Artificial Cells, Blood Substitutes, and Biotechnology, 34: 1125, 2006 Copyright Q Taylor & Francis Group, LLC

ISSN: 1073-1199 print/1532-4184 online DOI: 10.1080/10731190500428283

The Adhesive Properties of Endothelial Cells on Endovascular Stent Coated by Substrates of Poly-L-Lysine and Fibronectin
G.-X. Wang, X.-Y. Deng, C.-J. Tang, L.-S. Liu, L. Xiao, and L.-H. Xiang
Key Lab for Biomechanics and Tissue Engineering under the State Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, China

X.-J. Quan
Department of Bioengineering, Chongqing Institute of Technology, Chongqing, China

A. P. Legrand
Laboratoire de Physique Quantique, ESPCI, Paris, France

R. Guidoin
bec Biomaterials Institute, Laval University, Department of Surgery and Que bec, Canada and Key Lab for Biomechanics and Tissue Engineering Que under the State Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, China

Abstract: Optimizing endothelial cell growth and adhesion on the surface of metallic stents implanted in the vascular system is a fundamental issue in understanding and improving their long-term biocompatibility. The ability of
This research program was supported by the Chongqing Municipal Planning Committee, China; Chunhui Plan Project at the State Ministry of Education, China; the Chongqing Municipal Education Committee, China; MOE Key Laboratory of Biomechanics and Tissue Engineering, China and the Quebec Biomaterials Institute, Laval University, Quebec, Canada. The stents were kindly provided by Medtronic-AVE, Santa-Rosa, California, USA. The authors are indebted to Drs. Michel Letort and Randy Guzman for help and guidance. Address correspondence to Robert Guidoin, PhD, Department of Surgery, Faculty of Medicine, Vandry Building, Laval University, Quebec G1K 7P4, Canada. E-mail: rgg@biomedecine.org 11

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the endothelial cell to attach and adhere to the luminal stent surface as well as the capacity to withstand the significant shear stress associated with blood flow are important determinants. The adhesive characteristics of human umbilical vein endothelial cells (HUVEC) on stent surfaces coated with either Poly-L-Lysine (PLL) or fibronectin (FN) were compared with uncoated controls. Increasing concentrations of PLL and FN were measured using a micropipette aspiration system. The adhesive properties of HUVECs under static flow conditions were compared to a dynamic environment on endovascular stents using a parallel-plate-flow chamber. A scanning electron microscope picture was used to measure the number and the adhesive cell ratio as well as the percentage of surface coverage of stent by endothelial cells. The adhesive forces of HUVECs on foreign surfaces coated with PLL and FN were higher compared to uncoated surfaces, and were dependent on increasing concentrations. These coatings resulted in significant increase of the adhesive force of HUVECs. The influence of substrates on the adhesion of the endothelial cell monolayer under static or dynamic flow conditions was highly significant compared with controls (p < 0.01). No significant differences were observed between PLL and FN substrates. Both PLL and FN coated surfaces can significantly increase the adhesion and growth of HUVECs on metallic stent surfaces. Keywords: Poly-L-lysine; Fibronectin; Cell adhesion; Endothelial cells; Intravascular stent

INTRODUCTION Intravascular stents were been widely used to prevent restenosis following PTCA. Stents have represented a major advance in the treatment of obstructive coronary artery disease since the advent of balloon angioplasty [1]. However, subsequent thrombus formation and further instent restenosis following stent deployment have remained significant complications following stenting. Different approaches have been used in an attempt to improve the biocompatibility of stents including surface modification of the materials and=or grafting of biodegradable polymers with specific characteristics. On of the most intriguing approaches includes endothelialization of the foreign surface [2,3]. As this concept of the cell seeding matured, two important areas of research have also emerged. First is the area regarding the growth and adhesion of the endothelial cell on the foreign surface of the stent, with the second concerning the durability of the cell preservation on the foreign surface exposed to the blood flow [48]. The growth and adhesion of the endothelial cells on the foreign surfaces depend upon the morphology and

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the metal surface characteristics, including its thickness and edge angle. The influence of the extra cellular matrix to facilitate cell adhesion is also of great importance in determining endothelial cell attachment [913]. Ultimately, improving the ability of the endothelial cells to anchor on foreign surfaces can follow two approaches. The first one involves surface modification of the foreign material, which is important in particular reference to preventing thrombus formation. The surface chemistry, physics, and mechanical characteristics, including hydrophilic or hydrophobic properties, surface electric charge and conductivity will affect the interaction between the material and endothelial cells. Surface modification of endovascular stents through various physical, chemical, and biological techniques, may significantly increase the adherence of endothelial cells. This may further improve biocompatibility between metallic stent and human body, making it better adapted for long term function free of complications [1418]. The second approach involves improving the ability of endothelial cells to adhere to the artificial surface by cell modification. It is feasible to modify the characteristics of the cells by transferring a gene into those cells, which increases extra cellular matrix and thus strengthens cell adhesion. Zhu transferred a gene from transform growth factor-b1 (TGF b1) into endothelial cells, and found that the transfected group has significantly promoted synthesizing and secreting of the extra-cellular matrix [19]. This transfected cells increased the extra cellular matrix compared to the non-transfected group. This increased extracellular matrix may then further promote anchoring the endothelial cells adherence and cell coating. Alternatively one can identify substrates, which act as bridges to allow strong cell adhesion to the foreign surface. The micropipette aspiration technique used in this experiment was adapted to a parallel-plate flow system and designed to compare the adhesion ability of Poly-L-Lysine (PLL) with fibronectin (FN) compared to controls.

MATERIALS AND METHODS 1. The Stents The stents selected for this investigation were provided by Medtronic-Ave (Santa-Rosa, California, USA). These self-expanding devices were made of nitinol, whose diameter was 28 mm and length 8 mm following deployment. Such stents are currently used in the production of the AneuRx endovascular stent-graft [20].

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2. Cell Preparation HUVEC were harvested from human umbilical cords obtained from the 3rd Military Medical University (Chongqing, China) as described by Jaffe [21]. Cells were cultured on plastic dishes in M199 culture medium supplemented with 20% PBS, 75 mg=l ECGF and 0,1 g=l and maintained at 37C in a 5% carbon dioxide 95% air atmosphere. Cells were identified as endothelial cells by the presence of factor VIIIrelated antigen and by typical endothelial morphology at confluence. Using fluorescent microscopy, 100% of these cells were found to be factor VIII-related antigen positive. Cells were maintained in an incubator at 37C with 5% carbon dioxide. The final concentration of the cells for the micropipette experiments was approximatively 103 cells=ml. 3. Micropipette System and Analysis of the Adhesive Characteristics of ECV-304 Cell Seeds on Different Substrates The structure of the micropipette aspiration system (Figure 1) and the experimental procedures were described in previous papers [22,23]. Micropipettes were pulled from capillary glass tubes in a micropipette

Figure 1. Micropipette aspiration system measuring the adhesive force of a single endothelial cell.

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puller (P87, Sutler Instrument Co, USA). The average values of the internal radius of the pipette used in the present investigation was 2.45 0.90 mm. Fa was the adhesive force of a cell, Rp was the inner radius of micropipette, P was the negative pressure that pulled the adhesive cell away from the basement membrane coated by Poly-L-Lysine (PLL) or fibronectin (FN), and h was the angle of micropipette between basement membrane, Fa can be calculated from the following equation: Fa R2 p DP cosh. The adhesive forces were given as the mean standard deviation. Students t-test was used for statistical analysis. The differences were considered significant at p < 0.05 and highly significant at p < 0.01. 4. Adhesion of Endothelial Cells Cultured Under Static Flow Conditions on Stent Surface Coated with Different Substrates Second or third passage HUVECs were seeded onto stents coated with different substrates with increasing concentrations. The adhesive ratio was calculated by cell counting. The coverage of the stent by endothelial cells was observed by scanning electron microscopy (SEM). Cell morphology and retention were observed under microscope. The retention ratio was calculated by number of cells retained or present compared to the static culture. 5. Adhesion of Endothelial Cells Under Dynamic Culture on Stent Surface Coated with Different Substrates The adhesion of the endothelial cells cultured under dynamic on the surface of the stent coated with different substrates was done in the same way as for the static culture except that the effect of the shear stress on the morphology and the adhesion of endothelial cells was assayed in a parallel-plate-flow-chamber (Figure 2). Endothelial cells were exposed to shear stress of 24 dynes=cm2 for 24 h. The parallel-plate-flow-chamber consisted of a flow chamber, a flow meter, a roller pump, teflon tubings and recording parts. This design is well suited for studying the effects of shear stress on endothelial cells adhesion, retention on grafts surface and the change of endothelial cells morphology and function [2428]. RESULTS 1. Adhesive Properties of HUVECs in Different Substrates The adhesive forces of HUVECs on the surface of stent coated with different concentrations of PLL or FN are given in Table 1.

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Figure 2. Schematic drawing of the parallel-plate system. The stent coated by endothelial cells was mounted in the parallel plate flow chamber. The hydrostatic pressure head established between the two reservoirs provided a constant volume flow rate through the flow chamber.

Table 1. Effect of different concentrations of substrates on adhesive forces of HUVECs Sample size (n) VEC (CK) VEC 1 mg=ml VEC 2 mg=ml VEC 5 mg=ml VEC 2 mg=ml VEC 2 mg=ml VEC 2 mg=ml PLL PLL PLL PLL 1 mg=ml FN PLL 2 mg=ml FN PLL 10 mg=ml FN 35 25 25 25 25 25 25 Fs SD (1010N) 63 6 583 25 607 23 691 46 970 68 1164 121 1475 178

Treatments Control PLL only

PLL FN

PLL: Poly-L-Lysine, FN: Fibronectin, HUVEC: Human Umbilical Vein Endothelial Cells.

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Figure 3. Adhesive forces of the HUVEC on the surface of the stents coated with different concentration of the stents coated with different concentrations of PLL and=or FN. A: VEC (Control); B: VEC 1 mg=ml PLL; C: VEC 2 mg=ml PLL; D: VEC 5 mg=mlPLL; E: VEC 2 mg=ml PLL 1 mg=ml FN; F: VEC 2 mg=ml PLL 2 mg=ml FN; G: VEC 2 mg=ml PLL 5 mg=ml FN.

As is shown in Figure 3, the adhesive force of HUVECs on basement membrane coated with PLL was higher compared to HUVECs on uncoated basement membrane. With increasing PLL concentrations from 1 mg=ml to 5 mg=ml, the adhesive force of HUVECs on basement membrane increased significantly. Compared to the basement membrane coated by PLL, FN coated substrate also was associated with a significant increase of the adhesive force of HUVECs on basement membrane. However, these results come from the observation of single cells and may not be relevant with the endothelial cell monolayer in physiological conditions. Thus it is also important to determine the effect of substrates on the endothelial cell monolayer in terms of the adhesive force. 2. Adhesive Properties of Endothelial Cells Under Static Culture on Stent Surface Coated with Different Substrates A contiguous thin layer overlaying the whole stent by PLL or FN was observed in scanning electron microscopes following endothelial cells

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Figure 4. Endothelial cells grown on the surface of the intravascular stents exposed to the perfusion solution.

Figure 5. Endothelial cells grown on the surface of the intravascular stents coated by PBS, at the fifth day.

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Figure 6. Endothelial cells grown on the surface of the intravascular stents coated by fobronectin (20 mg=ml) at the fifth day.

seeding. However, no stent was overlaid completely by the endothelial cells (Figure 4). The endothelial cells were grown on stents coated by PBS, PLL and FN with different concentrations. The control group and an experiment group resulted in different amounts of cell surface coverage. Initially, the coated surfaces exhibited obvious cell attachment on the stent surface after day one of cell seeding, but no obvious cell growth and proliferation were observed. By day three, cell stretch and growth were present at the edges of the stent. This contrasted with the control group, which only had minimal amount of cell stretching and growth even at day five. At day five for the coated group, the edge and the center part of the stent were found to be almost completely overlaid by the endothelial cells (Figures 5, 6 and 7). Finally, the endothelial cells were digested down for cell counting, and the result was shown in Figure 8. With single cell culture, the benefit of substrate treatment (PLL and FN) on the adhesion of the endothelial cell monolayer on the stent was highly significant compared with the control (p < 0.01).

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Figure 7. Endothelial cells grown on the surface of the intravascular stents coated by the Poly-L-Lysine at the fifth day.

No significant differences were observed between substrates PLL and FN coated stents. 3. Adhesive Properties of Endothelial Cells Under Dynamic Culture on Stent Surface Coated with Different Substrates Endothelial cells coated on the stent surface were exposed to shear stress of 24 dyne=cm2 for 24 hours. The retention of endothelial cells on stent surface is presented in Figure 9. With static cell culture, the effect of substrate treatment on the adhesion of the endothelial cell monolayer exposed to shear stress on the stent was highly significant compared to the control stent (p < 0.01). No significant differences were observed between substrates PLL and FN coated stents. During this experiment, we observed on the stents coated with PLL and FN that endothelial cells had minimal shedding in the entrance and exit parts of the flow chamber. In addition, almost no endothelial cell shedding was observed in the middle part of the flow chamber. These observations contrasted significantly with the control group: the endothelial cells began shedding off at no longer than 4 hours of initiating

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Figure 8. Effect of substrate treatment on the adhesive ratio of endothelial cells under static culture on the stent.

the shear flow. This shedding was very marked at 12 hours, and almost completed at 24 hours when the experiment ended. Very few cells were preserved in the middle part of the chamber. DISCUSSION Following stent deployment in any artery, its luminal surface may become endothelialized, but complications such as acute thrombus formation and in-stent restenosis remain frequent. The primary objective of this study is to examine endothelialization of the stent in vitro under different surface and flow conditions. Endothelial cell stent surface coating can offer two theoretical advantages: one is to prevent the inherent thrombogenicity of metallic stents through the barrier of the endothelial cells and in-stent restenosis; the other is through the secretion of active substances by endothelial cells, which may modify cell migration, propagation and intimal hyperplasia.

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Figure 9. Effect of different substrate treatments on the adhesive ratio of endothelial cells exposed to shear stress on the stent surface.

Van der Giessen et al. [29] first reported the possibility of endothelialization of the stents. Subsequently, Dichek et al. [30] reported transferring the gene of t-PA into the sheep endothelial cells. This gene expression of t-PA of implanted endothelial cells resulted in reducing the inherent thrombogenicity of metallic endovascular stents to some extent. This emphasized the potential role for the endothelialized stent. However, exactly to what extent the endothelial cells adhere to the endovascular stent, as well as their ability to grow and stretch, and to withstand the shear stress from the blood flow, has been a worldwide significant problem. The research showed that seeding endothelial cells on uncoated biomaterials resulted in very low cell attachment and retention. Pretreatment of biomaterials with adhesive substrates has been beneficial to the cells attachment and retention [31,32]. Our investigations demonstrate that both PLL and FN surface coating promote cell adherence compared to uncoated controls. This influence may be secondary to the fact that PLL is a macromolecule chemical compound that holds positive charges in large quantities, while the cell membrane surface holds largely negative charges. As a result, PLL may increase the endothelial

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cell attachment rate by the physically electric charge adsorption. FN is found in the basement membrane, and it can promote the endothelial cell attachment by interaction of integrin receptor on the cell membrane. This interaction involves a receptor-ligand response with participation of the calcium ion [33]. CONCLUSION Both PLL and FN coated surfaces can significantly increase the adhesion and growth of HUVECs on metallic stent surfaces. However, in comparison with the endothelial cell seeding on the artificial heart valves and the artificial blood vessels, seeding of endothelial cells on endovascular stent appears to be more difficult. This may be a result of different shapes, structures and material properties, with higher requirements in the seeding method [34,35]. In addition, the relatively small surface area of the stent may not be ideal for the adhesion of any substrate or cell adhesion and growth. REFERENCES
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