Article pubs.acs.org/Langmuir
Interaction of Endothelial and Smooth Muscle Cells with Cobalt−Chromium Alloy Surfaces Coated with Paclitaxel Deposited Self-Assembled Monolayers Sujan Lamichhane, Susan Lancaster, Eagappanath Thiruppathi, and Gopinath Mani* Biomedical Engineering Program, The University of South Dakota, 4800 North Career Avenue, Sioux Falls, South Dakota 57107, United States ABSTRACT: The use of self-assembled monolayers (SAMs) as a polymer-free platform to deliver an antiproliferative drug, paclitaxel (PAT), from a stent material cobalt−chromium (CoCr) alloy has been previously demonstrated. In this study, the interaction of human aortic endothelial cells (ECs) and human aortic smooth muscle cells (SMCs) with CoCr alloy surfaces coated with SAMs- (SAMs-CoCr) and PAT-deposited SAMs (PAT-SAMs-CoCr) was investigated. A polished CoCr with no coatings was used as a control. The viability, proliferation, morphology, and phenotype of ECs and SMCs were investigated on these samples. SAMs-CoCr significantly enhanced the growth of ECs. Also, the ECs were well spreading with its typical morphological features and showed stronger PECAM-1 expression on SAMs-CoCr. This showed that the SAMs-CoCr surface is conducive to endothelialization. For PAT-SAMs-CoCr, although the adhesion of ECs was lower, the cells continued to proliferate with some degree of spreading and limited PECAM-1 expression. For SMCs, a significant decrease in the cell proliferation was observed on SAMs-CoCr when compared with that of Control-CoCr. PAT-SAMs-CoCr showed maximum inhibitory effect on the proliferation of SMCs. Also, the SMCs on PAT-SAMs-CoCr displayed a poorly spread discoid morphology with disarranged α-actin filaments. This showed that the PAT released from the SAMs platform successfully inhibited the growth of SMCs. Thus, this study showed the interaction of ECs and SMCs with SAMs-CoCr and PAT-SAMs-CoCr for potential uses in stents and other cardiovascular medical devices. used in stents cause adverse reactions which may lead to LST.6−9 The limitations of some of the currently used polymeric drug delivery carriers for stents include (a) mechanical defects of polymer coating during stent expansion10,11 and (b) inflammatory and hypersensitivity reactions induced by polymer coatings.6−9 Hence, the research in the area of creating novel drug delivery platforms for stents is focused on using either a more biocompatible polymer platform or a polymer-free platform. The use of self-assembled monolayers (SAMs) to deliver drugs from stents belongs to the latter category. SAMs are single-layered organic molecules that are chemically bound to material (typically metals and metal oxides) surfaces in an ordered manner.12 The three main components of SAMs include (a) a headgroup, which attaches to the material substrate; (b) a long hydrocarbon chain, which assists in the ordering process; and (c) a terminal group, which determines the functionality of the monolayer. A variety of biomolecules have been deposited on SAMs-coated metal surfaces through covalent or noncovalent (hydrogen bonding) interactions.13 We previously demonstrated the use of SAMs to deliver a model drug, flufenamic acid, from model substrates such as gold and titanium.14 The
1. INTRODUCTION Coronary artery disease (CAD) is the most common type of heart disease and the leading cause of death worldwide, accounting for more than 7 million deaths every year.1 CAD is caused by the narrowing of coronary arteries that supply oxygen-rich blood to the heart muscle, leading to heart attack. CAD is currently treated by opening the narrowed artery using balloon angioplasty, followed by implanting metallic stents to keep the artery open to provide continuous blood flow to the heart. When the stents are implanted, the endothelial cell lining in the artery is injured, which causes a series of molecular and cellular events resulting in the occlusion of stented arteries (instent restenosis).2 The event that is primarily responsible for causing in-stent restenosis is neointimal hyperplasia (NH). NH is the proliferation, migration, and extracellular matrix deposition of smooth muscle cells (SMCs) into the arterial lumen.3 Drugeluting stents (DESs) were developed to deliver antiproliferative drugs to inhibit the growth of SMCs for preventing NH. Although the DESs were successful in reducing the rate of restenosis, the occurrence of late stent thrombosis (LST) is a concern with DESs.4 LST is a major clinical event that results in heart attack or death. The delay in re-endothelialization of stented arteries is mainly believed to cause LST.5 DESs typically use polymers to coat drugs on stents and to deliver it for a period of time. However, some polymers (not all) © 2013 American Chemical Society
Received: September 11, 2013 Revised: October 21, 2013 Published: October 24, 2013 14254
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Figure 1. SEM (A−C), and AFM (D−F) images of Control-CoCr, SAMs-CoCr, and PAT-SAMs-CoCr. and fluorescein diacetate (FDA) were obtained from Sigma-Aldrich (USA). Anhydrous tetrahydrofuran (THF) and PAT were obtained from Alfa Aesar (USA) and ChemieTek (Indianapolis, IN), respectively. All chemicals were used as received without any further purification. 2.2. Preparation of Polished CoCr Alloy Specimens. CoCr alloy plates (1 cm × 1 cm) were mechanically polished using 1200, 2400, and 4000 grit SiC papers in a LaboPol-5 (Struers) polishing machine. The polished samples were cleaned by sonicating in organic solvents such as ethanol, acetone, and methanol twice for 10 min each. The samples were then dried using high purity nitrogen (N2) gas. Thus prepared polished and cleaned CoCr alloy specimens are denoted here as Control-CoCr. 2.3. SAMs Coating on Polished CoCr Alloy Specimens. A carboxylic-acid-terminated phosphonic acid SAM was coated on polished CoCr using a previously described procedure.17 In brief, the polished CoCr specimens were immersed in 3 mL of 1 mM solution of 16-PHDA in THF for 24 h. After that, the samples were immediately transferred to an oven and were subjected to heat treatment at 120 °C for 18 h. The samples were then removed from the oven and cleaned by sonication in THF and deionized water (diH2O) for 1 min each, followed by drying under N2 gas. Thus-prepared SAMs-coated alloy specimens are denoted here as SAMs-CoCr. 2.4. Paclitaxel Deposition on SAMs Coated CoCr Alloy Specimens. PAT was deposited on SAMs-CoCr specimens using a microdrop deposition method as previously described.17 In brief, PAT solution was prepared in ethanol at a concentration of 4 mg/mL. A 25 μL volume of the prepared solution was carefully placed on SAMs-CoCr specimens. The solvent was allowed to evaporate under ambient laboratory conditions for 3 h, leaving a thin PAT film on SAMs-CoCr. The amount of PAT deposited on SAMs-CoCr was 100 μg. Thus prepared PAT-deposited SAMs-coated CoCr alloy specimens are denoted here as PAT-SAMs-CoCr. 2.5. Surface Characterization. Control-CoCr, SAMs-CoCr, and PAT-SAMs-CoCr were characterized using scanning electron microscopy (SEM), atomic force microscopy (AFM), and Fourier
interaction of endothelial cells (ECs) with these model substrates has also been investigated.15 Later, the use of SAMs to deliver an antiproliferative drug, paclitaxel (PAT), from a stent material such as cobalt−chromium (CoCr) alloy was demonstrated.16 A carboxylic acid (−COOH)-terminated phosphonic acid SAM has been coated on CoCr alloy surfaces.16 PAT was then deposited on SAMs-coated CoCr alloy surfaces through extensive hydrogenbonding interactions.16 A biphasic release profile (an initial burst followed by a slow and sustained release for up to 5 weeks) has been observed for the delivery of PAT from SAMs-coated CoCr alloy surfaces.16 The effect of different processing methods on the release of PAT from SAMs-coated CoCr alloy surfaces has also been investigated.17 In this study, the behavior of two different cell types, ECs and SMCs, on SAMs-coated CoCr alloy and PATdeposited SAMs-coated CoCr alloy was investigated. In our previous studies, as-received nonpolished CoCr alloy surfaces have been used.16,17 However, in this study, the CoCr alloy samples were polished because the cell behavior strongly depends on the uniformity of the surface. Three different groups of samples including polished CoCr, polished CoCr coated with SAMs, and polished CoCr coated with SAMs followed by PAT deposition were prepared and characterized by SEM, AFM, and FTIR. The viability, proliferation, morphology, and phenotype of ECs and SMCs on these three different groups of samples were investigated.
2. MATERIALS AND METHODS 2.1. Materials. Cobalt−chromium (CoCr) alloy (HAYNES 25 alloy) was obtained from Haynes International (Kokomo, IA). Silicon carbide (SiC) polishing papers were obtained from Struers (Westlake, OH). Absolute ethanol (200 proof), methanol, acetone, 16-phosphonohexadecanoic acid (16-PHDA), Dulbecco’s phosphate-buffered saline (DPBS), 14255
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Figure 2. FTIR spectra of 16-PHDA powder (A), PAT powder (B), SAMs-CoCr (C), and PAT-SAMs-CoCr (D). transform infrared spectroscopy (FTIR). An SEM (Quanta 450, FEI, USA) was used in this study to obtain surface morphology of the specimens at an accelerating voltage of 30 KV. Prior to SEM characterization, all specimens were sputter-coated with a 15 nm thick gold−palladium film to avoid surface charging. An AFM (Nano-R2, Pacific Nanotechnology, Santa Clara, CA) was used in this study in tapping mode to obtain 3D surface topography of the specimens. All images reported here were flattened using a third-degree polynomial fit. The AFM determined root-mean-square (RMS) roughness represents an average of data obtained from at least three distinct spots (10 μm × 10 μm) on a sample along with its corresponding standard deviation. A Nicolet 6700 FTIR spectroscopy (Thermo Scientific, Madison, WI) equipped with an attenuated total reflection (ATR) accessory was used to characterize the SAMs- and PAT-coated specimens. The IR spectra were collected at a spectral resolution of 4 cm−1 with 512 scans acquired for SAMs-CoCr and 32 scans for PAT-SAMs-CoCr, 16-PHDA powder, and PAT powder. The reported IR spectra were baseline-corrected using OMNIC software. 2.6. Endothelial and Smooth Muscle Cell Cultures. Human aortic endothelial cells (HAECs, catalog no. 304−05a), human aortic smooth muscle cells (HASMCs, catalog no. 354−05a), EC growth medium (Catalog # 211−500), and SMC growth medium (catalog no. 311−500) were all obtained from Cell Applications (San Diego, CA).
The cells were cultured in their respective growth medium in a humidified incubator at 37 °C with 5% CO2. The cells from the passages four to six were used in this study. A density of 15 × 103 cells (in 100 μL of growth medium) was seeded on the surface of the alloy samples (1 cm × 1 cm). After day 1, the used media were removed from the sample surfaces, followed by briefly washing the samples with DPBS, and 1000 μL of fresh growth media was added. After that, the media were changed after days 3 and 5. 2.6.1. Cell Viability and Proliferation. Cell viability and proliferation were quantitatively measured using resazurin fluorometric assay (trademark name AlamarBlue). Resazurin cell viability assay kit was purchased from Biotium (Hayward, CA). After 1, 3, and 5 days, the used media were removed, and the specimens (cells cultured on alloy surfaces) were washed with DPBS. Then, a solution containing the mixture of resazurin (100 μL) and cell culture medium (900 μL) was added to the specimens. After the specimens were incubated in the solution at 37 °C for 6 h, the fluorescence of the solution was measured using a microplate reader at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. The fluorescence of the blanks (resazurin and growth medium mixture with no cells) was measured, and these values were subtracted from the fluorescence values of the experimental samples to obtain the corrected fluorescence values. To obtain the actual cell number on specimen surfaces, a series of known 14256
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Figure 3. Viability and proliferation of HAECs on Control-CoCr, SAMs-CoCr, and PAT-SAMs-CoCr (A). Percentage of increase in HAEC number from day 1 to day 5 on Control-CoCr, SAMs-CoCr, and PAT-SAMs-CoCr (B). * denotes statistical significance at p < 0.05. number of cells was prepared and its corresponding fluorescence values were measured. The calibration graphs were plotted with number of cells on the x axis and the corresponding fluorescence values on the y axis. For ECs, standard calibration curves were linear over the ranges from (1 to 75) × 103 and from (75 to 200) × 103 with correlation coefficients of R2 = 0.9918 and 0.9671, respectively. For SMCs, the standard calibration curve was linear over the range of (1 to 100) × 103 with a correlation coefficient of R2 = 0.9951. 2.6.2. Cell Morphology Study. A stock solution of FDA was prepared in acetone at a concentration of 1 mg/mL. The working solution of FDA was prepared by adding 100 μL of the FDA stock solution to 900 μL of DPBS. After 1, 3, and 5 days, the used media were removed, and the cells were washed with DPBS. Then, a solution containing the mixture of 60 μL of FDA working solution and 1000 μL of DPBS was added to the cells (cultured on the alloy samples) and incubated in the dark at 37 °C for 15 min. The cells were then characterized using Axiovert 200 M fluorescence microscopy (Carl Zeiss, Thornwood, NY). 2.6.3. Cell Phenotype Study. Platelet endothelial cell adhesion molecule (PECAM-1) antibody, goat antirabbit IgG-FITC, α-actin antibody (1A4), goat antimouse IgG-FITC, goat serum, DAPI dye,
tris-buffered saline with Tween-20 (TBST) and Triton X-100 were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA). After 3 days of culture, the cells were washed with DPBS and fixed with 4% paraformaldehyde (Affymetrix, Santa Clara, CA) for 10 min at room temperature. After that, the cells were washed with DPBS thrice for 5 min each and incubated in blocking buffer (10% of goat serum in TBST) for 30 min. After rinsing with DPBS, the ECs were incubated in primary antibody PECAM-1 diluted 1:50 with 1.5% goat serum in DPBS for 90 min at room temperature. The cells were then washed with DPBS five times for 5 min each, followed by incubating in secondary antibody goat antirabbit IgG-FITC diluted 1:100 with 1.5% goat serum in DPBS for 60 min at room temperature in dark. After washing the cells in DPBS five times for 5 min each, they were incubated in DAPI dye for 10 min to stain the nucleus. The cells were then imaged using a laser scanning confocal microscopy (Nikon, Melville, NY). For SMCs, the procedures of fixing, blocking nonspecific protein adsorption, antibody treatment conditions, staining nucleus, and confocal imaging were carried out as previously mentioned for ECs. The primary and secondary antibodies used for SMCs were α-actin antibody and goat antimouse IgG-FITC, respectively. Also, for SMCs, 14257
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Figure 4. Fluorescence microscopy images of FDA stained HAECs on Control-CoCr (A−C), SAMs-CoCr (D−F), and PAT-SAMs-CoCr (G−I) at 1, 3, and 5 days. after fixing, they were permeabilized by incubating in 0.1% Triton X100 in tris-buffered saline for 25 min. 2.7. Statistical Analysis. For the cell viability and proliferation assay, three samples were used for each Control-CoCr, SAMs-CoCr, and PAT-SAMs-CoCr at each time point (1, 3, and 5 days). Hence, 27 samples were used for each cell type in this part of the study. The experimental data collected in this study are presented as mean ± standard deviation. A one-way analysis of variance (ANOVA) was performed, and the statistical significance for difference was determined at p < 0.05. For the morphology study, three samples were used for each of the three groups of samples at each time point. Hence, 27 samples were used for each cell type in this part of the study. The fluorescence microscopy images were taken at at least three distinct spots on each sample used in this study. For the phenotype study, two samples were used for each of the three groups of samples at the time point day 3. Hence, six samples were used for each cell type for this part of the study. The confocal microscopy images were obtained at at least ten distinct spots on each sample.
CoCr alloy surfaces. PAT-SAMs-CoCr (Figure 1C,F) showed spherical and oval shaped PAT crystals deposited on SAMscoated CoCr alloy surfaces. The uniform distribution of PAT crystals was also evident from these images. A significant increase in the AFM RMS roughness value was observed for PAT-SAMs-CoCr (788.5 ± 103.8 nm) when compared with that of SAMs-CoCr or Control-CoCr. The FTIR spectra of 16PHDA powder, PAT powder, SAMs-CoCr, and PAT-SAMsCoCr are provided in Figure 2A−D, respectively. In the literature, for a well-ordered SAM, the IR peaks for the symmetric and asymmetric stretches of −CH2 groups of SAMs have been commonly observed at less than 2850 cm−1 and less than 2918 cm−1, respectively.18,19 In this study, the IR peaks for the symmetric and asymmetric stretches of −CH2 groups of SAMs were observed at 2849 and 2917 cm−1, respectively (Figure 2C). This strongly suggested the formation of well-ordered SAM on polished CoCr alloy surfaces. The peak for the CO stretches of −COOH terminal groups of SAM was observed at 1701 cm−1. Gawalt and coworkers19 have previously demonstrated the use of FTIR data to determine the bonding interactions between phosphonic acids and metal oxides. Such information was applied in this study to determine the nature of bonding between 16-PHDA and polished CoCr. The P−O region of 16-PHDA powder showed peak positions for PO, P−O, and P−OH bonds at 1206, 1075, and 931 cm−1, respectively (Figure 2A). The P−O region of SAMs-CoCr showed peaks at positions 1201, 1062, and 929 cm−1, respectively (Figure 2C). The broad peak at 1062 cm−1 is characteristic of P−O species that are chemically attached to a material surface. This suggested the covalent attachment of
3. RESULTS 3.1. Surface Characterization of Control-CoCr, SAMsCoCr, and PAT-SAMs-CoCr. Figure 1 shows the SEM (A−C) and AFM (D−F) images of Control-CoCr, SAMs-CoCr, and PAT-SAMs-CoCr specimens. The control sample surface appears smooth with a few grooves arisen from the polishing process (Figure 1A,D). The AFM image of the 3D surface topography of SAMs-CoCr (Figure 1E) appears similar to that of Control-CoCr (Figure 1D). Also, AFM showed no significant difference in the RMS roughness values measured for SAMs-CoCr (58.6 ± 5.3 nm) and Control-CoCr (52.2 ± 16.7 nm). This suggested that the SAM coating was homogeneous and followed the contour of underlying polished 14258
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Figure 5. Immunofluorescent microscopy images of HAECs on Control-CoCr (A,B), SAMs-CoCr (C,D), and PAT-SAMs-CoCr (E,F). White arrows in the images show the expression of PECAM-1.
16-PHDA on polished CoCr. The peaks at 1201 and 929 cm−1 showed the continued presence of PO and P−OH groups. This suggested the monodendate bonding19 between 16-PHDA and polished CoCr. Figure 2D shows the FTIR spectrum of PATSAMs-CoCr. The several peaks for the fingerprint region of PAT were observed at 1248, 1113, 1073, 1026, 981, and 716 cm−1. The peaks for the CO stretches of ester and amide bonds of PAT were observed at 1733 and 1647 cm−1, respectively. The broad peaks for −OH and C−H stretches of PAT were observed at 3447 and 2938 cm−1, respectively. Also, the peak for the −CH3 group of PAT was observed at 1373 cm−1. These peak positions were in
agreement with those of PAT powder (Figure 2B). Thus, FTIR confirmed the successful coating of PAT on SAMs-coated CoCr alloy surfaces. 3.2. EC Viability and Proliferation. The EC viability and proliferation for Control-CoCr, SAMs-CoCr, and PAT-SAMsCoCr are provided in Figure 3. An increase in EC viability and proliferation was observed for all three different sample groups used in this study from day 1 to day 5. On day 1, no significant difference in the number of cells attached was observed between Control-CoCr and SAMs-CoCr (Figure 3A). This suggests that the adhesion of cells on 14259
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Figure 6. Viability and proliferation of HASMCs on Control-CoCr, SAMs-CoCr, and PAT-SAMs-CoCr (A). Percentage of increase in HASMC number from days 1 to 5 on Control-CoCr, SAMs-CoCr, and PAT-SAMs-CoCr (B). * denotes statistical significance at p < 0.05.
SAMs-CoCr was equivalent to that of Control-CoCr. However, PAT-SAMs-CoCr showed lower cell attachment when compared with that of Control-CoCr or SAMs-CoCr. On the basis of these, the adhesion of ECs increased in the following order: PAT-SAMs-CoCr < Control-CoCr = SAMs-CoCr. On day-3, the number of viable cells on SAMs-CoCr was greater than that of Control-CoCr and PAT-SAMsCoCr. A similar trend continued on day 5 as SAMs-CoCr showed a maximum number of viable cells among the three different groups used in this study. Also, no significant difference in the number of cells was observed between Control-CoCr and PAT-SAMs-CoCr. The cell proliferation was determined by calculating the percentage of increase in cell number from days 1 to 5 (Figure 3B). The proliferation of ECs was higher for SAMs-CoCr when compared with that of Control-CoCr. The proliferation of cells on PAT-SAMs-CoCr was equivalent to that of Control-CoCr and SAMs-CoCr as there were no significant differences observed between PAT-SAMs-CoCr and Control-CoCr, and between PAT-SAMs-CoCr and SAMs-CoCr. 3.3. EC Morphology. The fluorescence microscopy images of FDA stained ECs on Control-CoCr, SAMs-CoCr, and
PAT-SAMs-CoCr specimens at days 1, 3, and 5 are shown in Figure 4. For all three different sample groups, an increase in cell number from days 1 to 5 was clearly evident from these images. The ECs were well spreading with typical polygonal shape on Control (Figure 4A−C) and SAMs coated CoCr alloy surfaces (Figure 4D−F). For PAT-SAMs-CoCr (Figure 4G−I), although an increase in cell number was observed from one time point to the other, the cells were not as spreading as on Control or SAMscoated surfaces. Although a few cells showed a polygonal shape on PAT-SAMs-CoCr, the majority of the cells were elongated on these surfaces. After 5 days, the cells reached more than 90% confluence on SAMs-CoCr, followed by 70−75% confluence on Control-CoCr and 50−60% confluence on PAT-SAMs-CoCr. 3.4. EC Phenotype. PECAM-1 is a membrane protein that is present on ECs. This protein plays a vital role in determining the adhesion of ECs on material substrates.20 Also, this protein involves in regulating EC−EC adhesion.20 In previous studies, when the ECs strongly expressed PECAM-1, it was considered as a sign of excellent endothelialization property.21 However, when the ECs poorly expressed PECAM-1, it was considered as 14260
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Figure 7. Fluorescence microscopy images of FDA-stained HASMCs on Control-CoCr (A−C), SAMs-CoCr (D−F), and PAT-SAMs-CoCr (G−I) at 1, 3, and 5 days.
3.6. SMC Morphology. The fluorescence microscopy images of FDA-stained HASMCs on Control-CoCr, SAMsCoCr, and PAT-SAMs-CoCr specimens at days 1, 3, and 5 are shown in Figure 7. On Control-CoCr (Figure 7A−C) and SAMsCoCr (Figure 7D−F), the SMCs were elongated and spindleshaped with typical hill-and-valley morphology. For PAT-SAMsCoCr (Figure 7G−I), not only was the cell number significantly reduced when compared with the other two groups but also the majority of the cells showed a discoid (flat circular) shape on these surfaces. After 5 days, the confluency of SMCs on ControlCoCr, SAMs-CoCr, and PAT-SAMs-CoCr was estimated to be 90−95, 70−75, and 10−15%, respectively. 3.7. SMC Phenotype. On Control-CoCr, the expression of SM α-actin was stronger in some cells as the well-defined α-actin filaments were oriented along the cell axis (contractile phenotype) (Figure 8A−C). A weaker expression (less intense staining) or no expression of α-actin was also observed in some cells (synthetic phenotype) (Figure 8A−C). Hence, the SMCs showed a mixture of contractile and synthetic phenotypes on Control-CoCr. Similar results were also observed for SAMs-CoCr (Figure 8D−F) and PAT-SAMs-CoCr (Figure 8G−I). Also, the SMCs on PAT-SAMsCoCr predominantly showed a disarrangement of α-actin filaments with circumferential orientation (Figure 8G−I).
possible cell damage.22 Immunofluorescence microscopy images confirmed the expression of PECAM-1 on ECs grown on all three different groups of samples used in this study (Figure 5, arrows are provided in the images to show PECAM-1 expression). The expression of PECAM-1 was stronger for Control-CoCr (Figure 5A,B) and SAMs-CoCr specimens (Figure 5C,D). No qualitative difference in the expression of PECAM-1 was observed between Control-CoCr and SAMs-CoCr. However, the expression was minimal on PAT-SAMs-CoCr specimens (Figure 5E,F). 3.5. SMC Viability and Proliferation. The viability and proliferation of SMCs on Control-CoCr, SAMs-CoCr, and PATSAMs-CoCr are provided in Figure 6. On day 1, the number of cells attached on PAT-SAMs-CoCr was significantly less than that of Control-CoCr or SAMs-CoCr. No significant difference in the number of cells attached was observed between Control-CoCr and SAMs-CoCr. On the basis of these, the adhesion of SMCs decreased in the following order: Control-CoCr = SAMs-CoCr > PAT-SAMs-CoCr. A similar trend was observed on days 3 and day 5 as well, with PAT-SAMs-CoCr showing the least number of cells when compared with that of the other two groups. The percentage of increase in SMC number from days 1 to 5 (SMC proliferation) was calculated and is provided in Figure 6B. The proliferation of SMCs on SAMs-CoCr was significantly inhibited when compared with that of Control-CoCr. PAT-SAMs-CoCr showed maximum inhibition of SMC proliferation when compared with the other two groups. On the basis of these, the proliferation of SMCs decreased in the following order: Control-CoCr > SAMs-CoCr > PAT-SAMs-CoCr.
4. DISCUSSION The interaction of ECs and SMCs with Control-CoCr, SAMsCoCr, and PAT-SAMs-CoCr samples was investigated in this study for their potential use in DES. 14261
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Figure 8. Immunofluorescence microscopy images of HASMCs on Control-CoCr (A−C), SAMs-CoCr (D−F), and PAT-SAMs-CoCr (G−I). The cells in the images were stained for smooth muscle α-actin.
sustained release of PAT in lower amounts (
