ring (Fig. 2h). Thus, shellac extraction products do not induce changes in the arrangement of IC molecules.
[FIGURE 1 OMITTED]
A further aspect in pro-inflammatory activation of the endothelium is the release of pro-inflammatory factors, among them interleukin 8 (IL8) which can be synthesized by EC and which is associated with chemotaxis of neutrophils (7). Exposure to different concentrations of shellac extraction product did not induce any changes in the release of IL8 compared to the untreated control, whereas the positive control (TNF) induced a clear inductive response (data not shown). Hence, no indications of proinflammatory activation by shellac extraction products, neither on IC nor on the IL8 release, were detectable. Thus, exposure of EC and SMC to shellac or its extraction products did not impair EC and SMC viability, did not activate proliferation of SMC and did not induce proinflammatory activation of EC in vitro.
[FIGURE 2 OMITTED]
DES have attracted considerable interest in the recent years. Many DES utilize long-term stable polymers as coatings. This long-term stability and thus related extended period of drug release is believed to be associated with impaired re-endothelialization and late stent thrombosis (1). Since application of a drug on the device's surface needs a carrier to attain controlled elusion, substantial efforts are currently underway to find alternative coating strategies. In general, biodegradable polymers provide promising options due to their adjustable elusion characteristics and the possibilities for further modifications (8).
We confirmed comprehensive cytocompatibility of the shellac biopolymer in all tested aspects. A recent investigation demonstrated that shellac-based coatings are suitable for drug delivery systems (9). Another recent study in a porcine coronary artery stent model revealed absence of increased inflammation for shellac coatings of synthetic polymer-coated rapamycin-eluting stents (10). Thus, shellac could give direction to the medical device-coating field.
This work financed by the European Union and the Federal State Mecklenburg-Vorpommern (Ref. No. V220630-08-TIFA-588). The authors would like to thank Stefanie Adam for technical assistance and Prof. Dr. G. Stuhldreier, Dr. M. Drewelow and Dr. I. Dittrich for the supply with tissue samples.
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Kirsten Peters  **, Cornelia Prinz , Achim Salamon  *, Joachim Rychly , Hans-Georg Neumann 
 Department of Cell Biology, * Junior Research Group, Biomedical Research Centre, Medi-cal Faculty, University of Rostock, Schillingallee 69, 18057 Rostock, Germany
 DOT GmbH, Charles-Darwin-Ring 1a, 18059 Rostock, Germany
Corresponding author: Kirsten Peters (firstname.lastname@example.org)
Received 13 February 2012; Accepted 20 February 2012; Available online 27 April 2012
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|Title Annotation:||Short Communication|
|Author:||Peters, Kirsten; Prinz, Cornelia; Salamon, Achim; Rychly, Joachim; Neumann, Hans-Georg|
|Publication:||Trends in Biomaterials and Artificial Organs|
|Date:||Apr 1, 2012|
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