Surface modification of coronary stents with SiCOH plasma nanocoatings for improving endothelialization and anticoagulation Qin Zhang,1 Yang Shen,1,2 Chaojun Tang,1 Xue Wu,1 Qingsong Yu,3 Guixue Wang1 1

Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education, Bioengineering College of Chongqing University, Chongqing 400030, China 2 Institute of Biomedical Engineering, School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China 3 Department of Mechanical and Aerospace Engineering, Center for Surface Science and Plasma Technology, University of Missouri, Columbia, Missouri 65211 Received 19 November 2013; revised 8 April 2014; accepted 22 May 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33229 Abstract: The surface properties of intravascular stent play a crucial role in preventing in-stent restenosis (ISR). In this study, SiCOH plasma nanocoatings were used to modify the surfaces of intravascular stents to improve their endothelialization and anticoagulation properties. SiCOH plasma nanocoatings with thickness of 30–40 nm were deposited by lowtemperature plasmas from a gas mixture of trimethysilane (TMS) and oxygen at different TMS:O2 ratios. Water contact angle measurements showed that the SiCOH plasma nanocoating surfaces prepared from TMS:O2 5 1:4 are hydrophilic with contact angle of 29.5 6 1.9 . The SiCOH plasma nanocoated 316L stainless steel (316L SS) wafers were first characterized by in vitro adhesion tests for blood platelets and human umbilical vein endothelial cells. The in vitro test results showed that the SiCOH plasma nanocoatings

prepared from TMS:O2 5 1:4 had excellent hemo- and cytocompatibility. With uncoated 316L SS stents as the control, the SiCOH plasma nanocoated 316L SS stents were implanted into rabbit abdominal artery model for in vivo evaluation of re-endothelialization and ISR inhibition. After implantation for 12 weeks, the animals testing results showed that the SiCOH plasma nanocoatings accelerated reendothelialization and inhibited ISR with lumen reduction of 26.3 6 10.1%, which were considerably less than the 41.9 6 11.6% lumen reduction from the uncoated control C 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: group. V Appl Biomater 00B:000–000, 2014.

Key Words: surface modification, coating, vascular stent, endothelialization, hemocompatibility

How to cite this article: Zhang Q, Shen Y, Tang C, Wu X, Yu Q, Wang G, Wang G. 2014. Surface modification of coronary stents with SiCOH plasma nanocoatings for improving endothelialization and anticoagulation. J Biomed Mater Res Part B 2014:00B:000–000.

INTRODUCTION

In-stent restenosis (ISR) is a serious problem after coronary stent implantation and remains a major concern with baremetal stent.1 Injured vascular intima and endothelial denudation during the stent implantation process could result in cascade reaction including thrombosis, inflammation and smooth muscle cells (SMCs) migration and proliferation.2,3 Some research results indicated that ISR could be decreased by rapid surface endothelialization of the injured vascular intima and has been regarded as an important means to prevent thrombogenicity, to reduce proliferation and migration of SMCs.4,5 Accordingly, the suitable surface properties

of coronary stents for endothelial cell adhesion, migration and proliferation are important and desirable for surface modification of implanted biomaterials. In order to improve hemocompatibility and endothelial cell adhesion, various types of coatings from diamond-like carbon (DLC),6,7 titanium-nitride-oxide (TiNOX),8 silicon carbide (SiC),9 plasma-activated coating,10 anti-CD34 antibody,11 and anti-VE-cadherin antibody12 have been used to modify coronary stents and some of them have been used in clinic applications. According to clinical data,13 however, most of these coatings provided ineffective or inconclusive performance in terms of reducing ISR. On the other hands,

Correspondence to: G. Wang (e-mail: [email protected]) Contract grant sponsor: National Natural Science Foundation of China; contract grant numbers: 11332003 and 30970721 Contract grant sponsor: National Key Technology R&D Program of China; contract grant number: 2012BAI18B02 Contract grant sponsor: Visiting Scholar Foundation of Key Laboratory of Biorheological Science and Technology (Chongqing University); contract grant number: CQKLBST-2012-006 Contract grant sponsor: National “111 Plan” Base; contract grant number: B06023 Contract grant sponsor: Public Experiment Center of State Bioindustrial Base (Chongqing), China

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the metallic sample holder, the 316L SS stents were fixed on the edge of a stainless steel plate by stainless steel wire and clips. Before plasma deposition, the 316L SS wafers and stents were pretreated and cleaned with O2 plasma for 2 minutes under conditions of 2 sccm oxygen flow, 25 mtorr pressure, and 5 W dc power input. Subsequently, SiCOH plasma nanocoatings were deposited with thickness controlled at 30–40 nm from a gas mixture of TMS and O2 with different TMS:O2 ratios by fixing TMS flow at 1 sccm and varing O2 flow at 0, 1, 2, 3 and 4 sccm. The gas pressure in the plasma reactor was controlled at 25 mtorr with 5 W of dc power input for plasma coating deposition. The surface wettability of the SiCOH plasma nanocoated and uncoated bare 316L SS wafers was then assessed by using a contact angle measurement system (KSV CAM 100, Finland).

FIGURE 1. Schematic illustration the DC bell jar plasma reactor system used for SiCOH plasma nanocoating deposition of 316L SS stents. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

SiCOH plasma nanocoatings prepared by low temperature plasma deposition could have well-controlled surface chemistry and wettability via mediating plasma gas compositions, for example, using a gas mixture of trimethysilane (TMS) and O2 with different molar ratios.14,15 The surface wettability, which had been recognized, would affect protein and cell adhesion behavior.16,17 In this study, therefore, SiCOH plasma nanocoatings prepared by low-temperature plasma deposition were used for surface modification of 316L stainless steel (316L SS) stents to examine their effects on re-endothelialization under in vivo conditions. SiCOH plasma nanocoatings deposited under different TMS:O2 ratios were used to explore the best plasma conditions for treatment of intravascular 316L SS stents by performing in vitro platelet and human umbilical vein endothelial cell (HUVEC) adhesion test. In vivo study was then performed by implanting the SiCOH plasma nanocoated 316L SS stentss into rabbit abdominal artery model to evaluate their effects on re-endothelialization and ISR inhibition. MATERIALS AND METHODS

316L SS wafers (15 mm in diameter and 2 mm in thickness) and 316L SS stents (1.6 mm in diameter and 18 mm in length) used in this study were kindly provided by Amsino Medical Co. (Beijing, China). SiCOH plasma nanocoating deposition and surface wettablity measurements The SiCOH plasma nanocoating deposition was performed at the Centre for Surface Science and Plasma Technology in University of Missouri (Columbia, MO) using an directcurrent (dc) plasma deposition reactor as schematically shown in Figure 1.18 To ensure the electrical conductivity to

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In vitro blood platelet adhesion test Fresh whole blood from healthy volunteers was centrifuged at 1500 rpm for 15 min to separate the blood corpuscles and to get platelet-rich plasma (PRP). For each SiCOH plasma nanocoated and uncoated bare 316L SS wafer sample, 1 mL of PRP was added and then incubated at 37 C for 1 h. After the incubation, these samples were first rinsed by phosphate-buffered saline (PBS) and then fixed by 2.5% glutaraldehyde solution under 4 C for 24 h. The samples were further washed for three times using PBS and subsequently immersed into 50, 75, 95, 100, and 100% aqueous ethanol solutions in sequence for 10 min. After dried in a desiccator, the samples were sputtering coated with gold before being imaged by scanning electron microscopy (SEM). Three 316L SS wafers from each sample group, and five different locations on each wafer were chosen randomly to obtain good statistical results. In vitro HUVEC adhesion test HUVECs were obtained from ATCC, and cultured in 1640 medium with 10% fetal bovine serum. All the SiCOH plasma nanocoated and uncoated bare 316L SS wafer samples were subjected to autoclaving at 120 C for 30 min before seeding the HUVECs. The sterile samples were placed into the wells of a 24-well flat-bottomed plate (Corning Costar, MA). A 1 mL of HUVECs suspension in medium at a density of 4 3 104 cells/ mL were seeded and incubation at 37 C in a humidified atmosphere containing 95% air and 5% CO2 for 8 h. At the end of the incubation, the samples were taken out of the 24well plate, and rinsed with PBS, then put into a new 24-well plate containing 1 mL fresh medium and 100 lL 3-(4,5-dimethylthiazol-2-yl)22,5-diphenyltetrazolium bromide (MTT, 5 mg/mL in PBS) in each well. After 4 h of incubation, the medium were withdrawn and replaced by 1 mL dimethyl sulfoxide (DMSO), followed by 2 h incubation in a humidified atmosphere (37 C, 5% CO2). DMSO solution of 100 lL from of the each samples was analyzed for absorbance at 490 nm in microplate reader (BioTek, Vermont). Five 316L SS wafers from each sample group were used for MTT assay. For SEM examination, the wafers after 8 h incubation were used. SiCOH plasma nanocoated stents implanted in rabbit abdominal artery model Animal feeding and surgical procedures were followed to conform to the Guide for the Chinese Animal Care and Use

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TABLE I. Sample Identification Codes for SiCOH Plasma Nanocoatings With the Corresponding Water Contact Angle Identification Codes of Samples #1 #2 #3 #4 #5 316LSS

Flow Ratio of TMS and O2 (sccm)

Water Contact Angle

1:0 1:1 1:2 1:3 1:4 –

100.5 6 2.6 86.3 6 4.7 58.0 6 3.9 40.2 6 3.5 29.5 6 1.9 39.3 6 5.4

Committee standards. Eighteen male New Zealand white rabbits were fed and housed in the Animal Laboratory Center (Chongqing Medical University, China). The rabbits weighing from 1.5 to 2.5 kg were randomly divided into two groups with group BMS received bare metal stents (n 5 9), group PCS received plasma coated stents (n 5 9), respectively. In order to avoid interaction between the implantation stents, each rabbit was subjected to one stent implantation. Before performing the stent implantation, all the rabbits were first anesthetized by administering ketamine (50 mg/kg) intramuscularly and sodium pentobarbital (30 mg/kg) intravenously. A 5F introducer sheath was positioned in the abdominal artery under surgical exposure. Heparin sodium (200 IU/kg) was intra-arterially injected. The stents were dilated at 10 atm with a 3-mm angioplasty balloon by making sure that no branches were present in the stent segment. Rabbits were fed a normal diet after the intervention. After the stent implantation, rabbits were sacrificed at weeks 1, 4, and 12 time points, for immediate excision of the aortic segment containing the stent. Aortic specimens were excised transversely into two parts. Because the percentage of ISR in the proximal and distal end has been demonstrated to be more obvious, we chose the proximal and distal ends as object for this study. The proximal end was fixed in 4% paraformaldehyde and embedded in light-cured resin. A representative number of 5 lm sections were cut perpendicularly to the long axis of the vessel by a precision microtome. Toluidine blue (TB) stain was used for pathology organization analysis. The distal end was fixed in 0.25% glutaraldehyde for SEM and morphologic examination of the intima surfaces. Morphometric analysis was performed with a computerized digital image analysis system by an independent operator. Histomorphometric analysis of each section included vessel area (mm2), internal elastic lamina area (IEL area, mm2), lumen area (mm2), neointima area (mm2), and the neointima thickness (mm) was performed using ImageTool (version 2.0). The percentage of stenosis was calculated by the following equation19: Percentage area of stenosis 5 [12(Lumen Area/IEL Area)] 3 100%. Statistical analysis The data obtained in this study were reported as the means 6 standard deviations. Data obtained under different treatment

groups were then statistically analyzed using statistical software SPSS 11.5 (SPSS, Chicago, IL). To reveal differences among the groups, one-way ANOVA followed by Tukey’s test was used. The differences were considered significant at p < 0.05. RESULTS

Surface wettability of the SiCOH plasma nanocoatings The sample identification codes and results of the water contact angle measurements of the SiCOH plasma nanocoated and uncoated bare 316L SS wafers were given in Table I. It was found that, with the increase of O2 flow ratio from 0 to 4 sccm in the plasma gas mixture, water surface contact angle of the SiCOH plasma nanocoatings showed a sharp decrease (from 100.5 6 2.6 to 29.5 6 1.9 ). However, further increasing the oxygen flow ratio (to 5 and 6 sccm) showed little effect on lowering the water contact angle (data not shown) of the resulted SiCOH plasma nanocoatings. In comparison, the water contact angle on bare 316L SS wafers without plasma coating was 39.3 6 5.4 . Blood platelet adhesion The platelet adhesion was usually used to evaluate the potential of implanted biomaterials for prevention of thrombosis. The morphology and amount of adhered platelet is a very important parameter in assessing the hemocompatibility of biomaterials. Figure 2 showed the representative SEM images of the platelets adhered on SiCOH plasma nanocoated 316L SS wafers as well as the uncoated bare 316L SS controls. The amount of adhered and activated platelets obtained from SEM images was shown in Figure 3. From the inserted SEM images in Figure 2, it can be seen that significant amount of blood platelets adhered on the surface of #1 SiCOH plasma nanocoatings with the platelets aggregated seriously in stacking states. It suggested that blood platelets on #1 SiCOH plasma nanocoatings had been highly activated. With the increase in surface hydrophilicity of the SiCOH plasma nanocoatings, the amounts of adhered platelets on samples #2 through #5 gradually decreased. There were few platelets found on samples #4 and #5. In addition, the spread pseudopodia of the platelets shown on samples #1, #2, and #3 could hardly be found on samples #4 and #5. Statistical analysis of SEM images indicated that the adhered platelet numbers on samples #2 and #3 were significantly different from samples 316L SS (p < 0.05). With increase in the surface hydrophilicity, the platelet numbers adhered to samples #4 and #5 decreased sharply. These platelet adhesion results indicated that hydrophilic SiCOH plasma nanocoatings were superior to hydrophobic coating in terms of hemocompatibility. Furthermore, the number of adherent platelets on the surface of #5 SiCOH plasma nanocoatings was 50% less than that on the uncoated bare 316L SS controls, indicating improvement in hemocompatibility by using SiCOH plasma nanocoatings. The blood platelets are usually small ellipse cellular tissues with a diameter of 2–4 lm. The platelets in sections of 1.74–4.54 lm are unactivated platelets, while those of larger than 4.54 lm are activated platelet. Based on their

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FIGURE 2. The representative SEM images (20003) of platelet adhesion to SiCOH plasma nanocoated and uncoated bare 316L SS wafers.

size difference, the activated or unactivated platelets on the surfaces of SiCOH plasma nanocoatings samples can be thus analyzed and distinguished by using SISC IASV6.0 software. From the statistical results shown in Figure 3, it can be seen that the #5 SiCOH plasma nanocoatings gave the best hemocompatibility.

FIGURE 3. Numbers of adhered platelets on the surfaces of SiCOH plasma nanocoated and uncoated bare 316L SS wafers. The adhered platelet number for #1 SiCOH plasma nanocoating was not shown because the aggregation of platelets on its surface was too serious for platelet number calculation. *p < 0.05.

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HUVECs adhesion results Figure 4 shows HUVECs adhesion test results on the SiCOH plasma nanocoated and uncoated bare 316L SS wafers by MTT assay. After 8 h cell culture, the HUVEC cell initial adhesion on the #4 and #5 SiCOH plasma nanocoating surfaces is significantly higher than that on the #1, #2, and #3 groups. It could also be seen that the cell initial attachment on the #4 and #5 SiCOH plasma nanocoating is significantly better than that on the uncoated bare 316L SS control wafers. Figure 5 shows the representative SEM images of the morphologies of HUVECs adhered on the SiCOH plasma nanocoatings and the uncoated bare 316L SS control surface. Compared with the cells on the 316L SS group, HUVECs spread better and exhibit much more pseudopodia on the #4 and #5 SiCOH plasma nanocoatings groups. From Figures 4 and 5, it can be seen that the #4 and #5 SiCOH plasma nanocoatings groups showed significantly improved compatibility with HUVECs.

SiCOH plasma nanocoated stents in rabbit abdominal artery model Eighteen SiCOH plasma nanocoated and uncoated bare 316L SS stents were deployed in the abdominal arteries of rabbits. All rabbits survived for the entire study period (after 1, 4, and 12 weeks). Rabbits were then sacrificed for histomorphometric,

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FIGURE 4. MTT assay results for HUVECs adhesion on the surfaces of various SiCOH plasma nanocoatings after 8-h cell culture. The absorbance intensity on the uncoated bare 316L SS control wafers has been normalized to 100%.*p < 0.05.

histopathologic analysis and the evaluation of endothelial regeneration. Angiograms obtained immediately after stent implantation and before euthanasia showed that all deployment procedures were successful and that all stented blood vessels were dilated. Figure 6 shows the re-endothelialization

areas of stent surface, which were longitudinal section of blood vessel with stent implantation for 1 week, 4 weeks and 12 weeks, respectively. From Figure 6, it could be found that SiCOH plasma nanocoated stents (PCS groups) were almost completely covered with cells after stent implantation for 1 week [Figure 6(B1)], in which the outline of implanted stent could be observed clearly. The surface of regenerative endothelium was compact and smooth without any hemocytes. The spindleshaped ECs arrayed orderly, being paralleled to the flow direction. In the case with the uncoated bare 316L SS stents (BMS groups) shown in Figure 6(A1), most part of the stent was exposed to vessel wall with significant amount of platelets and red cells covering the nonendothelialized areas could be found. After 4 week implantation, obvious neointimal could be found on both BMS and PCS as shown in Figure 6(A2,B2). The difference between the two groups from SEM images showed that the outline of BMS was clearly seen with some cracks observed on the fragile neointimal [Figure 6(A2)], while the outline of PCS was hardly to see, and the neointimal was healthy being similar to normal vessel wall [Figure 6(B2)]. As seen from Figure 6(A3,B3), after 12 weeks implantation, the surface of BMS and PCS was found to be almost the same. Figure 7 shows the representative arterial cross sections from BMS and PCS groups at each time points after the

FIGURE 5. The representative SEM images (30003) of HUVECs on the surfaces of SiCOH plasma nanocoated and the uncoated bare 316L SS wafers after 8-h cell culture.

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FIGURE 6. The representative SEM images of the uncoated bare 316L SS stents (A1, A2, and A3) and plasma nanocoated 316L SS stents (B1, B2, and B3) after 1 week (A1) and (B1), 4 weeks (A2) and (B2), and 12 weeks(A3) and (B3) implantation in the rabbit model.

implantation. The quantitative histomorphometric data for each of the stent groups are summarized in Table II. After 12 weeks, PCS groups showed significantly reduced neointima area (1.3 6 0.4 mm2) and percentage of stenosis (26.3%) as compared with those uncoated bare 316L SS stent controls with neointima area 5 2.1 6 0.6 mm2 and the percentage of stenosis 5 41.9%.

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DISCUSSION

The surface properties of intravascular stents, including surface roughness, surface chemistry, wettability and charge distribution determine the biocompatibility of implanted materials and devices.20 An ideal stent surface must have two important characters: (1) excellent hemocompatibility without thrombogenicity after stent implantation and (2)

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FIGURE 7. The representative histological sections (TB stain) of arteries at the sites of stent struts demonstrating neointimal growth after 1 week (A1 and B1), 4 weeks (A2 and B2), and 12 weeks (A3 and B3) implantation in rabbit model. (A1), (A2), and (A3) were from uncoated bare 316L SS stent implantation (BMS group); (B1), (B2), and (B3) were from SiCOH plasma nanocoated 316L SS stents (PCS group). Scale bar 5 500 lm. L stands for lumen; N stands for neointima; S stands for stent strut. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

good cytocompatibility in favor of adhesion, migration, and proliferation of endothelial cells on the stent surface for rapid surface endothelialization. Therefore, appropriate surface modification of intravascular stents should be performed to improve their surface properties. There are various techniques of surface modification, including physical and chemical modification methods.21 In this study, SiCOH plasma nanocoatings was applied to 316L SS stents in order to improve their biocompatibility. Plasma deposition could generate a variety of active groups on the surface of substrate materials and create a compact and nanoscaled plasma coatings for surface modification of various substrate materials.22 In plasma deposition process, the Si-CH3 structure of monomer TMS could be dissociated and then combine with O2 plasma species to generate oxygenous chemical functional groups in the resulted plasma nanocoat-

ings for improved surface hydrophilicity. Under the condition without O2 addition into the plasma deposition system, the deposited plasma coatings from TMS plasma are mainly covered by hydrophobic –CH3 groups, which results to a hydrophobic surface. With increase of O2 addition, O2 molecules could be dissociated into oxygen atoms and radicals that can be incorporated into the plasma nanocoatings in the forms such as –CH2–OH,–CH2–COOH to increase the coating surface hydrophilicity, that is, a decrease in the water contact angle shown in Table I. Surface characterization and analysis of SiCOH plasma nanocoatings, including the coating thickness, chemical composition and structures have been described in our previous reports.23,24 The adhesion and activation of blood platelets affects the hemocompatibility of implanted intravascular devices. The activation of platelets depends on the type, amount,

TABLE II. Results of Histomorphometric Measurement Treatment (n 5 4) 1 week BMS PCS 4 weeks BMS PCS 12 weeks BMS PCS

Vessel Area (mm2)

Lumen Area (mm2)

IEL (mm2)

Neointimal Area (mm2)

Percentage of Stenosis (%)

6.1 6 0.8 6.0 6 0.7

5.1 6 1.0 4.9 6 1.1

5.3 6 0.8 5.3 6 1.1

0.2 6 0.1 0.4 6 0.1

3.3 6 1.6 6.4 6 6.0

5.9 6 0.7 6.0 6 0.6

4.3 6 0.8 4.7 6 0.8

5.1 6 1.0 5.2 6 0.9

0.8 6 0.2 0.5 6 0.3

15.3 6 8.8 9.2 6 7.6

5.9 6 0.7 5.8 6 0.6

3.0 6 0.3 3.7 6 0.2

5.1 6 0.9 5.0 6 0.4

2.1 6 0.6 1.3 6 0.4

41.9 6 11.6 26.3 6 10.1a

Percentage of stenosis 5 1 2 (Lumen area/IEL)%. a p < 0.05.

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and conformational change of adhesive blood plasma proteins. Previous studies have illustrated that the wettability of a material surface can influence the adsorption behavior of blood plasma proteins. Sethuraman et al.25 has found that the proteins adsorbed on the surface of implanted biomaterials are the initial step when the surface contacts with blood. In addition, a hydrophobic surface enhanced absorption of fibrinogen, whereas a hydrophilic surface showed less absorption of proteins. Llopis-Hernandez et al.26 illustrated that the fibrinogen absorbed on the surface of hydrophobic materials experienced a conformational change, exposed the RGD sequence which can be recognized by the platelet membrane protein GPIIbIIIa, while the fibrinogen on the surface of the hydrophilic materials almost showed no conformational changes. Compared with the hydrophilic materials, more platelets were absorbed on the surface of hydrophobic materials. These previous reported research supports the results obtained in this study. The adhesion of endothelial cells on the stent surface is the key for the repair of vascular injury. In this study, a conclusion could be drawn based on the results of the in vitro tests: (1) the HUVECs adhesion and spreading on the material surface need a suitable wettability. Extremely hydrophilic or hydrophobic surfaces do not support cells adhesion and spreading, which is consisted with previous reports.16,27 (2) The chemical composition of material surface also influence the cell adherence and spreading. According to the water contact angle measurements, SiCOH plasma nanocoating #4 group has a similar surface wettability to the uncoated bare 316L SS wafers. As shown in Figure 4, however, cell adhesion test results have indicated that SiCOH plasma nanocoating #4 group gave significantly more adhesion of HUVECs than the uncoated bare 316L SS wafer group. According to the results of platelet adhesion and HUVECs adhesion, it can be concluded that the SiCOH plasma nanocoatings #4 group obtained with TMS:O2 ratio 5 1:4, showed excellent hemocompatibility and cytocompatibility. Therefore, these SiCOH plasma nanocoatings deposited a gas mixture with TMS:O2 ratio 5 1:4 was selected to modify 316L SS stents for in vivo examination using rabbit model to evaluate the effect of modified stent on ISR. The rabbit model has provided insights into the mechanisms of ISR and is widely used to evaluate candidate drug inhibitors of ISR.28 Based on the results of animal tests, it could be concluded that SiCOH plasma nanocoatings could inhibit the intimal hyperplasia. In the early period of stent implantation, it was found that the platelets aggregated on the surface of stent in control groups (BMS groups). It has been proved that aggregated platelets could release much cytokines such as PDGF, which could promote the SMCs migration and proliferation.29 In contrast the stent surface modified with SiCOH plasma nanocoatings (PCS groups) showed excellent hemocompatibility and accelerated initial endothelization in the first week of implantation [Figure 6(B1)]. There are no obvious aggregated platelets found on the surface of the SiCOH plasma nanocoated stent. After stent implantation for 4 weeks, the stents in PCS

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group were found being completely coated with endothelium. In conclusion, the SiCOH plasma nanocoatings applied to PCS group were much more effective in preventing ISR than the uncoated BMS group. Various coatings on stents using different surface modification techniques showed ISR inhibition effects. For examples, DLC coating showed 24 6 13% lumen reduction at 30 days after implantation,30 and 41 6 17% lumen reduction at 6 weeks,31 TiNOX showed 44% lumen reduction at 6 weeks,32 while gold showed 26% lumen reduction at 28 days,33 and silicon-carbide showed 15.8 6 4.7% lumen reduction at 4 weeks.34 Compared with ISR inhibition effect of the afore mentioned surface modification techniques, 26.3% lumen reduction after 12 weeks stent implantation obtained using SiCOH plasma nanocoatings suggests that such SiCOH plasma nanocoatings are excellent candidates with huge application potential for surface modification of implanted biomaterials including vascular stents. Although surface properties of materials play an important role in determining adhesion behaviors of endothelial cells and blood platelets, it must be acknowledged that a single surface coating itself is hard to completely prevent ISR due to the intricate mechanism of ISR. However, surface modification has contributed to vast body of knowledge which has been gained in the field of materials performance in biological environments.13 Combined with other techniques,35–38 such as drug delivery and cell-seeding, the improved stents through appropriate surface modification and correlative adjuvant therapy could inhibit crucial links of ISR including inflammation, SMCs proliferation and thrombosis, and eventually prevent ISR. CONCLUSION

Low-temperature plasma deposition technology exhibits its capability in surface modification, which can be applied to various biomedical implants. The rational design of surface wettability depends on different demands and implanted sites. 12 weeks animal model results obtained in this study indicate that surface modification of 316L SS stents with appropriate SiCOH plasma nanocoatings could significantly accelerate re-endothelialization, effectively inhibit ISR, and show its great promise in clinical treatment of various cardiovascular diseases. ACKNOWLEDGMENTS

The authors are grateful to Mr. Young Jo Kim and Mr. Andrew Ritts for their helpful discussion and assistance in this study. Thanks are due to M.D. Zhenggong Li of Chongqing Zhongshan Hospital for animal experiment. REFERENCES 1. Saito T, Hokimoto S, Oshima S, Noda K, Kojyo Y, Matsunaga K. Metal allergic reaction in chronic refractory in-stent restenosis. Cardiovasc Revasc Med 2009;10:17–22. 2. Costa MA, Simon DI. Molecular basis of restenosis and drugeluting stents. Circulation 2005;111:2257–2273. 3. Mitra AK, Agrawal DK. In stent restenosis: Bane of the stent era. J Clin Pathol 2006;59:232–239. 4. Tang CJ, Wang GX, Cao Y, Wu X, Xie X, Xiao L. Adhesion and endothelialization of endothelial cells on the surface of

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5.

6.

7.

8. 9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

endovascular stents by the novel rotational culture of cells. Appl Surf Sci 2008;255:315–319. Jakabcin J, Bystron M, Spacek R, Veselka J, Kvasnak M, Kala P. The lack of endothelization after drug-eluting stent implantation as a cause of fatal late stent thrombosis. J Thromb Thrombolysis 2008;26:154–158. Roy RK, Choi HW, Yi JW, Moon MW, Lee KR, Han DK. Hemocompatibility of surface-modified, silicon-incorporated, diamond-like carbon films. Acta Biomater 2009;5:249–256. Ma WJ, Ruys AJ, Mason RS, Martin PJ, Bendavid A, Liu ZW. DLC coatings: Effects of physical and chemical properties on biological response. Biomaterials 2007;28:1620–1628. Kornowski R. The titanium nitride oxide coated stent. Catheter Cardio Inte 2010;76:288–290. Atar E, Avrahami R, Koganovich Y, Litvin S, Knizhnik M, Belenky A. Infrapopliteal stenting with silicon carbide-coated stents in critical limb ischemia: A 12 month follow-up study. Israel Med Assoc J 2009;11:611–614. Waterhouse A, Wise SG, Yin YB, Wu BC, James B, Zreiqat H. In vivo biocompatibility of a plasma-activated, coronary stent coating. Biomaterials 2012;33:7984–7992. Lin QK, Ding X, Qiu FY, Song XX, Fu GS, Ji J. In situ endothelialization of intravascular stents coated with an anti-CD34 antibody functionalized heparin-collagen multilayer. Biomaterials 2010;31: 4017–4025. Lee JM, Choe W, Kim BK, Seo WW, Lim WH, Kang CK. Comparison of endothelialization and neointimal formation with stents coated with antibodies against CD34 and vascular endothelialcadherin. Biomaterials 2012;33:8917–8927. O’Brien B, Carroll W. The evolution of cardiovascular stent materials and surfaces in response to clinical drivers: A review. Acta Biomater 2009;5:945–958. Weikart CM, Miyama M, Yasuda HK. Surface modification of conventional polymers by depositing plasma polymers of trimethylsilane and of trimethylsilane plus O-2 II. Dynamic wetting properties. J Colloid Interf Sci 1999;211:28–38. Choukourov A, Pihosh Y, Stelmashuk V, Biederman H, Slavinska D, Kormunda M. Rf sputtering of composite SiOx/plasma polymer films and their basic properties. Surf Coat Technol 2002;151:214–217. Arima Y, Iwata H. Effect of wettability and surface functional groups on protein adsorption and cell adhesion using welldefined mixed self-assembled monolayers. Biomaterials 2007;28: 3074–3082. Tzoneva R, Faucheux N, Groth T. Wettability of substrata controls cell–substrate and cell–cell adhesions. Biochim Biophys Acta 2007;1770:1538–1547. Wang GX, Shen Y, Zhang H, Quan XJ, Yu QS. Influence of surface microroughness by plasma deposition and chemical erosion followed by TiO2 coating upon anticoagulation, hydrophilicity, and corrosion resistance of NiTi alloy stent. J Biomed Mater Res A 2008;85:1096–1102. Wang K, Kessler PD, Zhou ZM, Penn MS, Forudi F, Zhou XR. Local adenoviral-mediated inducible nitric oxide synthase gene transfer inhibits neointimal formation in the porcine coronary stented model. Mol Ther 2003;7:597–603. Nazneen F, Herzog G, Arrigan DWM, Caplice N, Benvenuto P, Galvin P. Surface chemical and physical modification in stent technology for the treatment of coronary artery disease. J Biomed Mater Res B 2012;100B:1989–2014. Mani G, Feldman MD, Patel D, Agrawal CM. Coronary stents: A materials perspective. Biomaterials 2007;28:1689–1710.

22. Favia P, Lopez LC, Sardella E, Gristina R, Nardulli M, d’Agostino R. Low temperature plasma processes for biomedical applications and membrane processing. Desalination 2006;199:268–270. 23. Shen Y, Wang G, Chen L, Li H, Yu P, Bai M. Investigation of surface endothelialization on biomedical nitinol (NiTi) alloy: Effects of surface micropatterning combined with plasma nanocoatings. Acta Biomater 2009;5:3593–3604. 24. Shen Y, Wang G, Huang X, Zhang Q, Wu J, Tang C, Yu Q, Liu X. Surface wettability of plasma SiOx:H nanocoating-induced endothelial cells’ migration and the associated FAK-Rho GTPases signalling pathways. J R Soc Interface 2012;9(67):313–327. 25. Sethuraman A, Han M, Kane RS, Belfort G. Effect of surface wettability on the adhesion of proteins. Langmuir 2004;20:7779–7788. 26. Llopis-Hernandez V, Rico P, Ballester-Beltran J, Moratal D, Salmeron-Sanchez M. Role of surface chemistry in protein remodeling at the cell–material interface. PLoS One 2011;6. 27. Yang D, Lu X, Hong Y, Xi T, Zhang D. The molecular mechanism of mediation of adsorbed serum proteins to endothelial cells adhesion and growth on biomaterials. Biomaterials 2013;34:5747– 5758. 28. Lowe HC, Oesterle SN, Khachigian LM. Coronary in-stent restenosis: Current status and future strategies. J Am Coll Cardiol 2002; 39:183–193. 29. Millette E, Rauch BH, Defawe O, Kenagy RD, Daum G, Clowes AW. Platelet-derived growth Factor-BB-induced human smooth muscle cell proliferation depends on basic FGF release and FGFR1 activation. Circul Res 2005;96:172–179. 30. Castellino M, Stolojan V, Virga A, Rovere M, Cabiale K, Galloni MR. Chemico-physical characterisation and in vivo biocompatibility assessment of DLC-coated coronary stents. Anal Bioanal Chem 2013;405:321–329. 31. De Scheerder I, Szilard M, Huang YM, Ping XB, Verbeken E, Neerinck D. Evaluation of the biocompatibility of two new diamond-like stent coatings (Dylyn (TM)) in a porcine coronary stent model. J Invasive Cardiol 2000;12:389–394. 32. Windecker S, Mayer I, De Pasquale G, Maier W, Dirsch O, De Groot P. Stent coating with titanium-nitride-oxide for reduction of neointimal hyperplasia. Circulation 2001;104:928–933. 33. Edelman ER, Seifert P, Groothuis A, Morss A, Bornstein D, Rogers C. Gold-coated NIR stents in porcine coronary arteries. Circulation 2001;103:429–434. 34. Unverdorben M, Schywalsky M, Labahn D, Bolz A, Amon M, Langer F. Stents coated with hypothrombogenic amorphous silicon carbide—Preliminary results in the New Zealand white rabbit. Perfusion 2000;13:124. 35. Sun D, Zheng Y, Yin T, Tang C, Yu Q, Wang G. Coronary drugeluting stents: From design optimization to newer strategies. J Biomed Mater Res Part A 2014;102(5):1625–1640. 36. Wang HG, Yin TY, Ge SP, Zhang Q, Dong QL, Lei DX, Sun DM, Wang GX. Biofunctionalization of titanium surface with multilayer films modified by heparin–VEGF–fibronectin complex to improve endothelial cell proliferation and blood compatibility. J Biomed Mater Res Part A 2013;101A:413–420. 37. Yin TY, Wang GX, Zhang DC, Du DY, Li ZG, Luo LL, Hou YB, Wang YZ, Zhao JB. Endothelialization and in-stent restenosis on the surface of glycoprotein IIIa monoclonal antibody eluting stent. J Biomed Mater Res Part A 2012;100A:1398–1406. 38. Luo LL, Wang GX, Li YL, Yin TY, Jiang T, Ruan CG. Layer-by-layer assembly of chitosan and platelet monoclonal antibody to improve biocompatibility and release character of PLLA coated stent. J Biomed Mater Res Part A 2011;97A:423–432.

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | MONTH 2014 VOL 00B, ISSUE 00

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Surface modification of coronary stents with SiCOH plasma nanocoatings for improving endothelialization and anticoagulation.

The surface properties of intravascular stent play a crucial role in preventing in-stent restenosis (ISR). In this study, SiCOH plasma nanocoatings we...
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