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Surface functionalization of Ti6Al4V via self-assembled monolayers for improved protein adsorption and fibroblast adhesion Abshar Hasan, Varun Saxena, and Lalit M Pandey Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03152 • Publication Date (Web): 28 Feb 2018 Downloaded from http://pubs.acs.org on March 2, 2018
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Abstract Graphic 279x114mm (150 x 150 DPI)
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Surface functionalization of Ti6Al4V via self-assembled monolayers for improved protein adsorption and fibroblast adhesion
Abshar Hasan1, Varun Saxena1, Lalit M. Pandey1* 1
Bio-Interface & Environmental Engineering Lab, Department of Biosciences and Bioengineering,
Indian Institute of Technology Guwahati, Assam, 781039, India *Corresponding author: Tel. +91-361-258-3201; Fax +91-361-258-2249 Email addresses:
[email protected],
[email protected],
[email protected] 1 ACS Paragon Plus Environment
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Abstract Although metallic biomaterials find numerous biomedical applications, their inherent low bioactivity and poor osteointegration had been a great challenge since decades. Surface modification via silanization can serve as an attractive method for improving aforementioned properties of such substrates. However, its effect on protein adsorption/conformation and subsequent cell adhesion and spreading, has rarely been investigated. This work reports the indepth study of the effect of Ti6Al4V surface functionalization on protein adsorption and cell behaviour. We prepared self-assembled monolayers (SAMs) of five different surfaces (amine, octyl, mixed [1:1 ratio of amine:octyl], hybrid and COOH). Synthesized surfaces were characterized
by
Fourier
transform
infrared-attenuated
total
reflection
(FTIR-ATR)
spectroscopy, contact angle goniometer, profilometer, and field emission scanning electron microscopy (FESEM). Quantification of adsorbed mass of bovine serum albumin (BSA) and fibronectin (FN) was determined on different surfaces along with secondary structure analysis. The adsorbed amount of BSA was found to increase with increase in surface hydrophobicity with the maximum adsorption on octyl surface while the reverse trend was detected for FN adsorption, having the maximum adsorbed mass on COOH surface. The α-helix content of adsorbed BSA increased on amine and COOH surfaces while decreased for other surfaces. Whereas increasing β-turn content of the adsorbed FN with the increase in the surface hydrophobicity was observed. In FN, RGD loops are located in β-turn and consequetevely the increase in ∆ adhered cells (%) were predominantly increased with the increasing ∆ β-turn content (%). We found hybrid surfaces to be the most promising surface modifier due to maximum cell adhesion (%) and proliferation, larger nuclei area and the least cell circularity. Bacterial density increased with the increasing hydrophobicity and was found maximum for amine surface (θ=63±1°) which further decreased with the increasing hydrophobicity. Overall, modified surfaces (in particular hybrid surface) showed better protein adsorption and cell adhesion properties as compared to unmodified Ti6Al4V and can be potentially used for tissue engineering applications.
Keywords: Ti6Al4V alloy; Cell adhesion and spreading; Silanization; Protein adsorption; Wettability; Surface energy; Fibronectin
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1. Introduction Various types of biomaterials based on polymers, ceramics, and metals have been reported till date for numerous applications such as tissue engineering, drug delivery, wound healing, biosensors and environmental cleanup.1-5 Although polymeric and ceramic biomaterials have been widely studied in homogenous system, polymer-polymer composites and polymer-ceramic composites, but metal based biomaterials are less explored due to their inherent limitations and can find potential applications in various fields. Titanium (Ti) and its alloys (Ti6Al4V and TiNbHf etc.) are widely used in orthopedics and dental applications due to their excellent mechanical properties like low elastic modulus and good fatigue strength. Ti and its alloy have superior bulk physical properties but they suffer from poor osseointegration and osteoconductivity.6-7 Moreover, implants made from bare Ti and its alloys after implantation exhibited aseptic loosening and ion release, which can be deleterious to health and lead to implant rejection.8 Hence, biomimetic surface modification becomes important for improving physio-chemical properties that can regulate and tune various biological interactions/processes at bio-interface. Various modification approaches at micro and nano-level had been implemented to accelerate osseointegration for proper bonding between implant surfaces and surrounding tissue to ultimately reduce implant rejection. Different techniques like plasma treatment,9 deposition of calcium phosphates,10 and hydroxyapatite coatings11 have been used but suffers from one or the other disadvantages. For example, thin coating of calcium phosphate on Ti undergoes degradation under in vivo conditions resulting in the loss of interaction between tissue and implant.12 Moreover, such coatings suffer from mechanical failure which results in crack formation at the interface between the titanium surfaces and coatings.13 Among various reported techniques,9-11 self-assembled monolayers (SAMs) had also been explored to functionalize metals and metal oxides surfaces.14 SAMs are nano-thick coatings of ordered molecules formed due to spontaneous arrangement via chemisorption on surfaces. SAMs of alkanethiols for modification of Au (111) surface was first introduced by Allara and Nuzzo15, had attracted lot of attention of scientific community and was utilised for various applications in surface science.16 SAMS of phosphonic acids are of great interest due to the flexibility in forming SAMs on wide range of metallic surfaces.17-18 Different metal surfaces such as stainless steel and titanium alloys have been tested for the functionalization using organophosphorus
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compounds.19-20 Alkene-based SAMs have shown promising role in platinum surface modification for enzyme immobilization in biosensor applications.21 Structure and stability (thermal and hydrolytic) of various monolayers on different substrates like silicon, silicon carbide, gold, platinum and stainless steel have been investigated and monolayers on oxide surfaces exhibited thermal stability upto ~500ºC.22 Recently, Shaheen et al.23 reported alkanethiol SAMs formation on a ruthenium surface, which exhibited good stability under nitrogen atmosphere but decayed within few hours when exposed to ambient conditions. Indepth details on covalent surface modification using silanes, amines, phosphonates, carboxylates and catecols monolayers and their stability on various surfaces have been extensively reviewed.24 Organosilane SAMs offer well ordered, highly robust, good thermal and chemical stable nanocoatings on metal oxide surfaces.25-26 A detailed description of the SAMs for modifying metal oxide surfaces can be found elsewhere.16, 27 SAMs of organosilanes are formed by using silane coupling agents on hydroxylated surfaces wherein silanes form covalent bond with surfaces using hydroxyl groups in a condensation reaction.28 Organosilane molecules basically comprise of three parts (i) head group, that attaches covalently to surface, (ii) alkyl linker chain, and (iii) terminal group, having functional moieties that regulate surface properties. SAMs modified Ti surfaces have been reported for various applications such as drug delivery, formation of antibacterial layers, mineral deposition for improving osteoconductivity, regulating non-specific protein adsorption and cell adhesion, and immobilization of biological molecules 2934
etc. Though, very few reports exist in which silane and phosphate based SAMs have been
investigated on sputtered TiO2 substrates rather than commercially available Ti sheets.14, 35 Liu et al. demonstrated the formation of different functionalities (such as –OH, –COOH, –NH2, – PO4H2, –CHCH2, –CH3) on Ti substrates and their role in hydroxyapatite deposition and calcium phosphate nucleation.35-36 Marin-Pareja et al. recently demostrated the effect of silanized Ti surfaces on the organization of type-1 collagen which regulated the fibroblast adhesion, spreading and fibronectin secretion.37 Collagen organized into globular clusters on hydrophilic surface enhanced fibroblast adhesion and spreading. Authors also stressed on the concentration of collagen used, as on increasing its value above threshold, resulted in masking of collagen conformation, hence, similar behavior of fibroblast was observed on all the surfaces.37 Although some efforts have been made to determine the effect of physio-chemical properties at the interface on the behavior of adsorbed FN and initial cell response,38-39 the effect of silanized
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Ti/Ti-alloy on FN secondary structure and subsequent fibroblast adhesion have not been reported yet to our best knowledge. In our previous reports, we have successfully described the silaneSAMs based modifications on glass and silicon surfaces for regulating protein and polymer behavior.40-43 In this study, we implemented same silanization technique using different silanes for controlling Ti6Al4V surface properties and determining their effect on FN adsorption and conformation, which in turns regulated fibroblasts adhesion and spreading. We have prepared Ti6Al4V surfaces with wide range of wettability by forming hydrophilic (carboxylic and amine), hydrophobic (Octyl) and moderate hydrophobic (mixed and hybrid) SAMs, which were tested for protein adsorption and cell adhesion. We used various techniques like Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy, field emission scanning electron microscopy (FESEM), energy-dispersive X-ray spectroscopy (EDX) and contact angle goniometer to confirm surface modifications. Adsorbed proteins (BSA and FN) were quantified using bicinchoninic acid (BCA) assay while their secondary structures were analysed using FTIR-ATR. Fibroblast adhesion and spreading were analysed using fluorescence technique. In addition, we have investigated the antimicrobial properties of the modified surfaces against the clinically significant Staphylococcus aureus (Gram positive) and Escherichia coli (Gram negative) bacteria. Since, silanization using organosilanes involves covalent bond formation between surface and silane molecules, they can provide very stable and robust coatings for long term performance of biomaterials, particularly in the surgical and dental implants as well as in medical devices.
2. Materials and Methods 2.1. Materials Triethoxy(octyl)silane (TEOS, cat. no. 440213), (3-Aminopropyl)triethoxysilane (APTES, cat. no. 440140), anhydrous toluene (cat. no. 244511), bovine serum albumin (BSA) and fibronectin (FN), propidium iodide (PI, cat. No. P4170) were purchased from Sigma Aldrich, India. Methanol, toluene, Sodium chloride (NaCl), potassium chloride (KCl), monobasic potassium phosphate (KH2PO4), dibasic sodium phosphate (Na2HPO4), sulfuric acid (H2SO4), and hydrogen peroxide (H2O2) were procured from Himedia, India. Ti6Al4V alloy with 1 mm thickness was purchased from Narendra Steel, India. Mili-Q water (18 MΩ) was used throughout the work.
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2.2. Surface modification Ti6Al4V alloy was cut into 1x1 cm2 chip size by wire-electro discharge machining (WEDM) process and polished using 2000 µm grit silicon carbide emery papers. Pre-modification substrates washing was carried out using method described previously by our group.42, 44 Briefly, chips were washed in piranha solution (H2SO4:H2O2=7:3) for 1 h followed by washing for 30 min each in ammonia solution (Water:H2O2:NH3=5:1:1 v/v) and in HCl solution (Water:H2O2:HCl=3:1:1 v/v). Later, chips were washed with water and acetone and dried overnight at 50ºC. We synthesized monotype (amine, octyl and carboxylic), mixed, and hybrid SAMs on Ti6Al4V surfaces. We aimed to design surfaces with different physio-chemical properties derived from positively charged group (amine, moderately hydrophilic nature), negatively charged group (carboxylic, hydrophilic nature) and hydrophobic group (octyl). Mixed surfaces contained randomly mixed and non-homogeneous assembly of hydrophobic and hydrophilic moieties whereas hybrid SAMs carries hydrophobic and hydrophilic groups on the same molecule.
Figure 1. (a) Schematic representation of silanization process on Ti surface, (b) formation of urea linkage on amine SAM during hybrid surface preparation and (c) acidified oxidation of CH3 group of octyl SAM to carboxylic acid during COOH SAM preparation.
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Dried substrates were modified via formation of monotype self-assembled monolayers (SAMs) of amine (NH2) and octyl (CH3) silanes using APTES and TEOS, respectively. Cleaned surfaces were dipped in 1% (v/v) silane solution prepared in anhydrous toluene for 24 h at room temperature (25ºC) under inert N2 atmosphere. Mixed SAMs of NH2-CH3 silane was prepared by mixing APTES and TEOS in 1:1% ratio (v/v) under similar environmental conditions. Schematic shown in Figure 1 describes the preparation of various SAM surfaces. Hybrid SAMs was prepared by immersing the NH2 modified surface in p-Tolyl isocyanate solution (1%, v/v in anhydrous toluene) for 4 h in the presence of catalyst dibulyltin dilaurate, as shown in reaction scheme in Figure 1b. Similarly, carboxylic (COOH) SAMs was prepared by oxidizing CH3 modified surface in 5% acidic (w/w) KMnO4 for 30 min at room temperature.35,
45
Post
modification, surfaces were washed with toluene, toluene: methanol (1:1, v/v) and methanol for 5 min each and dried overnight at 37ºC. 2.3. Characterization of the modified surfaces 2.3.1. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) Silanization of surfaces was confirmed using ATR-FTIR (Spectrum TWO, Perkin Elmer) instrument at a scanning rate of 15 scans per second with resolution 1 cm−1. Unmodified Ti6Al4V surface was taken as background and was subtracted from the sample spectra to record only modified spectra. 2.3.2. Contact angle (θ) and surface energy (γSV) analysis Contact angle (θ) analysis was carried out with Holmarc instrument, India, using sessile drop method reported previously.42, 44 De-ionized water and diiodomethane (Himedia, India) liquids were used to measure static contact angles at 22ºC and the drop image was captured using inbuilt microscope. Contact angle was recorded for at least five different points on the same surface. θ primarily depends on interfacial tensions at the air−liquid, solid−liquid, and solid−air interfaces and is related through Young’s equation: − =
(1)
Where , , and are interfacial tensions between solid−vapor (SV), solid−liquid (SL), and liquid−vapor (LV) phases, respectively.
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The surface energy (γSV) was calculated based on contact angles of both the liquids, using geometric mean expression (shown below) reported previously by our group.41 Others researchers have also previously reported the surface energy of Ti and stainless steel with different microtextures.46-47
= + − 2∅[( ) + ]
(2)
Where, and are dispersive and polar component of liquid surface energy while and
are the dispersive and polar component of solid surface energy, respectively. ∅ is the
interaction parameter, whose value is taken as 1 for most of the similar types of molecules. 2.3.3. Surface roughness and morphology analysis Roughness of the SAM modified surfaces was analyzed using high precision non-contact computerized surface profilometer (Taylor Hobson, UK). Instrument was fitted with 20X lens that scanned an area of 850×850 µm2 at a focal length of 4.7 mm. Profilometer is based on optical light interference principle that provides surface information such as topography and roughness. Due to its non-contact mode of operation, it does not damage actual surface features. Surface morphologies of unmodified and modified Ti6Al4V substrates were distinguished using FESEM (Zeiss, Model: Sigma) instrument which was operated at an accelerating voltage of 2-4 kV and at a magnification of 150 KX. Energy-dispersive X-ray spectroscopy (EDX) equipped with the instrument was used to determine the distribution of elements on the modified surfaces. 2.4. Protein adsorption and secondary structure analysis Physico-chemical properties of biomaterial implant surfaces regulate protein adsorption and cell adhesion and proliferation.28 We investigated protein adsorption on different modified surfaces using bicinchoninic acid (BCA) assay. Protein samples of BSA (100 µg/ml) and FN (10 µg/ml) were prepared in phosphate buffer saline (PBS, pH 7.4) and incubated separately with the surfaces for 1 h at room temperature. Adsorbed proteins were desorbed in 5% SDS solution (prepared in PBS) for 1 h in shaking incubator (120 rpm) at 37ºC and protein was estimated using BCA kit (Sigma, India). Experiments were carried out in triplicate (n=3) for calculating the standard error.
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Change in secondary structures of the adsorbed proteins (BSA and FN) was also investigated using ATR-FTIR particularly in the amide I range 1600-1700 cm-1. Briefly, protein adsorbed surfaces were dried at 37ºC before recording spectra by FTIR-ATR instrument. Peak positions in Amide I range were resolved and fitted with a Gaussian shape using second derivative. 2.5. Cell culture studies Mouse fibroblast cell line, L929 was cultured in Dulbecco's modified Eagle's medium (DMEM, HiMedia, India) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% antibiotic (Pen Strep, Invitrogen) and maintained in CO2 incubator at 37ºC, 5% CO2 and 85% humidity. Cells were grown to 90% confluency, trypsinised, centrifuged and counted using hemocytometer before performing cell culture experiments. 2.5.1. Cytotoxicity assay Cytocompatibility of modified surfaces was assessed by performing MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay using mouse fibroblast L929 cells. Cells were seeded onto samples (UV sterilized, 30 W, 30 min) at a density of 5×104cells/cm2 and incubated for 2, 4 and 6 days. After each specified time interval, media was replaced with fresh media containing 20 µl of MTT solution (5mg/ml, PBS pH 7.4) and incubated at 37 ºC for 4 h. Post incubation, media was replaced with 500 µl of DMSO and incubated for another 10 min to dissolve formazan crystals formed due to metabolic activity of live cells. Optical density of the dissolved formazan was recorded at 570 nm (Infinite 200 Pro, Tecan). Cell proliferation (%) on modified surfaces was calculated with respect to unmodified surface which was considered as reference exhibiting 100% proliferation. Assay was performed in triplicates to calculate standard deviation. 2.5.2. Cell morphology using FESEM Effect of surface functionalities and wettability were also analyzed for understanding their role in promoting cell adhesion. Cell spreading or cell morphology of the adhered L929 cells was studied using FESEM after 6 h of incubation on modified surfaces. Post incubation, surfaces were washed thrice with PBS (pH 7.4) to remove non-adhered cells whereas adhered cells were fixed using 2.5% (v/v) glutaraldehyde solution for 2 h at room temperature. Surfaces were later washed with PBS followed by graded dehydration with 40%, 60%, 80%, 90% ethanol for 10 min, respectively and with 95% and 100% ethanol for 30 min each. Surfaces were then critical 9 ACS Paragon Plus Environment
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point dried using hexamethyldisilazane (Sigma, India) for 10 min, gold sputtered, and examined using FESEM. 2.5.3. Fluorescent imaging Fibroblast cells were seeded at density 5x104 cells/cm2 on modified surfaces and incubated for 6 h under two different conditions: (a) surfaces without pre-adsorbed protein, in presence of FBS, (b) surfaces pre-adsorbed with FN. Samples with pre-adsorbed FN was incubated with cells which were dispersed in FBS free DMEM media for 6 h at 37ºC in CO2 incubator. Post incubation, surfaces were washed thrice by filter sterilized PBS (pH 7.4) to remove non-adhered cells. Adherent cells were fixed with 4% (v/v) paraformaldehyde solution (HiMedia, India) overnight at 4ºC. Cells were later washed and treated with 2% (w/v) BSA and 0.2% (v/v) triton X100 for 6 h followed by fluorescent staining of actin filaments with FITC-Phalloidin (Sigma, India) for 12 h. Cells nuclei were stained by incubating substrates in 20 µg/ml of DAPI (Sigma, India) for 10 min at room temperature. Post staining, substrates were washed thrice with PBS and imaging was done by Nikon (Nikon Eclipse Ti-S) fluorescent microscope. Using image processing software (ImageJ), we calculated the % cell adhered, cell spreading area, nuclei area and circularity of the adhered cells on different modified surfaces.
2.6. Bacterial adhesion studies Effect of surface functional groups against bacterial cells adhesion was also investigated to determine their antimicrobial properties. We investigated the adhesion of both Gram positive (Staphylococcus aureus, ATCC 6538) and Gram negative (Escherichia coli, MTCC 1610) bacteria on modified surfaces for 2 h at 37ºC. Functionalized surfaces were UV sterilized (30W) for 30 min and were seeded with 1x107 CFU/mL in sterilized phosphate buffer saline (PBS). Post incubation, nonadhered bacterial cells were removed and surfaces were washed three times with PBS. Cells were later fixed with 4% (v/v) paraformaldehyde solution (Himedia, India) for 2 h followed by washing with PBS thrice. Fixed cells were permeabilized with 0.2% (v/v) triton X100 (Himedia, India) for 30 min and fluorescent stained overnight with propidium iodide (PI, Sigma India) at room temperature. Cells were visualized under inverted fluorescent microscope with 100X lens and atleast 10 images were captured at different places on the same surface to determine the average number of adhered cells. ImageJ software was utilized for counting cells on different surfaces. 10 ACS Paragon Plus Environment
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2.7. Statistical analysis All the experiments were carried out in triplicate and results are expressed as mean standard deviation for atleast n=3. Software SigmaPlot version 14.0 was used to determine the statistically significant differences (p
