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Original Contribution
Polymer Stent Coating for Prevention of Neointimal Hyperplasia
September 2006
Attempts to reduce neointimal proliferation following coronary artery stent implantation have focused on stent coatings with or without local drug delivery. Polymers provide some unique features, which determine their use as coating material: (1) the ease of surface application by dip coating; (2) the potential for biodegradability;1 and (3) the passivation of the arterial wall by paving of the stent surface.2,3 However, previous data using polymers as stent coating material have shown mixed results, with a strong inflammatory and thrombogenic reaction enhancing neointimal proliferation and thus in-stent restenosis.4 In contrast, polymeric gel paving with polyethylene-diacrylate following conventional balloon angioplasty has been shown to reduce platelet-derived growth factor (PDGF)-mediated neointimal proliferation and to limit basic fibroblast growth factor (bFGF) release.2 These data suggest that the prevention of platelet and inflammatory cell recruitment following stenting could reduce the extent of neointimal hyerplasia.
Poly(L-lysine)-graft-poly(ethyleneglycol) (PLL-g-PEG) (Figure 1) is a new, bifunctional polymer: the positively charged amines provide stable electrostatic binding to the negatively charged metal oxide stent surface, whereas the hydrophilic, nonionic and mobile polyethylene glycol chains prevent adhesion of circulating proteins and cells.5,6 The adsorption of the PLL domains forces the pendant PEG chains into a so-called “brush” above the stent surface, which thermodynamically repels adsorption of plasma proteins in a phenomenon named steric stabilization. In vitro, adsorption of proteins from whole serum was demonstrated to be less than 1% of a monolayer.7 The purpose of the present study was to investigate the effect of polymeric steric stabilization of the stent surface with PLL-g-PEG on neointimal hyperplasia in the porcine restenosis model.
Methods
Animal instrumentation. The local Institutional Animal Care and Use Committee approved the study. It conforms to the guidelines established in the “Position of the American Heart Association on Research Animal Use” adopted by the American Heart Association on November 11, 1984. A total of 7 domestic farm pigs of either sex (mean weight: 48 ± 8 kg) were anesthetized according to our standard protocol.8Poly(L-Lysine)-graft-Poly(ethylene Glycol). PLL-g-PEG utilized in the present study was synthesized and purified following chemical reaction between PLL and an activated PEG, as described elsewhere.5–7Stent coating and implantation. A custom-made stainless steel, slotted-tube stent (Biotronik, Berlin, Germany) of 15 mm length and 2.5 to 3.5 mm expanded diameter was used in the present study. Stent coating was performed with PLL-g-PEG by dipping the stents for 30 seconds into the polymer solution (1% in phosphate-buffered saline) at room temperature under sterile conditions. Thereafter, the stents were allowed to dry for approximately 60 seconds and then manually crimped on a standard balloon for intracoronary placement.
Arterial access was established via the right carotid artery under sterile conditions. Heparin sodium (100 IU/kg) was administered intravenously. Coronary artery stenting was performed by positioning a 0.014 inch guidewire in the distal coronary artery and advancing the balloon-mounted stent under fluoroscopic control. Each pig was implanted with 2 stents: the PLL-g-PEG-coated stent and an uncoated control stent of otherwise identical design. The 2 stents in each pig were randomly implanted in either the left anterior descending artery, the circumflex artery, or right coronary artery in a vessel segment with a diameter of 2.5 to 3.5 mm. Inflation pressure ranged between 8 and 12 bar depending on the size of the vessel, approximating a stent-to-artery ratio of 1.1 to 1. Quantitative evaluation of coronary angiograms was performed off-line with the help of an automated, edge-detection system (CAAS-II, Pie Medical, The Netherlands). Vessel diameter and cross-sectional area of the proximal and distal stent segment were determined in all animals.
Histological examination. The animals were sacrificed 6 weeks after stent implantation by injection of 20 ml KCl into the aortic root. Then, the heart was excised and fixed in 300 ml buffered 4% formaldehyde. The coronary arteries were carefully dissected and embedded in poly(methyl-methacrylate). The fixed coronary artery segments were sectioned transversally and polished to a thickness of 100 µm. The tissue was prepared for light microscopy by staining with paragon.8 Next, the sections were analyzed on a digital system for quantitative histomorphometry (Image Pro Plus, Media Cybernetics, Silver Spring, Maryland). Histological evaluation was performed by two observers unaware of the location or coating status of the stent. Within the stented coronary segments, 3–5 sections were examined. Area of the lumen, intima and media were determined by digital histomorphometry.8 The extent of stent-mediated arterial injury was assessed according to the method of Schwartz et al.9Statistical analysis. Data are expressed as mean ± standard deviation (SD). Continuous variables were compared by the paired, two-sided Student’s t-test since each animal served as its own control. Data were analyzed using SPSS statistical software, Version 12.0 (SPSS, Inc., Chicago, Illinois). Statistical significance was assumed with a p-value Results
The distribution of PLL-g-PEG-coated and uncoated stents in the coronary artery tree were similar. Balloon-to-artery ratio and injury scores were comparable in both stent groups. Representative histological samples of a PLL-g-PEG coated and uncoated stent are shown in Figure 2.
Angiographic data. Quantitative coronary angiographic data before and immediately after stent implantation were similar for PLL-g-PEG-coated and uncoated stents (Table 1). Follow-up angiography 6 weeks after stent implantation revealed no significant differences in minimal luminal diameter and restenosis between the 2 stent types. Late luminal loss was significantly lower for PLL-g-PEG-coated stents than uncoated stents (Table 1).
Histomorphometric data. Neointimal area (1.15 ± 0.59 vs. 2.33 ± 1.01; p Discussion
The key players of vascular proliferation following stent implantation are platelets, leukocyte and smooth muscle cells.10,11 As a first response to vascular injury, platelets aggregate and fibrin adheres to the subendothelial matrix, resulting in thrombus formation.10 Activated neutrophils12 and monocytes invade the arterial wall through the expression of local adhesion molecules13 and generate oxygen radicals and metalloproteinase inhibitors, thus contributing to smooth muscle cell proliferation and migration. Efforts to reduce neointimal proliferation concentrate on the interruption of this cascade by stent coating with biocompatible materials8 and antiproliferative drugs14 directly deposited on the metallic surface of the stent or bonded to polymeric substances.
The present approach to limit neointimal hyperplasia was based on the concept of steric stabilization of the metallic stent surface, reducing adsorption of cell-adhesive proteins from the serum, such as fibrinogen, to nearly zero and thereby reducing platelet and leukocyte adhesion.5–7 Coating of the stent surface with PLL-g-PEG resulted in a significant reduction of neointimal hyperplasia and thus in-stent restenosis in the porcine restenosis model.
Polymers as stent coating materials. Previous investigations with polymers such as poly(glycolic acid-co-lactic acid), poly(e-caprolactone), poly(hydroxybutyrate-co-valerate), poly(orthoester), and poly(ethyleneoxide)/poly(butylene terephthalate) revealed an excessive inflammatory and thrombogenic reaction, which was attributed to a variety of factors such as polymeric load, degradation products, release kinetics and metallic salts bound to the polymer.4 More recently, Lincoff and colleagues15 reported a strong inflammatory reaction with a low-molecular weight poly(L-lactic acid) (PLLA) polymer, leading to enhanced neointimal proliferation, as opposed to reduced neointimal proliferation with a high-molecular weight PLLA. These investigators suggested that the differences in inflammatory response and neointimal proliferation are related to the increased degradation time and the amount of degradation products.
PLL-g-PEG appears to be one of the first polymers diminishing neointimal hyperplasia in the absence of an antiproliferative drug in the experimental setting. Furthermore, PLL-g-PEG did not provoke any inflammatory or prothrombotic response. Similar findings have been reported with gel paving of the arterial wall following conventional balloon angioplasty.2,3 These investigators suggested that gel paving insulates the injured arterial wall from blood-borne growth factors and chemokines. Thus, gel paving prevented the release of bFGF from the media and PDGF-mediated neointimal proliferation following balloon angioplasty. The novel design of the present polymer combined the poly(L-lysine) backbone as a strong anchor to the metallic stent surface and the anti-aggregatory properties of the PEG brush to resist plasma protein adsorption and, thus, prevent platelet aggregation and cell adhesion. The bifunctional nature of the present polymer is essential for the concept of polymeric steric stabilization of stents. This approach led to a 50% reduction in neointimal hyperplasia, probably related to the reduced cellular adherence with a decreased thrombotic and inflammatory reaction as observed with gel paving following conventional balloon angioplasty.
Conclusions
The advantages of polymeric steric stabilization with PLL-g-PEG are: (1) the ease of application by dip-coating; (2) the significant reduction of neointimal proliferation; and (3) the lack of an inflammatory and prothrombotic reaction in the experimental restenosis model. Further studies need to explore the potential of this polymer for stent-based drug elution.
References
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