Surface Modification of CoCr Alloy Using Varying Concentrations of

Dec 10, 2014 - Biomedical Engineering Program, The University of South Dakota, 4800 N. Career Avenue, Sioux Falls, South Dakota 57107, United States...
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Surface Modification of CoCr Alloy Using Varying Concentrations of Phosphoric and Phosphonoacetic Acids: Albumin and Fibrinogen Adsorption, Platelet Adhesion, Activation, and Aggregation Studies Eagappanath Thiruppathi,† Mark K. Larson,‡ and Gopinath Mani*,† †

Biomedical Engineering Program, The University of South Dakota, 4800 N. Career Avenue, Sioux Falls, South Dakota 57107, United States ‡ Department of Biology, Augustana College, 2001 S. Summit Avenue, Sioux Falls, South Dakota 57197, United States ABSTRACT: CoCr alloy is commonly used in various cardiovascular medical devices for its excellent physical and mechanical properties. However, the formation of blood clots on the alloy surfaces is a serious concern. This research is focused on the surface modification of CoCr alloy using varying concentrations (1, 25, 50, 75, and 100 mM) of phosphoric acid (PA) and phosphonoacetic acid (PAA) to generate various surfaces with different wettability, chemistry, and roughness. Then, the adsorption of blood plasma proteins such as albumin and fibrinogen and the adhesion, activation, and aggregation of platelets with the various surfaces generated were investigated. Contact angle analysis showed PA and PAA coatings on CoCr provided a gradient of hydrophilic surfaces. FTIR showed PA and PAA were covalently bound to CoCr surface and formed different bonding configurations depending on the concentrations of coating solutions used. AFM showed the formation of homogeneous PA and PAA coatings on CoCr. The single and dual protein adsorption studies showed that the amount of albumin and fibrinogen adsorbed on the alloy surfaces strongly depend on the type of PA and PAA coatings prepared by different concentrations of coating solutions. All PA coated CoCr showed reduced platelet adhesion and activation when compared to control CoCr. Also, 75 and 100 mM PA-CoCr showed reduced platelet aggregation. For PAA coated CoCr, no significant difference in platelet adhesion and activation was observed between PAA coated CoCr and control CoCr. Thus, this study demonstrated that CoCr can be surface modified using PA for potentially reducing the formation of blood clots and improving the blood compatibility of the alloy. (cracks, fissures, and fractures) in the coating and the detachment and delamination of coating still exist.5,6 In the latter approach, the alloy surfaces have been physically modified to obtain different surface textures.7 However, the physical alteration not only affects the mechanical properties of the alloy but also increases the chances for blood platelets to attach and activate on the textured surfaces due to its greater surface area.7 The chemical modification involves the deposition of molecular coatings on the alloy surface.8,9 The molecular coatings are small organic molecules chemically attached to the metal surface through a functional group that is present at one end of the organic molecule with an excellent affinity toward the metal surface, while the functional group at the other end of the organic molecule remains free at the material−air interface to decide the surface chemistry of the metal. We have previously demonstrated the use of phosphoric acid (PA) and phosphonoacetic acid (PAA) molecular coatings for incorporating hydroxyl (−OH) and carboxylic acid (−COOH) functionalities on CoCr alloy surfaces, respectively.8,9 In this study, the CoCr

1. INTRODUCTION Cobalt−chromium (CoCr) alloy is one of the metallic biomaterials commonly used in cardiovascular medical devices such as stents, stent grafts, heart valves, and pacemakers.1 Although the alloy has excellent physical and mechanical properties, the blood compatibility is always an issue as with most other cardiovascular biomaterials.2,3 Specifically, when the blood platelets interact with biomaterial surfaces, they can adhere, activate, and aggregate to ultimately incorporate into thrombi (blood clots).4 The thrombus can occlude arteries that supply blood to different organs and can cause fatal complications such as heart attack, stroke, and embolism. Hence, it is vital to develop approaches for improving the surface properties of cardiovascular biomaterials such as CoCr alloy for preventing or reducing thrombus formation. The approaches available to surface modify CoCr alloy to improve its blood compatibility can be grouped under two major categories: (a) coating the existing surface of the alloy with a blood-compatible material; (b) altering the original surface of the alloy either physically or chemically. In the former approach, although several different biocompatible polymers and ceramics have been explored as coatings, the inherent limitations including the formation of physical defects © 2014 American Chemical Society

Received: September 29, 2014 Revised: December 8, 2014 Published: December 10, 2014 358

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2.6. Protein Adsorption Studies. The protein adsorption studies carried out in this study are grouped under three categories: (a) single protein adsorption study, (b) dual protein adsorption study, and (c) protein displacement study. 2.6.1. Single Protein Adsorption Study. The solutions of albumin and fibrinogen each were prepared at a concentration of 200 μg/mL in 0.9% sodium chloride (NaCl). A 60 μL aliquot of each of the protein solutions prepared was microdrop deposited on control, PA, and PAA coated CoCr specimens (n = 3) and incubated at 37 °C. After 15 min, the protein solution deposited was carefully removed from the specimens by aspiration and transferred to a vial. The specimens were then washed with 300 μL of NaCl solution to remove any nonadherent proteins. After that, the NaCl solution used for washing was also removed and added to the aspirated protein solution stored in the vial. The solutions were mixed well by shaking. A 150 μL of this solution was then added to 150 μL of bicinchoninic acid (BCA) protein assay kit (Thermo Scientific) solution and incubated at 37 °C for 2 h. After that, the absorbance of the solution was measured using a microplate reader at a wavelength of 562 nm. The blanks were prepared from the NaCl solution (without proteins). The blank NaCl solution was also subjected to the exact same protocol as described above for protein containing solutions. The absorbance of the blanks was measured, and these values were subtracted from the values of experimental samples to obtain the corrected absorbance values. Calibration plots for albumin and fibrinogen were obtained by plotting the known concentrations of proteins in x-axis and their corresponding absorbance values in y-axis. The plots were linear for the proteins in the concentration range of 2−40 μg/mL with a correlation coefficient of R2 = 0.99. Using the calibration plot, the amount of extracted protein in the collected solutions was determined. Also, the amount of protein used for initial loading on the alloy surface was determined by adding 60 μL of protein solutions to NaCl and measuring the absorbance of the solutions. Using the calibration plot, the amount of protein used for initial loading was also measured. Then, the difference between the initial amount of protein loaded and the amount of protein extracted was calculated to determine the amount of protein adsorbed on alloy surfaces. 2.6.1.1. Fluorescence Microscopy Images of Proteins Adsorbed on Alloy Surfaces. For imaging the proteins adsorbed on the alloy surfaces, the fluorescent dye conjugated proteins such as bovine serum albumin Alexa Fluor 555 conjugate (BSA-AF-555) and human plasma fibrinogen Oregon Green 488 conjugate (HF-OG-488) were used in this study. These fluorescent protein conjugates were deposited on control, PA, and PAA coated CoCr alloy surfaces as per the protocol described in section 2.6.1. The proteins adsorbed on the alloy surfaces were then imaged using a Axiovert 200 M inverted fluorescence microscope (Carl Zeiss Microscopy, Thornwood, NY). TRITC and FITC filters were used to image BSA-AF-555 and HF-OG-488, respectively. 2.6.2. Dual Protein Adsorption Study. For dual protein adsorption study, a solution mixture of BSA-AF-555 (200 μg/mL) and HF-OG488 (200 μg/mL) was prepared. The dual protein solution was then deposited on control, PA, and PAA coated CoCr alloy surfaces as per the protocol described in section 2.6.1. The amount of proteins adsorbed on the alloy surfaces was determined by measuring the difference between the fluorescence of the protein solution used for initial loading and the fluorescence of the solution collected after washing the specimens. Linear calibration plots were obtained for both BSA-AF-555 and HF-OG-488 in the concentration range of 2−100 μg/mL with linear coefficients of R2 = 0.99. A Tecan Infinite M200 microplate reader (Tecan Group Ltd, Mannedorf, Switzerland) was used to measure the fluorescence of the solution. For determining BSA-AF-555, the fluorescence of the solution was measured with an excitation wavelength of 555 nm and an emission wavelength of 585 nm (20 μs integration time, gain 100). For determining HF-OG-488, the fluorescence of the solution was measured with an excitation wavelength of 496 nm and an emission wavelength of 524 nm (20 μs integration time, gain 50). The fluorescence microscopy images of proteins adsorbed on the alloy surfaces were determined as per the details provided in section 2.6.1.1.

alloy was surface modified using varying concentrations (1, 25, 50, 75, and 100 mM) of PA and PAA to generate various surfaces with different surface wettability, chemistry, and roughness. Then, the adsorption of blood plasma proteins such as albumin and fibrinogen and the adhesion, activation, and aggregation of platelets on the various surfaces generated in this study were investigated.

2. MATERIALS AND METHODS 2.1. Materials. CoCr alloy (HAYNES 25 alloy) was purchased from Haynes International (Kokomo, IA). Ethanol, methanol, acetone, phosphoric acid, phosphonoacetic acid, sodium chloride, bovine serum albumin, and human plasma fibrinogen were all purchased from Sigma-Aldrich and used as received. 2.2. Preparation of Control CoCr Alloy Specimens. The CoCr alloy specimens were polished and chemically cleaned as described previously.8 Briefly, the CoCr alloy specimens (1 cm × 1 cm) were polished in a Labopol-5 (Struers Inc., Cleveland, OH) machine using 600, 800, and 1200 grit SiC papers. The polished specimens were cleaned by sonicating in organic solvents such as ethanol, acetone, and methanol for 10 min twice and dried using nitrogen (N2) gas. The polished and chemically cleaned CoCr alloy specimens are denoted in this study as control CoCr. 2.3. Preparation of Phosphoric Acid Coated CoCr Alloy Specimens. Phosphoric acid (PA) solutions were prepared in deionized water (di-H2O) at five different concentrations of 1, 25, 50, 75, and 100 mM. The polished and chemically cleaned CoCr specimens were immersed in 3 mL of different concentrations of phosphoric acid solutions prepared. After 24 h, the specimens were transferred to an oven without rinsing and heated at 120 °C in air for 19 h. The specimens were then cleaned by sonicating in di-H2O for 1 min and dried using N2 gas. The CoCr alloy specimens coated with 1, 25, 50, 75, and 100 mM concentrations of PA solutions are denoted in this study as 1 mM PA-CoCr, 25 mM PA-CoCr, 50 mM PA-CoCr, 75 mM PA-CoCr, and 100 mM PA-CoCr, respectively. 2.4. Preparation of Phosphonoacetic Acid Coated CoCr Alloy Specimens. Phosphonoacetic acid (PAA) solutions were prepared in deionized water (di-H2O) at five different concentrations of 1, 25, 50, 75, and 100 mM. The CoCr alloy specimens were coated with five different concentrations of PAA using the same coating protocol as described in the above section 2.3 for PA coating. The CoCr alloy specimens coated with 1, 25, 50, 75, and 100 mM concentrations of PAA are denoted in this study as 1 mM PAA-CoCr, 25 mM PAA-CoCr, 50 mM PAA-CoCr, 75 mM PAA-CoCr, and 100 mM PAA-CoCr, respectively. 2.5. Surface Characterizations of PA and PAA Coated CoCr Alloy Specimens. The PA and PAA coated CoCr specimens were characterized using contact angle goniometry, Fourier transform infrared spectroscopy (FTIR), and atomic force microscopy (AFM). 2.5.1. Contact Angle Goniometry. A VCA optima system (AST Products, Inc) system was used in this study. A 6 μL volume of di-H2O was placed on the specimens, and the contact angles were measured after 15 s. The contact angle values reported here represent the mean ± standard deviation of the values measured from three different specimens for each sample group. 2.5.2. Fourier Transform Infrared Spectroscopy. A Nicolet 6700 FTIR spectroscopy (Thermo Scientific) equipped with a specular apertured grazing angle (80°) accessory was used in this study to characterize PA and PAA coatings on CoCr specimens. The IR spectra were collected using 1024 scans at 4 cm−1 spectral resolution. The PA and PAA compounds in powder form were characterized using attenuated total reflection (ATR) using 32 scans and 4 cm−1 spectral resolution. 2.5.3. Atomic Force Microscopy. A Nano-R2 AFM (Pacific Nanotechnology) was used in this study. All the images were collected in tapping mode. The images presented here were flattened according to third degree polynomial fit. The RMS roughness values reported here represent the mean ± standard deviation of the values measured at three different spots on a specimen for each sample group. 359

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Figure 1. Contact angles (A, B) and AFM roughness values (C, D) of CoCr alloy specimens coated with different concentrations (1, 25, 50, 75, and 100 mM) of PA and PAA. Asterisk denotes statistical significance at p < 0.05. 2.6.3. Protein Displacement Study. In this study, a 60 μL of BSAAF-555 (200 μg/mL) was deposited on control CoCr alloy as per the protocols described in section 2.6.1. The amount of BSA-AF-555 adsorbed on the alloy surface was determined as per the details provided in section 2.6.2. Then, a 60 μL of HF-OG-488 (200 μg/mL) was deposited on BSA-AF-555 adsorbed control CoCr alloy. After that, the amount of HF-OG-488 adsorbed, the amount of BSA-AF-555 desorbed from the alloy surface, and the amount of BSA-AF-555 retained on the alloy surface were determined as per the details provided in section 2.6.2. Fluorescence microscopy images were also obtained before and after the deposition of HF-OG-488 on BSA-AF555 adsorbed control CoCr alloy as per the details provided in section 2.6.1.1. 2.7. Platelet Interaction Studies. The interaction of platelets with control, PA, and PAA coated CoCr specimen surfaces was studied by investigating the adhesion, activation, and aggregation of platelets on the specimens both quantitatively and qualitatively. 2.7.1. Platelet Isolation from Human Blood. The blood was collected from healthy donors who have not consumed aspirin or any other anticoagulants 1 week prior to blood donation and drawn into 3.2% sodium citrate tubes. The collected blood was mixed with an anticoagulant acid citrate dextrose at a ratio of 7:1. Then, the blood was centrifuged at 180g for 20 min at room temperature to separate out the platelet-rich plasma (PRP). Prostacyclin I2, an inhibitor for platelet activation and aggregation, was added to PRP at a concentration of 50 ng/mL, and the mixture was centrifuged at 550g for 10 min. The plasma was then completely removed from PRP to obtain only a platelet pellet. The platelet pellet was then resuspended in Tyrode’s buffer, which is an iso-osmotic phosphate

buffer at pH 7.4 containing bovine serum albumin protein (0.1%, w/ v), glucose (0.1%, w/v), HEPES (10 mM), NaCl (144.9 mM), KCl (2.9 mM), and MgCl2 (0.96 mM). The number of platelets in the solution was determined by flow cytometry (Accuri C6, Becton Dickinson). The platelets were further diluted in Tyrode’s solution to bring the platelet number to 2 × 108/mL. 2.7.2. Platelet Adhesion Study. The adhesion of platelets was measured by quantifying the total amount of protein released by disrupting the adhered platelets by sodium dodecyl sulfate (SDS) lysis buffer. The protein released from the platelets was quantified by the BCA kit. The control, PA, and PAA coated CoCr alloy specimens (n = 3) were placed in a 24-well plate. The specimens were immersed in 350 μL of platelets suspension and allowed to incubate at 37 °C. After 1 h, the platelet suspension from the wells was removed and used for the activation study (see section 2.7.3) and aggregation study (see section 2.7.4), while the CoCr specimens used in the experiments were used for the adhesion study. For determining the adhesion of platelets, the CoCr specimens were washed in Tyrode’s solution twice to remove the nonadhered platelets. Then, 300 μL of lysis buffer was added to each of the wells that contains specimens and rigorously agitated at 4 °C for 30 min. After that, 25 μL of the lysis buffer was withdrawn and mixed with 250 μL of BCA kit. The mixture was incubated at 37 °C for 30 min, and the absorbance was measured using a spectrophotometer at 562 nm. The amount of protein released by a known number of platelets in the suspension was also determined. This information was then used to convert the amount of protein released by the adhered platelets to the number of platelets/mm2. 2.7.3. Platelet Activation Study. The platelet activation was quantified by flow cytometry using an antibody against P-selectin 360

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Figure 2. FTIR spectra of PA and PAA in powder forms (A), PA coated CoCr (B), and PAA coated CoCr (C). (FITC antihuman CD62P, Fisher Scientific). A 25 μL of platelet suspension collected (see section 2.7.2) was added to 5 μL of Pselectin antibody diluted in 25 μL of tyrode’s solution. The solution was incubated in dark at room temperature for 10 min. After that, the platelets were fixed by adding 500 μL of 0.2% paraformaldehyde and then incubated the solution in dark at room temperature for 10 min. The level of activation was determined using a flow cytometer by quantifying the fluorescence intensity of 10 000 platelet specific events per sample. 2.7.4. Platelet Aggregation Study. The aggregation of platelets was determined by analyzing the changes in turbidity (opacity) of platelet suspension collected before and after immersion of alloy specimens. The turbidity was determined using a spectrophotometer by measuring the light absorbed through the platelet suspension at wavelength 650 nm. When the platelets in the suspension exist as individual particles (with no aggregation), the suspension will be cloudy and will not allow the light to pass through. However, when the platelets are aggregated, the cloudiness of the suspension will decrease and will allow the light to pass through. In this study, as-prepared platelet suspension (i.e., the suspension not used for immersing CoCr specimens) was used as the reference. The experimental samples were the platelet suspension (250 μL) collected (see section 2.7.2). The percentage of aggregation was determined using the following formula:

aggregation % ⎛ absorbance of the experimental sample ⎞ ⎟ × 100% = ⎜1 − ⎠ ⎝ absorbace of the reference 2.7.4. Qualitative Analysis of Platelet Adhesion, Activation, and Aggregation, and the Degree of Activation Determined by SEM. The control, PA, and PAA coated CoCr alloy specimens were immersed in the platelet suspension followed by washing the specimens in Tyrode’s solution twice as described in the section 2.7.2. The platelets adhered on CoCr alloy surfaces were fixed by immersing the specimens in 700 μL of 2% glutaraldehyde solution at 4 °C for 4 h. After fixation, the specimens were dehydrated by immersing in a series of ethanol (30, 50, 70, 80, 90, and 100%) washes at room temperature for 10 min each. The specimens were then sputter coated using gold−palladium and imaged using SEM. The images were acquired at a minimum of 15 different spots on each specimen to qualitatively analyze the adhesion, activation, and aggregation of platelets as well as to determine the degree of platelet activation on the alloy specimen surfaces. 2.8. Statistical Analysis. The experimental data collected in this study are presented as mean ± standard deviation. A one-way analysis of variance (ANOVA) was used to determine the statistical significance at p < 0.05. 361

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3. RESULTS 3.1. Contact Angle Analysis of Control, PA, and PAA Coated CoCr Specimens. Contact angle is used to measure the wettability (hydrophilic and hydrophobic properties) of a substrate. When a material is coated with molecules that contain functional groups such as −OH and −COOH groups, it can provide hydrophilic property to the material surface. Also, the degree of hydrophilicity depends on the density of −OH and −COOH groups on the material surface. In this study, contact angle analysis was used to determine the hydrophilic properties of PA and PAA coated CoCr alloy surfaces. Figures 1A and 1B show the contact angles measured for PA and PAA coated CoCr specimens, respectively. The contact angle of control CoCr was determined as 59 ± 3°. All the PA coated CoCr prepared using different concentrations of PA (from 1 to 100 mM) showed significantly lesser contact angle when compared to that of control CoCr (Figure 1A). This suggested the successful deposition of PA on CoCr. For PA coatings, a gradual decrease in contact angle was observed for up to 50 mM PA-CoCr, and after that no significant difference in the contact angle was observed between 50, 75, and 100 mM PACoCr. Similar to PA coated surfaces, all the PAA coated CoCr showed significantly lesser contact angle when compared to that of control CoCr (Figure 1B). This suggested the successful deposition of PAA on CoCr. For PAA coatings, a gradual decrease in contact angle was observed for up to 75 mM PAACoCr, and after that no significant difference in the contact angle was observed between 75 and 100 mM PA-CoCr. 3.2. FTIR Characterization of PA and PAA Coated CoCr Specimens. FTIR is commonly used to determine the structure of molecules on a material surface. The presence of specific chemical functionalities as well as their bonding information on the surface can be determined by FTIR. In this study, FTIR was used to determine the structure of PA and PAA coatings on CoCr alloy surface. The FTIR spectra of PA and PAA in powder forms are shown in Figures 2A-1 and 2A-2, respectively. The chemical structures of PA and PAA are shown as the subsets of Figures 2A-1 and 2A-2, respectively. Several references from the literature were used to interpret all the FTIR spectra collected in this study.10−14 In the spectrum of PA powder (Figure 2A-1), the two major peaks and a shoulder peak observed in the P−O region at 963, 1099, and 1197 cm−1 were assigned to P−OH, P−O, and PO species, respectively. The two broad peaks at 2719 and 2319 cm−1 were assigned to symmetric and asymmetric stretches of OP−OH groups, respectively. Multiple peaks were observed for the presence of −OH groups at positions 3371, 3489, and 3534 cm−1 (−OH stretching) and 733 and 887 cm−1 (−OH bending). The peaks for the −OH stretching at lower wavenumbers (3371−3534 cm−1) suggested hydrogen bonding interactions between the multiple −OH groups of PA. The peak at 1622 cm−1 indicated the presence of water molecules in the PA powder due to its hygroscopic property. The FTIR spectra of PA coated CoCr prepared using 1, 25, 50, 75, and 100 mM concentrations of PA are shown in Figure 2B. Several changes were observed in the spectra of PA coated CoCr when compared to that of PA powder. The peak position for the P−O bond was shifted to a higher wavenumber at 1117 cm−1 for 1 mM PA-CoCr. This peak was significantly broadened with doublet peaks at 1117 and 1160 cm−1 in the spectra obtained for 25, 50, 75, and 100 mM PA-CoCr. Such a shift in the P−O peak position to a higher wavenumber has

been commonly attributed to the formation of P−O−metal bonds.8 These results suggested that PA was covalently bound to CoCr alloy for all the different concentrations of PA used in this study. For 1 mM PA-CoCr, no peak was observed at ∼970 cm−1, which further suggested that most of the P−OH bonds were converted into P−O−metal bonds. Also, no peak for P O bonds at ∼1260 cm−1 was observed for 1 mM PA-CoCr. These results suggested the formation of tridentate bonding in 1 mM PA-CoCr. For 25 mM PA-CoCr as well, no peak for the P−OH groups was observed at ∼970 cm−1. However, a peak observed at 1264 cm−1 suggested the presence of PO groups. These results suggested the formation of bidentate bonding in 25 mM PA-CoCr. For 50, 75, and 100 mM PA-CoCr, the small peaks observed at ∼970 and ∼1260 cm−1 suggested the presence of some P−OH and PO groups, respectively. These results suggested that PA was bonded to CoCr primarily through monodentate bonding in 50, 75, and 100 mM PACoCr. The multiple peaks observed in the 1650−1750 cm−1 region were assigned to phosphate species. The peak at 1517 cm−1 was assigned to the presence of unavoidable hydrocarbon contamination. The multiple peaks observed for the −OH stretching were shifted to higher wavenumbers between 3566 and 3861 cm−1. This result suggested the presence of free −OH groups (not hydrogen bonded) of PA coated CoCr. In the spectrum of PAA powder (Figure 2A-2), the peaks observed in the P−O region at 943, 1012, and 1217 were assigned to P−OH, P−O, and PO species, respectively. A major peak observed at 1739 cm−1 was assigned to CO stretching of the −COOH group. The peak for C−O stretching in −COOH group was observed at 1295 cm−1. The peak observed at 3461 cm−1 was assigned to −OH stretching while the multiple peaks observed at 855, 756, and 694 cm−1 were assigned to −OH bending vibrations. The peaks for the stretching and deformation vibrations of C−H group were observed at 2970 and 1366 cm−1, respectively. After the PAA was coated on CoCr, several changes occurred in the FTIR spectrum (Figure 2C). In the P−O region, a new broad peak appeared at 1133 cm−1, which was assigned to the P−O−metal bond. This suggested that the PAA was covalently bound to CoCr irrespective of the concentrations of PAA used in this study. The peak for the P−OH group was observed at 935 cm−1 in 50, 75, and 100 mM PAA-CoCr. However, this peak was absent in 1 and 25 mM PAA-CoCr. Also, the peak for PO bond in the region 1200−1270 cm−1 was not present in any of the PAA coated CoCr. These results suggested the formation of tridentate bonding in 1 and 25 mM PAA-CoCr and bidentate bonding in 50, 75, and 100 mM PAA-CoCr. The multiple peaks observed in the region 1650−1750 cm−1 for phosphonate species might have overlapped with a peak expected to observe at ∼1700 cm−1 for CO of the −COOH group. The peak for −OH bending vibration was shifted to a lower wavenumber 676 cm−1. Also, the peaks for multiple −OH groups in PAA were observed in the region 3619−3863 cm−1. 3.3. AFM Characterization of Control, PA, and PAA Coated CoCr Specimens. AFM is commonly used to determine the uniformity of coatings on a substrate. Specifically, the homogeneity or heterogeneity of molecular coatings on a material surface can be determined by AFM. In this study, AFM was used to determine the homogeneity of PA and PAA coatings on CoCr alloy surface. Figure 3 shows the AFM tapping mode images of 3D surface topography of control, PA, and PAA coated CoCr specimens. In the images, 362

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heterogeneous coating. The AFM determined RMS roughness values of PA and PAA coated CoCr are provided in Figures 1C and 1D, respectively. All the PA and PAA coated CoCr prepared in this study showed roughness values either lesser than or equivalent to that of control CoCr. This further confirmed the formation of homogeneous PA and PAA coating on CoCr. In Figure 1C, no significant difference in the roughness values was observed between control, 1 mM, 25 mM, and 100 mM PA-CoCr. However, there was a significant decrease in the roughness observed for 50 and 75 mM PACoCr when compared to that of other sample groups. Also, the roughness value of 75 mM PA-CoCr was the least among the values of all the other sample groups. This suggested that the CoCr specimens coated with 50 and 75 mM PA were smoother than the other sample groups, with 75 mM PA being the smoothest surface. A similar trend in the roughness values was observed for PAA coated specimens as well, except that there was no significant difference between the roughness values of 50 and 75 mM PAA-CoCr (Figure 1D). 3.4. Protein Adsorption on Control, PA, and PAA Coated CoCr Specimens. 3.4.1. Adsorption of Single Protein. Figure 4 shows the amount of proteins adsorbed on control, PA, and PAA coated CoCr alloy surfaces. In Figure 4A, when compared to control CoCr, the amount of albumin adsorbed on PA coatings gradually decreased for up to 25 mM PA-CoCr and then increased gradually from there to show maximum amount of protein adsorption on 100 mM PA-CoCr. In Figure 4B, no significant difference in the amount of fibrinogen adsorbed was observed between control CoCr and 1 mM or 25 mM PA-CoCr. However, the amount of fibrinogen adsorbed significantly decreased on 50 and 75 mM PA-CoCr to show negligible amount of protein adsorption on these surfaces. Similar to albumin, the amount of fibrinogen adsorbed on 100 mM PA-CoCr was greater than that of all other sample groups (Figure 4B). For PAA coatings (Figure 4C,D), irrespective of the type of protein, more protein was adsorbed on 25, 50, 75, and 100 mM PAA-CoCr, whereas the protein adsorbed on 1 mM PAA-CoCr was equivalent to that of control CoCr. Figure 5 shows the fluorescence microscopy images of BSAAF-555 and HF-OG-488 adsorbed on control, PA, and PAA coated CoCr alloy surfaces. These qualitative data were in excellent agreement with the quantitative results (Figure 4) described in the above paragraph. 3.4.2. Adsorption of Dual Proteins. For PA coatings (Figure 6A), irrespective of the type of protein, the amount of proteins adsorbed significantly decreased for all the PA coated CoCr when compared to that of control CoCr. Within the PA coated specimens, the amount of albumin adsorbed gradually decreased for up to 25 mM PA-CoCr and then increased gradually from there to show maximum adsorption on 75 mM PA-CoCr and saturated after that (Figure 6A). The amount of fibrinogen adsorbed significantly decreased at 1 mM PA-CoCr and then gradually increased from there to show maximum protein adsorption on 50 mM PA-CoCr and saturated after that (Figure 6A). For PAA coatings (Figure 6B), irrespective of the type of protein, the amount of proteins adsorbed significantly decreased for all the PAA coated CoCr when compared to that of control CoCr. Also, no significant difference in the amount of proteins adsorbed was observed between any of PAA coated CoCr specimens (1−100 mM PAA-CoCr). The fluorescence microscopy images (Figure 6C−X) obtained were also in agreement with the quantitative results (Figure 6A,B).

Figure 3. AFM images of control, PA, and PAA coated CoCr. Note that the vertical scales are 0−140 nm in all cases with the exception of (D), (E), (I), and (J), where the scale is 0−45 nm.

the vertical scales are 0−140 nm in all cases with the exception of Figure 3D,E,I,J, where the scale is 0−45 nm. The reason is that the surfaces of 50 mM PA-CoCr, 75 mM PA-CoCr, 50 mM PAA-CoCr, and 75 mM PAA-CoCr are ultrasmooth when compared to all the other surfaces. Hence, it is essential to keep a smaller vertical scale (0−45 nm) in these images in order to clearly observe the topography; otherwise, the surfaces will look completely flat at larger vertical scale (0−140 nm). The images of PA and PAA coated specimens appear similar to that of control CoCr with no evidence of micelle or island formed on the coated surfaces. These results suggested that both PA and PAA formed uniform molecular coatings that followed the contour of underlying polished CoCr surface. In the literature, the AFM roughness values of metal oxide surfaces measured before and after the deposition of molecular coatings have been commonly used to determine the homogeneity of coatings.11,15 If the roughness value of coated specimens is lesser than or equal to that of control specimens, then it was attributed to the formation of homogeneous coating. However, if the roughness value of coated specimens is significantly greater than that of control specimens, then it was attributed to the formation of 363

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Figure 4. Amount of albumin and fibrinogen adsorbed on PA coated (A, B) and PAA coated (C, D) CoCr alloy specimens. Asterisk denotes statistical significance at p < 0.05.

3.4.3. Displacement of Albumin by Fibrinogen. When HFOG-488 was allowed to deposit on BSA-AF-555 adsorbed control CoCr, it displaced ∼60% of the BSA-AF-555 adsorbed on the alloy surface (Figure 7A). Fluorescence microscopy images (Figure 7B−D) were also obtained to show fibrinogen displaced albumin. These results were in excellent agreement with the literature to show that fibrinogen effectively displaces albumin on the material surface.16 3.5. Interaction of Platelets with PA Coated CoCr Specimens. All PA coated specimens, irrespective of the concentrations of PA (1−100 mM) used for coating, showed significantly reduced adhesion of platelets when compared to that of control CoCr (Figure 8A). However, no significant difference in platelet adhesion was observed between the PA coated specimens prepared using different concentrations of PA. The activation of platelets was also significantly reduced on all PA coated CoCr specimens when compared to that of control CoCr (Figure 8B). Similar to adhesion, no significant difference in platelet activation was observed between the PA coated specimens prepared using different concentrations of PA (Figure 8B). With regard to platelet aggregation, no significant difference was observed between control and PA coated specimens prepared using 1, 25, and 50 mM PA (Figure 8C). However, both 75 and 100 mM PA-CoCr showed significantly reduced aggregation when compared to that of control. The 100 mM PA showed the least aggregation of platelets among

the different sample groups used in this experimental set (Figure 8C). For PAA coating, no significant difference in the adhesion (Figure 8D) or activation (Figure 8E) of platelets was observed between the control and PAA coated specimens prepared using different concentrations of PAA (1−100 mM). With regard to platelet aggregation (Figure 8F), although there was a tendency for increased aggregation observed on the specimens coated with higher concentrations of PAA (25−100 mM), only 75 mM PAA showed significantly increased aggregation than that of the control. The degree of platelet activation was determined by SEM using Goodman’s method.17 This method categorizes the activation degree of adhered platelets from lower to higher level in the following order: (a) round or discoid; (b) dendritic or early pseudopodial; (c) spread-dendritic or intermediate pseudopodial; (d) spreading or late pseudopodial; and (e) fully spread. In this study, SEM images were obtained at 1000× for qualitatively studying the adhesion of platelets and at 10000× for studying the activation and aggregation of platelets on the different sample groups. The low magnification SEM images (Figure 9A−F) of platelets adhered on control and PA coated CoCr showed that the number of platelets adhered on PA coated CoCr surfaces was lesser than that of the control. The high magnification SEM images (Figure 9G) showed that the platelets were completely activated and present in fully 364

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Figure 5. Fluorescence microscopy images of BSA-AF-555 and HF-OG-488 adsorbed on control, PA, and PAA coated CoCr alloy specimens.

It has been shown in the literature that phosphonic acid molecules can form one of the following three covalent bonding configurations on metal oxide surfaces depending on the number of oxygen atoms the molecule uses to coordinate to the surface: (a) monodentate (through one oxygen atom); (b) bidentate (through two oxygen atoms); (c) tridentate (through three oxygen atoms).18 In this study, FTIR showed the formation of tridentate bonding on 1 mM PA-CoCr, bidentate bonding on 25 mM PA-CoCr, and monodentate bonding on 50, 75, and 100 mM PA-CoCr. The type of bonding formed is directly related to the packing density of molecules.19 The monodentate bonding results in higher molecular packing density whereas the tridentate bonding results in lower molecular packing density. This suggests that the density of PA molecules coated on CoCr increased in the following order: 1 mM PA-CoCr < 25 mM PA-CoCr < 50 mM PA-CoCr = 75 mM PA-CoCr = 100 mM PA-CoCr. It was interesting to observe that the contact angles obtained for PAA coated CoCr was lesser than that of PA coated CoCr for all the different concentrations (1−100 mM) of solutions used for coating. This showed that the PAA coated surfaces were more hydrophilic than PA coated surfaces. Also, this result suggested that PAA formed better molecular packing than PA on CoCr alloy surfaces. FTIR showed that tridentate bonding was formed in 1 and 25 mM PAA-CoCr, while bidentate bonding was formed in 50, 75, and 100 mM PAA-CoCr. Unlike PA coated CoCr, no monodentate bonding was observed in PAA coated CoCr. This could be due to the reason that the terminal carboxylic acid groups in PAA coating is relatively larger in size than the terminal hydroxyl groups in PA coating. However, the presence of short alkyl chain (one −CH2 group)

spread form on control CoCr. Also, the platelets were severely aggregated to form platelet clumps on control CoCr. Unlike control CoCr, only some of the platelets adhered on PA coated CoCr surfaces were fully spread (Figure 9H−L). Most other platelets showed dendritic (partially activated) morphology on PA coated CoCr surfaces (Figure 9H−L). Also, the platelet clumps were not observed on any of the PA coated CoCr surfaces, with aggregation primarily occurred between two or three individual platelets (Figure 9H−L). The number of fully activated platelets was lesser on 75 and 100 mM PA-CoCr when compared to other PA coated specimens. For PAA coating, the number of platelets adhered was similar to that of control CoCr (Figure 10A−F). The platelets were fully activated with clumps of aggregated platelets observed on control CoCr (Figure 10G). A similar result was also observed for 1 mM PAA-CoCr (Figure 10H). The platelets adhered on 25 and 50 mM PA-CoCr showed a mixture of dendritic and fully spread morphologies (Figure 10I,J). On 75 mM PAA, the platelets adhered formed multiple small clumps (Figure 10K). On 100 mM PAA-CoCr, few dendritic shaped and fully spread platelets were observed (Figure 10L). The number of fully spread platelets on 100 mM PAA-CoCr was less when compared to that of control or other PAA sample groups.

4. DISCUSSION The goal of this study was to surface modify CoCr alloy using varying concentrations of phosphoric and phosphonoacetic acids to prepare alloy surfaces with variable hydrophilicity, chemistry, and roughness and to investigate albumin and fibrinogen adsorption and platelet adhesion, activation, and aggregation on the modified alloy surfaces. 365

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Figure 6. Dual protein adsorption studies: amount of BSA-AF-555 and HF-OG-488 adsorbed on PA coated (A) and PAA coated (B) CoCr alloy specimens and fluorescence microscopy images of BSA-AF-555 and HF-OG-488 adsorbed on control, PA, and PAA coated CoCr alloy specimens (C−X).

in between phosphonic acid and carboxylic acid functionalities in PAA might have assisted in better packing of these molecules on alloy surfaces when compared to that of PA. The PA and PAA molecules coated on the alloy surface attained saturation when 75 mM concentration of the coating solutions was used. Increasing the concentration of coating solutions further for up to 100 mM may provide additional molecules that could be inserted between the molecules that were already coated on the surface. This could cause an increase in surface roughness at the nanoscale level. Although this did not affect the overall homogeneity of 100 mM PA or PAA coatings on CoCr, it is important to determine the concentration of coating solutions that provide saturation of molecules on the alloy surface. This factor should be taken into

consideration when the concentration of coating solutions is optimized. The adsorption of blood plasma proteins on cardiovascular biomaterials plays a crucial role in determining the platelet responses and thereby deciding the blood compatibility of the material.16 Although over 150 proteins are present in the blood, albumin and fibrinogen belong to the group of most important proteins that mediate platelet responses.16 Since the albumin is present in high concentration (40 mg/mL) in the blood plasma and is moderately sized (66 kDa), it is one of the proteins that adsorb onto the material surface initially.16 Albumin adsorption is commonly considered to be favorable as it prevents the adhesion of platelets on the material surface.20 However, due to the Vroman effect, the proteins that are adsorbed initially are 366

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Figure 7. Quantitative data (A) and fluorescence microscopy images (B−D) for albumin displaced by fibrinogen on control CoCr alloy.

exchanged continuously until the high affinity proteins are adsorbed on the material surface.16 Fibrinogen is one the proteins that has greater affinity toward material surface. Although fibrinogen is present in low concentration (3 mg/ mL) in blood plasma and is largely sized (340 kDa), it can still effectively replace several other adsorbed proteins on the material surface mainly due to its greater affinity.16 Fibrinogen adsorption is not favorable due to its prominent role in

Figure 9. SEM images of platelets adhered on control and PA coated specimens at magnifications 1000× (A−F) and 10000× (G−L).

initiating platelet adhesion and paving the way for thrombosis.21 Hence, it is important to investigate the adsorption of

Figure 8. Adhesion, activation, and aggregation of platelets on PA coated (A−C) and PAA coated (D−F) CoCr alloy. Asterisk denotes statistical significant at p < 0.05. 367

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albumin), it can show a similar trend to other types of proteins such as fibrinogen as well. Hence, techniques need to be developed for making surfaces that show protein-specific responses. For PAA coating, irrespective of the type of protein (albumin or fibrinogen) deposited, a greater amount of protein was adsorbed on most of the PAA coated specimens (from 25 to 100 mM PAA-CoCr) when compared to the control. Rodrigues et al.22 showed a linear decrease in fibrinogen adsorption with the increase in hydrophilicity of material surfaces by increasing the number of hydroxyl (−OH) groups. In our study, although the PAA coated surfaces were very much hydrophilic when compared to the control, the amount of fibrinogen adsorbed on PAA coated surfaces was still greater. Also, it is important to note that although a gradual increase in hydrophilicity was observed for the PAA coated surfaces, no significant difference in fibrinogen adsorption was observed between the different PAA coated specimens. This suggests that effect of surface chemistry is more dominating than surface wettabilitiy in determining protein adhesion. Sivaraman et al.23 also showed that although the contact angles observed for −OH and −COOH terminated SAMs coated metal surfaces were almost the same, a greater amount of protein was adsorbed on −COOH terminated SAMs than −OH terminated SAMs. Although both −OH and −COOH functionalized surfaces are hydrophilic, the difference in protein adsorption to these two surfaces can be attributed to the role of water molecules that are inherently present on all material surfaces.1 The water molecules are weakly bound to −COOH functionalized surfaces than to −OH functionalized surfaces. Hence, when proteins interact with these different surfaces, they can replace the water molecules present on −COOH functionalized surfaces and adsorb on these surfaces more easily than doing the same on −OH functionalized surfaces. Also, the −COOH groups exist as negatively charged species (COO−) under physiological conditions (pH = 7.4).16 Most proteins contain both positively and negatively charged functional groups.16 Hence, an electrostatic attraction can occur between the negatively charged COO− groups on the material surface and the positively charged functional groups of proteins. Hence, more protein adsorbs on negatively charged −COOH functionalized surfaces than on neutrally charged −OH functionalized surfaces. The formation of thrombi on a biomaterial surface is typically initiated by adhesion, activation, and aggregation of platelets.4 In this study, a reduced adhesion and activation was observed for all the PA coated specimens when compared to the control. Although the amount of albumin adsorbed on 100 mM PACoCr was significantly greater than that of 1−75 mM PACoCr, all these surfaces showed similar reduction in platelet adhesion and activation. This could suggest that the platelet adhesion and activation are independent of the amount of albumin adsorbed on the PA coated CoCr alloy surfaces. Also, no significant difference in the adsorption of proteins was observed between control CoCr and 1 mM PA-CoCr. However, a significant reduction in platelet adhesion was observed for 1 mM PA-CoCr when compared to that of control CoCr. This could suggest that the conformation of proteins adsorbed on PA coated surfaces might be different in such a way that it resists platelet adhesion. Although all PA coated surfaces showed reduced platelet adhesion and activation, only 75 and 100 mM PA-CoCr showed reduced platelet aggregation. This could be due to the difference in the degree of activation

Figure 10. SEM images of platelets adhered on control and PAA coated specimens at magnifications 1000× (A−F) and 10000× (G− L).

proteins such as albumin and fibrinogen on novel surface modified cardiovascular biomaterials. The adsorption of proteins is a complex process, and it depends on various surface properties of the material including surface chemistry, surface wettability, surface roughness, and surface charge.16 In this study, a reduced amount of albumin was adsorbed on 25 and 50 mM PA-CoCr when compared to that of control. However, a greater amount of albumin was adsorbed on 100 mM PA-CoCr. Although there was no difference in the wettability observed between 100 mM PACoCr and 50 mM PA-CoCr, the amount of protein adsorbed was different. Also, the molecular packing density that is directly related to the density of terminal hydroxyl groups was expected to be not significantly different between 100 mM PACoCr and 50 mM PA-CoCr. This could suggest that the increased surface roughness observed in 100 mM PA-CoCr might have played a crucial role in greater protein adsorption. An increase in surface roughness can cause more proteins to adsorb since rough surfaces provide more surface area, which in turn provides more surface sites for the protein to adsorb. However, no significant difference in the surface roughness was observed between 100 mM PA-CoCr and control or 1 or 25 mM PA-CoCr. This clearly shows that the adsorption of proteins on material surfaces is not determined by a single parameter, but rather a combination of multiple parameters including surface chemistry, wettability, roughness, and charge. Although the adsorption trend of fibrinogen on PA coated CoCr was similar to that of albumin, it was interesting to observe that 50 and 75 mM PA-CoCr completely resisted fibrinogen adsorption. Also, these results underline a fundamental issue that when a material is surface modified to adsorb more amount of a specific protein (for example, 368

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of albumin and fibrinogen adsorbed on PA and PAA coated surfaces was less when compared to that of control CoCr. However, the adsorption of dual proteins was strongly dependent on the type of PA coatings (1, 25, 50, 75, and 100 mM of PA-CoCr) used whereas no such effect was observed for PAA coatings. In platelet interaction studies, all PA coated CoCr showed reduced platelet adhesion and activation when compared to control CoCr. Also, the platelet aggregation on 75 and 100 mM PA-CoCr was at a reduced level when compared to the other sample groups. For PAA coating, there was no difference in the platelet adhesion and activation observed between PAA coated CoCr and control CoCr. The platelet aggregation was slightly greater on 75 mM PAA-CoCr when compared to other sample groups. Thus, this study demonstrated that PA coating on CoCr alloy was superior to PAA coating for reducing platelet adhesion, activation, and aggregation. Hence, CoCr alloy can be surface modified using PA for potentially reducing blood clots on the alloy and improving its blood compatibility.

of platelets on these surfaces. When there are not many fully activated platelets on the surfaces to release its granules to initiate the aggregation process, then the number of aggregated platelets will be lesser. This suggests that the high density of −OH groups on the surface may influence the degree of platelet activation to decrease aggregation. Hence, 75 and 100 mM PA coatings provide optimal surfaces for reducing platelet adhesion, activation, and aggregation. For PAA coating, no significant difference in adhesion or activation or aggregation (except 75 mM PAA-CoCr, which showed slightly greater aggregation) was observed between PAA coated and control specimens. Lee et al.24 have prepared polyethylene substrates with a wettability gradient by incorporating multiple oxygen containing functional groups (hydroxyl, ether, ketone, aldehyde, and carboxylic acid) on the surfaces using a coronary discharge treatment. In their study, the platelet adhesion increased on the polyethylene surfaces with increased hydrophilicity. In another study, Rodrigues et al.22 have prepared gold substrates with a wettability gradient by coating with different ratios of −OH and −CH3 terminated thiol molecules. In their study, the platelet adhesion and activation decreased on gold substrates with increased hydrophilicity. These contradictory results in the literature show that the nature of functional groups is more important than wettability in determining platelet responses. In our study, although a gradient in hydrophilicity was observed for PAA coated surfaces, no significant difference in platelet adhesion or activation was observed for these surfaces. Also, although the wettability of some of the PA coated surfaces was in the same range of PAA coated surfaces, a reduction in platelet adhesion and activation was observed only for the PA coated surfaces when compared to the control. This shows that the −OH groups in PA are better than −COOH groups in PAA for reducing platelet adhesion and activation. Chuang et al.25 have showed that the functional groups with nearly neutral in surface charge can reduce platelet adhesion and activation. This suggests that the neutral charge of −OH groups in PA coating might have played an important role in reducing platelet adhesion and activation in our study as well. Sivaraman et al.21 have showed that although the wettabilities of −OH and −COOH functionalized surfaces were almost the same, a lesser number of platelets was adhered on −OH functionalized surfaces when compared to that of −COOH functionalized surfaces. Thus, the platelet responses to surface modified CoCr alloy observed in our study are in excellent agreement with previous studies on studying platelet responses to various other surface modified biomaterials. Also, this study showed that the nature of surface functional groups (i.e., surface chemistry) is the critical determinant of platelet responses to biomaterial surfaces rather than surface wettability.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (G.M.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was partially supported by American Heart Association (AHA) National Scientist Development Grant (10SDG2630103), Sigma Xi Grants-in-Aid of Research, and University of South Dakota’s Graduate Student Research and Creativity Activity Grant.

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REFERENCES

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5. CONCLUSIONS CoCr alloy was successfully surface modified using varying concentrations of PA and PAA at 1, 25, 50, 75, and 100 mM to obtain various surfaces with gradients of hydrophilicity, different surface chemistry, and surface roughness. The single protein adsorption studies showed the amount of albumin or fibrinogen adsorbed on 25, 50, and 75 mM PA-CoCr was less when compared to that of control CoCr. However, more proteins were adsorbed on 100 mM PA-CoCr when compared to control CoCr. For PAA coating, a greater amount of albumin or fibrinogen was adsorbed on 25, 50, 75, and 100 mM PAACoCr. The dual protein adsorption studies showed the amount 369

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