In vitro evaluation of cytocompatibility of shellac as coating for intravascular devices.
Abstract: Shellac is used for decades as food additive and in pharmaceutical industry. Since characteristics of shellac are well known, we considered shellac as a coating for drug-eluting intravascular devices. Endothelial cells (EC) and smooth muscle cells (SMC) are important in arteriosclerotic progression, and both cell types come into direct contact with intravascular devices during intervention. Therefore, we examined shellac regarding its in vitro cytocompatibility by utilization of primary human cells. We could show that shellac-coated materials did not impair viability of EC and SMC, did not induce proliferation of SMC, and did not change the inflammatory status of EC in vitro. Thus, shellac is particularly suitable for coating of drug-eluting intravascular devices.
Article Type: Report
Subject: Drug-eluting stents (Materials)
Shellac (Health aspects)
Shellac (Testing)
Biomedical materials (Testing)
Biomedical materials (Health aspects)
Authors: Peters, Kirsten
Prinz, Cornelia
Salamon, Achim
Rychly, Joachim
Neumann, Hans-Georg
Pub Date: 04/01/2012
Publication: Name: Trends in Biomaterials and Artificial Organs Publisher: Society for Biomaterials and Artificial Organs Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2012 Society for Biomaterials and Artificial Organs ISSN: 0971-1198
Issue: Date: April, 2012 Source Volume: 26 Source Issue: 2
Topic: Event Code: 330 Product information
Geographic: Geographic Scope: Germany Geographic Code: 4EUGE Germany
Accession Number: 304842713
Full Text: Introduction

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

Materials

All chemicals, enzymes, antibiotics and biological factors were, if not indicated otherwise, supplied by Sigma Aldrich. Cell culture plastics were from Nunc and Greiner.

Sample preparation

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.

Cell culture

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).

IL8 ELISA

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.

Results

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]

Conclusions

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.

Acknowledgments

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.

References

(1.) E. Van Belle, S. Susen, B. Jude and M.E. Bertrand, Drug-eluting stents: trading restenosis for thrombosis?, J. Thromb. Haemost., 5 Suppl 1, 238-245 (2007).

(2.) B. Qussi and W.G. Suess, The influence of different plasticizers and polymers on the mechanical and thermal properties, porosity and drug permeability of free shellac films, Drug Dev. Ind. Pharm., 32, 403-412 (2006).

(3.) M.W. Webster and J.A. Ormiston, Drug-eluting stents and late stent thrombosis, Lancet, 370, 914-915 (2007).

(4.) T. Inoue and K. Node, Vascular failure: A new clinical entity for vascular disease, J. Hyperten., 24, 2121-2130 (2006).

(5.) K. Peters, H. Schmidt, R.E. Unger, M. Otto, G. Kamp and C.J. Kirkpatrick, Software-supported image quantification of angiogenesis in an in vitro culture system: application to studies of biocompatibility, Biomaterials, 23, 3413-3419 (2002).

(6.) K. Peters, R.E. Unger, S. Stumpf, J. Schafer, R. Tsaryk, B. Hoffmann, E. Eisenbarth, J. Breme, G. Ziegler and C.J. Kirkpatrick, Cell type specific aspects in biocompatibility testing: the intercellular contact in vitro as an indicator for endothelial cell compatibility, J. Mater. Sci. Mater. Med., 19, 1637-1644 (2008).

(7.) P. Proost, A. Wuyts and J. van Damme, The role of chemokines in inflammation, Int. J. Clin. Lab. Res., 26, 211-223 (1996).

(8.) C. Engineer, J. Parikh and A. Raval, Review on hydrolytic degradation behavior of biodegradable polymers from controlled drug delivery systems, Trends Biomater. Artif. Organs, 25, 79-85 (2011).

(9.) S. Limmatvapirat, C. Limmatvapirat, S. Puttipipatkhachorn, J. Nunthanid, M. Luangtana-Anan and P. Sriamornsak, Modulation of drug release kinetics of shellac-based matrix tablets by in-situ polymerization through annealing process, Eur. J. Pharm. Biopharm., 69, 1004-1013 (2008).

(10.) K. Steigerwald, S. Merl, A. Kastrati, A. Wieczorek, M. Vorpahl, R. Mannhold, M. Vogeser, J. Hausleiter, M. Joner, A. Schomig and R. Wessely, The pre-clinical assessment of rapamycin-eluting, durable polymer-free stent coating concepts, Biomaterials, 30, 632-637 (2009).

Kirsten Peters [1] **, Cornelia Prinz [2], Achim Salamon [1] *, Joachim Rychly [1], Hans-Georg Neumann [2]

[1] Department of Cell Biology, * Junior Research Group, Biomedical Research Centre, Medi-cal Faculty, University of Rostock, Schillingallee 69, 18057 Rostock, Germany

[2] DOT GmbH, Charles-Darwin-Ring 1a, 18059 Rostock, Germany

Corresponding author: Kirsten Peters (kirsten.peters@med.uni-rostock.de)

Received 13 February 2012; Accepted 20 February 2012; Available online 27 April 2012
Gale Copyright: Copyright 2012 Gale, Cengage Learning. All rights reserved.