Octadecyl Chains Immobilized onto Hyaluronic Acid Coatings by Thiol

Subscriber access provided by Washington University | Libraries

Article

Octadecyl chains immobilized onto hyaluronic acid coatings by thiolene "click chemistry" increase the surface antimicrobial properties and prevent platelet adhesion and activation to polyurethane. Helena P. Prado Felgueiras, Li-Mei Wang, Ke-Feng Ren, Micaela M. Querido, Qiao Jin, Mário Barbosa, Jian Ji, and M. Cristina L. Martins ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16415 • Publication Date (Web): 06 Feb 2017 Downloaded from http://pubs.acs.org on February 8, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 44

ACS Applied Materials & Interfaces Page 1 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Octadecyl chains immobilized onto hyaluronic acid coatings by thiol-ene "click chemistry" increase the surface antimicrobial properties and prevent platelet adhesion and activation to polyurethane

H.P. Felgueiras1,2, L.M. Wang3, K.F. Ren3, M.M. Querido1,2,4, Q. Jin3, M.A. Barbosa1,2,5, J. Ji3, M.C.L. Martins1,2,5*

1

i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal

2

INEB, Instituto de Engenharia Biomédica, Universidade do Porto, Portugal

3

Department of Polymer Science & Engineering, Zhejiang University, Hangzhou, China

4

FEUP, Faculdade de Engenharia da Universidade do Porto, Portugal

5

ICBAS, Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Portugal

*Corresponding author: Email: [email protected] Tel.: (+351) 220 408 800

Keywords: polyurethane; C18 alkyl chains; hyaluronic acid; click chemistry; albumin; antimicrobial; hemocompatible; biomaterial

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

Page 2 of 44 Page 2 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Infection and thrombus formation are still the biggest challenges for the success of blood contact medical devices. This work aims the development of an antimicrobial and hemocompatible biomaterial coating through which selective binding of albumin (passivant protein) from the blood stream is promoted and, thus, adsorption of other proteins responsible for bacterial adhesion and thrombus formation can be prevented. Polyurethane (PU) films were coated with hyaluronic acid, an antifouling agent, that was previously modified with thiol groups (HA-SH), using polydopamine as the binding agent. Ocatdecyl acrylate (C18) was used to attract albumin since it resembles the circulating free fatty acids and albumin is a fatty acid transporter. Thiol-ene "click chemistry" was explored for C18 immobilization on HA-SH through covalent bonding between the thiol groups from the HA and the alkene groups from the C18 chains. Surfaces were prepared with different C18 concentrations (0%, 5%, 10% and 20%) and successful immobilization was demonstrated by scanning electron microscopy (SEM), water contact angle determinations, x-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). Surfaces ability to bind albumin selectively was determined by quartz crystal microbalance with dissipation (QCM-D). Albumin adsorption increased in response to the hydrophobic nature of the surfaces, which augmented with C18 saturation. HASH coating reduced albumin adsorption to PU. C18 immobilized onto HA-SH at 5% promoted selective binding of albumin, decreased Staphylococcus aureus adhesion and prevented platelet adhesion and activation to PU in the presence of human plasma. C18/HA-SH coating was established as an innovative and promising strategy to improve the antimicrobial properties and hemocompatibility of any blood contact medical device.


Page 3 of 44


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1. Introduction Blood infections and platelet adhesion and activation remain important obstacle to the development of blood compatible medical devices, including intravascular catheters. Bacteremias and fungemias resultant from catheter infections are estimated between 0.1 and 2.7%,1 with mortality varying among 12 and 25% in critically ill patients.2 These infections are mostly caused by biofilm forming bacteria, like Staphylococcus aureus (S. aureus, commonest pathogen responsible for 40% of the infections), that, after biofilm formation, are difficult to treat with local/systemic antibiotics, forcing implant substitution and revealing the need for new antimicrobial coatings.3 Thrombus formation, which is catalyzed by the adsorption of proteins, like human fibrinogen (HFg), that stimulate the adhesion and activation of platelets and blood coagulation factors, are also very frequent.4-5 In the USA, incidence of catheter-induced thrombus varies between 2.0 and 5.5%, with an estimated mortality risk of 1 to 2%.6 Coatings with albumin have been thought to “passivate” biomaterial surfaces. In its native conformation, albumin prevents platelet adhesion and activation (absence of specific binding-sequences to platelet integrins) and the adsorption of other proteins involved in thrombus formation, like HFg, FXII, FXI, prekallikrein, and high-molecular-weight kininogen (initiation of coagulation cascade).7-8 Also, albumin coatings have been reported to exhibit an inhibitory effect on bacterial adhesion, including various strains of Staphylococcus.9-10 Albumin is the most abundant protein in the blood serum, representing near 60% of the total protein content, and exhibits high affinity for circulating free fatty acids, primarily formed of 16-18 carbons (C18).7, 11 Because of the limitations associated with albumin immobilization onto biomaterial surfaces, denaturation, degradation and displacement, the covalent binding of n-alkyl chains with C18, that resemble the circulating free fatty acids, to polymers has been researched. It was previously demonstrated by our team that the immobilization of octadecyl isocyanate (C18) onto OH-terminated self-assembled monolayers (SAMs) and poly(2hydroxyethyl methacrylate) (pHEMA) surfaces was able to induce human serum albumin (HSA) adsorption in a selective and reversible way, increasing the antithrombotic nature of the surfaces.12-14 However, that work was performed as proof-of-concept using SAMs and pHEMA




1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

films as model surfaces, which do not possess adequate mechanical properties to be used, per se, as biomaterials for blood contact. In the present study, an antimicrobial and hemocompatible coating, capable of being used as a coating of most medical devices was engineered. We proposed to coat medical grade polyurethane (PU) films, with a C18/hyaluronic acid (HA) complex, using polydopamine, a derived synthetic eumelanin polymer that possesses the ability to deposit via oxidative self-polymerization at slightly basic pH onto virtually any type and shape of surface,15 to bind HA to PU. PU is one of the most widely used materials in the production of intravascular devices because of its excellent mechanical properties, high elongation capacity, good abrasion resistance, high flexibility and hardness, and blood compatibility.16 HA, an extracellular matrix glycosaminoglycan, was selected because of its exceptional antifouling, angiogenic and antithrombotic properties.17-18 HA is also known to reduce bacterial adhesion.19 A new strategy for C18 immobilization was explored using thiol-ene "click chemistry". For that HA was firstly modified with thiol (-SH) groups (HA-SH) to allow the coupling of ocatdecyl acrylate (C18) by its alkene (-CH2=CH2) group using a thermal trigger. Thiol-ene "click chemistry" is a fast, simple and user-friendly radical-based reaction that offers versatility. Using this approach, homogeneous polymer networks are acquired through a controllable combination of step-growth and chain-growth processes, with significantly simplified polymerization kinetics, reduced shrinkage and stress, and insensitivity to oxygen or water inhibition.20-21 The functionalized surfaces were characterized in terms of morphology, wettability, elemental composition and molecular structure. HSA single and competitive adsorption behavior with HFg were followed by quartz-crystal microbalance with dissipation (QCM-D), a fast and accurate technique that monitors frequency and energy dissipation responses of the freely oscillating sensor, frequently used in the study of complex biomolecular systems.22 The antimicrobial efficacy of the C18/HA-SH complex was determined against S. aureus bacteria, and its ability to prevent platelet adhesion and activation was observed using human platelets. HSA, HFg and 1% plasma proteins were used at the interface.


Page 5 of 44


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2. Materials and Methods 2.1 PU Films Production PU pellets (Pellethane 2363-80 AE, Dow Chemical) were initially sonicated (15 min) twice with hexane (Millipore) and once with ethanol (Millipore) to eliminated silicone casing. This process was repeated twice. After, PU pellets were rinsed with deionized water (ddH2O) and left to dry overnight in a vacuum oven at room temperature (RT). PU was dissolved at 12.5% w/v in tetrahydrofuran (THF, Sigma) and 80 mL of the solution were casted onto clean glass petri dishes of 140 mm of diameter. These were covered with aluminum foil and small holes were punched with a needle for a very slow evaporation of THF.23 Films were left to dry in a hood for 48 h. PU films were then cut in small disks of 13 mm of diameter and washed in hexane, ethanol and 3 times with ddH2O (5 min sonication) to remove possible impurities resultant from the cutting process. PU films were dried with argon (Ar) and stored protected from light.

2.2 Chemical Modification of HA with -SH Groups HA-SH (Freda, MW 330 kDa) were synthesized according to

24-25

. Briefly, HA was

dissolved at 1% w/v concentration in ddH2O, overnight. N-(3-dimethylaminopropyl)-n'ethylcarbodiimide hydrochloride (EDC, Aladdin) and hydroxybenzotriazole (HOBt, Sigma) at 1.5 mM were added to HA solution and stirred for 2 h to activate the carboxyl (-COOH) groups. For a 40% substitution of -COOH groups into -SH, 1.15 g of EDC and 0.85 g of HOBt were added to a 80 mL solution. Then, cystamine dihydrochloride (Cys, Sigma) at 1.5 mM was added to the mixture and stirred overnight at RT to form HA-Cys conjugates. The final solution was exhaustively dialyzed for 24 h in ddH2O (MW cut-off: 8000-14000 Da, Thermo Scientific) to remove the unreacted HOBt, EDC and Cys. HA-Cys was treated for 4 h with 5-fold excess of dithiothreitol (DTT, Aladdin) to reduce dissulfide bonds (pH 8.5). Finally, thiol functionalized HA, or HA-SH, was dialyzed against ddH2O at pH 3.5 for 2 days. The resultant product was lyophilized for 2 days and stored at -20ºC until use. The degree of SH substitution was confirmed by 1H nuclear magnetic resonance spectroscopy (NMR) in deuterium oxide (D2O).




1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2.3 Immobilization of HA-SH on PU Films Dopamine hydrochloride (DOPA, Sigma) was prepared at 2 mg/mL in 10 mM trizma hydrochloride (Tris-HCl, Sigma) at pH 8.5. Cleaned PU films were coated with DOPA by immersion for 24 h at RT (under agitation). For optimal polymerization conditions (conversion of DOPA into polydopamine or pDOPA), this step was conducted protected from light. pDOPA-coated films were then sonicated twice with Tris-HCl and once with ddH2O (2 min) to remove pDOPA precipitates, followed by several washes with ddH2O. Surfaces were dried with Ar. pDOPA worked as a binding agent between PU and HA-SH. HA-SH was prepared at 1 mg/mL in ddH2O. pDOPA coated films were immersed in HA-SH solution for 24 h at RT (under agitation). In the end, HA-SH coated films were sonicated three times in ddH2O (2 min) and rinsed several times. Surfaces were dried with Ar and stored protected from light.

2.4 Thiol-Ene "Click Chemistry" of C18 Thermal "click chemistry" was used to covalently immobilize octadecyl acrylate (C18, Sigma) on HA-SH coated films. First, dissulfide bonds (S-S) generated on HA-SH coated films (contact with air) were reduced with DTT at 10 mM in ddH2O (overnight at RT). Films were sonicated twice with toluene and once with ddH2O to eliminate the DTT. This was followed by several washes with ddH2O. 0%, 5%, 10% and 20% w/v C18 solutions in toluene (Sigma) were prepared. These were transferred to round bottom beakers and the reduced HA-SH films were immersed. Each solution was degasified for 20 min with Ar and hermetically closed with silicon caps. Solutions were heated in a oil bath at 80ºC for 4 h to induce the "click" reaction between the -CH2=CH2 groups from the C18 and the -SH groups from the HA-SH. During the 4 h reaction, the beakers atmosphere was continuously saturated with Ar. At last, films were sonicated twice with toluene and once with ddH2O (2 min) to eliminate unreacted C18, and thoroughly rinsed with ddH2O.


Page 7 of 44


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Surfaces were hydrated for 24 h to eliminate toluene (ddH2O was changed four times), dried with Ar and stored protected from light.

2.5 Surface Characterization Various techniques, including scanning electron microscopy (SEM), contact angle, xray photoelectron spectroscopy (XPS) and Fourier transformed infrared spectroscopy in attenuated total reflectance mode (ATR-FTIR), were applied to characterize the films' properties in every stage of modification: PU, pDOPA, HA-SH, HA-SH_DTT (after reduction with DTT), 0%C18, 5%C18, 10%C18 and 20%C18 (a schematics of the many layers composing the engineered film is provided in Supporting Information: Figure S1).

2.5.1 SEM Micrographs of the films' surfaces were taken using a high resolution SEM with X-ray Microanalysis, JEOL JSM 6301F/Oxford INCA Energy 350. Electron beam intensity of 5 kV (accelerating voltage) and magnification of 5000x were applied. To increase the surfaces conductivity, films were sputtered with Au/Pd for 60 seconds and 15 mA current using the SPI Module Sputter Coater equipment.

2.5.2 Water Contact Angle Water contact angle measurements were performed using a Data Physics, Model OCA 15, equipped with video CCD-camera. 4 µL ddH2O droplets (syringe) were placed on top of each film and the angle made with the surface measured using the sessile drop method. To prevent droplet evaporation, measurements were conducted in a closed thermostated chamber (25ºC) saturated with ddH2O. Angle variations with time were recorded 30 times per second during 3 min using the SCA 20 software. Droplet profiles were fitted using the Young-Laplace mathematical function. The contact angle respective to each film was calculated by extrapolating the time-dependent curve to zero.26 6 measurements were made per coating.




1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2.5.3 XPS XPS measurements were performed using a Kratos Axis Ultra HSA spectrometer with an Al monochromatic X-ray source operating at 15 kV (90W). Photoelectrons were analyzed at 100 Å from the outermost layer and 70º takeoff angle. Survey scans were acquired with a pass energy of 80 eV. High-resolution spectra of carbon (C), nitrogen (N) and sulfur (S) were collected with an analyzer energy of 40 eV. These were resolved into individual Gaussian peaks using the CASAXPS version 2.3.17 software and fitted by setting the maximum of the resolved C(1s) peak to 285.0 eV.23

2.5.4 ATR-FTIR ATR-FTIR spectra were recorded using a Nicolet 6700 Class 1 Laser Product from Thermo Scientific. Data was collect at 4 cm-1 resolution with a total of 32 scans. Films were uniformly pressed against a diamond tip using a Smart Omni Sampler during readings. Measurements were acquired in the mid-IR frequency range from 4000 to 400 cm-1 and treated using the Omnic software.

2.6 Biological Testing 2.6.1 Protein Adsorption Studies: QCM-D Gold-coated QCM-D sensors (fundamental frequency of 5 MHz), purchased from Biolin Scientific, were spin coated with 0.1% w/v PU in THF for 1 min at 9000 rpm, and dried for 1 h in a vacuum oven at RT. The thin films produced were coated with HA-SH (section 2.3) and, then, immobilized with different concentrations of C18 in toluene (section 2.4). QCM-D system (Q-Sense E4 instrument, Biolin Scientific) was used to monitor the frequency (∆f) and dissipation (∆D) shifts during protein single and competitive adsorptions. Voigt model was used to calculate the adsorbed protein masses by including the changes in both the frequency and dissipation, and thus taking into account the viscoelastic contributions of the hydrated layer. The Sauerbrey equation does not consider these contributions and, as a result, is not recommended when more complex or fibrous-like proteins, i.e. HFg, are used, or when


Page 9 of 44


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

sequential protein studies are conducted. Data from the 3rd to the 11th harmonics were collected and used in the analysis. The density and viscosity of the protein solutions were established at 1.35 g/cm3 and 0.0014 kg/ms (values commonly used for low concentrated protein solutions2728

), respectively. During testing, the temperature was kept constant at 37ºC and the flow rate

was established at 25 µL/min. Results were reported as mass per area (ng/cm2).

2.6.1.1 Albumin Adsorption (Single) HSA at 4000 µg/mL in phosphate buffered saline solution (PBS, pH 7.4) was individually injected in the QCM-D modules at a rate of 25 µL/min (37ºC). The flow was maintained until saturation was reached (≈ 30 min). PBS solution was used as baseline (10 min) and to remove unattached protein molecules after saturation (10 min).

2.6.1.2 Protein Competitive Adsorption This experiment was designed to understand the influence of albumin on the adsorption of HFg to C18-modified surfaces. To this purpose, proteins were presented to the surface sequentially and PBS was used as baseline (10 min). Bovine serum albumin-fluorescein isothiocyanate conjugate (BSA-FITC, Sigma) at 1000 µg/mL in PBS was used in the place of HSA, so its quantification would be possible. The adsorption of BSA-FITC was followed until a plateau was reached (≈ 30 min). Then, HFg at 20 µg/mL was introduced and left until a new adlayer reached saturation (≈ 90 min). The entire experiment was conducted protected from light at 37ºC and flow rate of 25 µL/min. 27 min after the introduction of the HFg protein (time required for a protein solution to travel the entire system), the waste was collected and its absorbance red at 280 nm using a lambda 35 UV/VIS spectrometer (PerkimElmer). Data acquired from spectrometer readings were normalized against a BSA-FITC calibration curve ranging from 0.1 to 50 µg/mL. Between protein injections, PBS was used to clean the system and remove unattached fractions of the proteins. Individual adsorption of HFg (≈ 90 min) was followed to determine the level of BSA-FITC interference on HFg adsorption during sequential adsorption studies.




1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2.6.2 Staphylococcus aureus Adhesion The antimicrobial properties of the PU, HA-SH, 0%, 5%, 10% and 20%C18 films were evaluated by following the adhesion of S. aureus (American Type Culture Collection 49230) for 2 h at 37ºC. Films were sterilized with UV light (30W) and immersed in 70% v/v ethanol (twice), for 15 min each. Surfaces were washed 3 times with PBS and then immersed for 30 min at 37ºC in PBS (control), 1% v/v plasma (provided by Hospital de São João, Porto), 4000 µg/mL HSA and 20 µg/mL HFg, all in PBS. The surfaces were washed again 3 times with PBS, and bacteria prepared at 1x107 CFUs/mL in PBS were seeded. After the 2 h incubation, the bacterial solution was removed, the surfaces washed 3 times with PBS and the adhered S. aureus fixated with 4% v/v paraformaldehyde (Sigma) in PBS for 20 min at RT. Eventually, the films were washed with PBS and mounted with vectashield supplemented with DAPI (Baptista Marques-Diagnóstico e Reagentes, Lda) for fluorescent microscopy observation (Axiovert 200 M, Zeiss). Five images per film were collected. Number of adhered bacteria per mm2 was determined using the Image J software.

2.6.3 Platelets Adhesion and Activation Platelets from an Intermediary Platelet Unit (IPU), provided by Hospital de São João, Porto, were used. PU, HA-SH, 0%, 5%, 10% and 20%C18 films were sterilized as described in the previous section, immersed in PBS, 1% plasma, 4000 µg/mL HSA and 20 µg/mL HFg, all in PBS, for 30 min at 37ºC, and finally washed 3 times with PBS. Simultaneously, 24-well tissue culture polystyrene plates (TCPS, Sarstedt) were incubated at 37ºC with 1% w/v bovine serum albumin (BSA) in PBS for 1 h, to reduce platelet activation in response to the oxidized TCPS. Protein-adsorbed surfaces were transferred to the BSA-treated plates, previously washed 5 times with PBS, and incubated with IPU at 3x108 platelets/mL in PBS for 30 min at 37ºC and 70 rpm. Surfaces were rinsed with PBS and the adhered platelets fixated with 1.5% v/v glutaraldehyde (Merck) in 0.14 M sodium cacodylate (Merck) buffer for 30 min at RT. Finally, platelet-adhered surfaces were dehydrated in a growing ethanol/water gradient, 50%, 60%, 70%, 80%, 90% and


Page 11 of 44


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

99% (v/v), for 10 min each. To avoid superficial tension effects and preserve the materials and the adhered platelets structural integrity during SEM visualization, critical point drying was applied. This process includes a series of temperature variations from 4ºC to 33-38ºC until a maximum pressure of 1000-1400 psi is reached. The films conductivity was enhanced by sputtering with Au/Pd for 60 seconds and 15 mA current. Five micrographs of each film were taken by SEM using an electron beam intensity of 5 kV and magnification of 2000x. The number of platelets per surface condition was determined using the Image J software and reported as platelets/mm2. Adhered platelets were divided per degree of activation in round, dendritic, spread dendritic, spread and fully spread, according to a pre-established scale,29 and reported as percentage per degree of activation.

2.7 Statistical Analysis Experiments were conducted in triplicate. Numerical data were reported as mean ± standard deviation (SD). Statistical significance was determined by two-way analysis of variance (ANOVA) or one-way ANOVA followed by the Friedman-Dunn's, Bonferroni and Tuckey's multiple comparisons tests, using the GraphPad Prism 6.0 software. Significance was defined as having p

Octadecyl Chains Immobilized onto Hyaluronic Acid Coatings by Thiol

Recommend Documents