ab_2018_00879q Designing nanostructured Ti6Al4V bioactive

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Bio-interactions and Biocompatibility

Designing nanostructured Ti6Al4V bioactive interfaces with directed irradiation synthesis towards cell stimulation to promote host tissue-implant integration Ana Civantos, Alethia Barnwell, Akshath R. Shetty, Juan Pavon, Osman El-Atwani, Sandra L. Arias, Eric Lang, Lisa M Reece, Michael Chen, and Jean Paul Allain ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b00469 • Publication Date (Web): 13 May 2019 Downloaded from http://pubs.acs.org on May 18, 2019

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ACS Biomaterials Science & Engineering

Ref. Manuscript ID: ab_2018_00879q

Designing nanostructured Ti6Al4V bioactive interfaces with directed irradiation synthesis towards cell stimulation to promote host tissue-implant integration

Ana Civantos1, 2*, Alethia Barnwell, Akshath R. Shetty1,2, Juan Jose Pavón1, 2, 4 Osman El-Atwani5, Sandra L. Arias2, 3, Eric Lang1, Lisa M. Reece6, Michael Chen7, Jean Paul Allain1, 2, 3*

1Department

of Nuclear, Plasma and Radiological Engineering, College of Engineering, University of Illinois at Urbana-Champaign, 104 S Wright St, Urbana, IL 61801, USA

2Micro

and Nanotechnology Laboratory, University of Illinois at Urbana-Champaign, 208 N Wright St, Urbana, IL 61801, USA

3Department

4Group

of Bioengineering, University of Illinois at Urbana-Champaign, 1406 W Green St, Urbana, IL 61801, USA

of Advanced Biomaterials and Regenerative Medicine, Bioengineering Program, University of Antioquia, Cl. 67 ##53-108, Medellín, Antioquia, Colombia

5Materials

6University

Science and Technology Division, Los Alamos National Laboratory, New México 87545, USA of Texas Medical Branch at Galveston Sealy Center for Vaccine Development, 301 University Blvd, Galveston, TX 77555, USA

7City

of Hope National Research Medical Center, 1500 E Duarte road, CA USA

Keywords: Regenerative Medicine, Nano-biomaterials, Directed Irradiation Synthesis (DIS), Cells Stimulation, Titanium, Advanced Biointerfaces, Nano-medicine.

ACS Paragon Plus Environment

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ACS Biomaterials Science & Engineering 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

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ABSTRACT A new generation of biomaterials are evolving from being biologically inert towards bioactive surfaces which can further interact with biological components at the nano-scale. Here, we present Directed Irradiation Synthesis (DIS) as a novel technology to selectively apply plasma ions to bombard any type of biomaterial and tailor the nanofeatures needed for in vitro growth stimulation. In this work, we demonstrate for the first time, the influence of physio-chemical cues (e.g. selforganized topography at nano-scale) of medical grade Ti6Al4V results in control of cell shape, adhesion and proliferation of human aortic smooth muscle stem cells. The control of surface nanostructures was found to be correlated to ion-beam incidence angle linked to a surface diffusive regime during irradiation synthesis with argon ions at energies below 1 keV and a fluence of 2.5 x 1017 cm-2. Cell viability and cytoskeleton morphology were evaluated at 24h, observing an advance cell attachment state on post-DIS surfaces. These modified surfaces showed 84% of cell biocompatibility and an increase in cytoplasmatic protusions ensuring a higher cell adhesion state. Filopodia density was promoted by a 3-fold change for oblique incidence angle DIS treatment compared to controls (e.g. no patterning) and lamellipodia structures were increased more than a factor of two, which are indicators of cell attachment stimulation due to DIS modification. In addition, the morphology of the nanofeatures were tailored, with high fidelity control of the main DIS parameters that control diffusive and erosive regimes of self-organization. We have correlated the morphology and the influence in cell behavior, where nano-ripple formation is the most active morphology for cell stimulation. 1. INTRODUCTION Millions of Americans undergo procedures each year involving medical implant devices, including heart valves replacements and stents. Between 2012 and 2015 nearly one million people suffer


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from infections each year in the United States alone due to medical implants1,2. Additionally, an estimation of the healthcare costs for cardiovascular disease has been reported of USD $300 billion per year in the USA3. Currently treatments for cardiac disease vary ranging from drug administration to surgical interventions, most of which involve bypass grafts or tissue transplantation.1 However, several limitations associated with human donor tissue have prompted the search for alternative approaches based on the use of synthetic and natural biomaterials to repair and or regenerate cardiac defects. In this context, the most frequent materials applied for the latter purpose have been used in stents, to open the obstructed vessels, and in heart valves design, highlighting Nitinol, titanium, stainless steel or cobalt-chromium among others3,4.

However, some materials are associated with allergic reactions, infections or even lack of integration hindering their use in cardiovascular regeneration.

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Biomimetic and bioinspired

concepts have emerged based on the principle of construction of artificial materials as native cardiac tissue that can actively interacting with the surrounding cells.5 One of the most critical challenges to the success of the implant device is designing an active interface which induce the required cell interactions to promote tissue healing. For example, the absence of an endothelial cell layer on artificial vascular grafts results in the occlusion of the vessel by thrombosis. Minimizing adhesion of platelets on vascular graft material surfaces reduces hemostasis and cloth formation whereas increasing endothelial cell population are key factors to restore properly the damage cardiac tissue.6 Designing surface properties of biomaterials mimicking the nano-to-micro level surface structure of the target tissue has shown to potentially promoted endothelial bioactivation. Therefore, one smart strategy to stimulate endothelial cells interactions at the interface is to reproduce cardiac


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extracellular matrix (ECM) nano-to-micro level topography. In order to achieve that, surface modification of biomaterials has emerged as a potential tool to induce faster integration, modulate immuno-response, and reduced blood coagulation 7. Numerous approaches at material design have been attempted to control properties that enhance tissue integration, without any detrimental or reduction of bulk properties. For example, in blood vessels, stent materials are designed to have high resilience to accommodate the local pulsating blood vessel environment while designing surface properties that mitigate hemostasis and thrombosis.4

The role of surface nanostructuring on clinically-relevant materials (e.g. Ti alloys) has been studied extensively, yet high-fidelity control of bioactive properties remain elusive.7 Reports summarizing in vitro cell behavior of nanostructured surfaces have shown an improvement of some specific cell processes such as cell adhesion and differentiation.8–10 However, the processes that mediate the cellular reaction with nano-scale surface structures are also not well understood.10 For example, does the role of nanotopography derive directly from surface contact, or indirectly with surface structures possibly affecting the composition, orientation, or conformation of the adsorbed ECM components?11,12 Several studies have shown improvements of cell adhesion due to interactions with nano-patterned surfaces, as regulators of cellular functions of human mesenchymal stem cells13 and rat aortic endothelial cells14, through the upregulation of integrin signaling pathway and the increased of focal adhesion points. Another important point is the surface modification of “as-is” clinically-relevant materials has been limited to only vary the random surface roughness or in other words “kinetic roughening”.15– 18

The control of nano-scale topography has been dominated by the development of advanced top-

down fabrication methods such as lithography-based techniques including focused (electron or


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ion) beam lithography and scanning probe lithography. 19,20 The drawbacks of these conventional techniques have mainly been attributed to their physical limitations in fabricating nanostructures between 5-100 nm at scale and only on a few classes of materials. Therefore, bottom-up techniques that rely on self-assembly, self-organization, and local patterning, have become emergent technologies to fabricate biocompatible surface nanostructures. Furthermore, energetic chargedparticle beams can be used to induce patterned structures with unique topography at the nano-scale by means of sputtering and other surface-related processes.21–29 Some of these surface-related phenomena involve ion-induced defect dynamics at temperatures above the thermal vacancy heat of formation (e.g. ~ > 0.3Tm) such as the case for random nanotendril formation at sub-threshold sputtering energies below 80 eV for tungsten surfaces bombarded by helium (He) ions. 30 In other instances, the inherent energy distribution density and inherent thermodynamic properties and phase separation of the material components (i.e. in the case of III-V semiconductors) can have significant effects on the topography with respect to incident ion energy and angle.31 Further examples the energy deposition from incident ions could have a strong dependence on crystallographic orientation whereby polycrystalline metals could exhibit a variety of surface patterns.32 An important limitation in nano-manufacturing approaches is a dependence on chemical-driven surface modification processes which require very high temperature processes or toxic chemicals.33 Consequently, many of the biomaterials used in biomedicine cannot be processed with conventional bottom-up techniques. In addition, techniques that result in self-organized structures in the mesoscale combining nano-to-micro hierarchical scales in one single exposure on complex 3D geometries such as those found in biomedical devices remain elusive. Directed Irradiation Synthesis (DIS) address those limitations by introducing a synthesis process that is


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scalable to high-value manufacturing by virtue of intrinsic large-area exposure of materials surfaces and interfaces that delivers 3D hierarchical architected structures at both nano and micro scales with unprecedented control of physico-chemical properties. DIS consists of taking a gradient energetic ion beam combined with energetic neutrals (e.g. from a sputtered source target) that enables a morphology gradient that can tune hierarchical structures combining additive/subtractive fabrication sequential steps [US Patent # US9932664B2].

In this work we demonstrate the role of DIS in nanostructuring Ti6Al4V surface and we analyze the influence of these nanofeatures in in vitro cells behavior. We report a detailed characterization of successfully nanostructured Ti6Al4V surface and correlate the effect of incidence angle in surface modification and nanofeatures morphology. Additionally, cell biocompatibility and adhesion using human aortic smooth muscle cells (HASMCs) have been evaluated to study cells interaction with nanostructured Ti6Al4V surface in the context of vascular tissue models. These new nanostructured surfaces were biologically evaluated for cytotoxicity and cell adhesion. In vitro cellular response to these new surfaces has enable us, for the first time, to establish correlations between nanostructuring Ti6Al4V by DIS and cellular behavior, as well as to show DIS techniques as a highly surface processing approach to modify clinically-relevant materials.

2. EXPERIMENTAL SECTION

2.1. Directed Irradiation Synthesis (DIS) of Ti6Al4V samples Medical grade Ti6Al4V alloy (ASTM F136, F1472) samples were used for surface modification by DIS. Samples were initially prepared by grinding and polishing up to mirror finish, before


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exposure to irradiation processing. DIS synthesis conditions for various Ti6Al4V samples are summarized in Table 1. Conditions were selected after an exhaustive revision of our previous DIS nanostructuring results on metals.34,35 Argon (Ar+) source at 1 keV was used to irradiate discs of Ti6Al4V; the sample were irradiated at several angles of incidence, as shown in Table 1. Table 1. Irradiation Parameters (1 keV Ar+) on Ti6Al4V Samples Samples Energy

Flux

Fluence

Incidence angle

Time

(keV)

(ions sec cm-2)

(cm-2)

()

(s)

S1

1.0

6.51E14

2.5E17

0

384.0

S2

1.0

3.29E14

2.5E17

30

758.8

S3

1.0

6.44E14

2.5E17

60

387.9

S4

1.0

6.55E14

2.5E17

80

381.6

2.2. Structural and energy characterization of DIS modified surfaces Morphological features of samples were analyzed by Scanning Electron Microscopy, SEM (Philips XL40 field emission, FEI, Hillsboro, Oregon, USA). Surface topography and roughness quantification were evaluated by AFM using Asylum Cypher, in tapping mode using BSTap300Al tapping tips (Budget sensors). Chemical composition of the surface was examined by X-ray photoelectron spectroscopy (XPS). These XPS measurements were carried out in the IonGas-Neutral Interactions with Surfaces (IGNIS) facility using an Al Kɑ (1486.6 eV) x-ray source. Full spectrum scans were taken with 1 eV step size, whereas the analyzed region scans corresponding to C1s, O1s, Ti2p, Al2p and V3p were taken with 0.1 eV step size. Resulting spectra were analyzed using Casa XPS software. X-ray diffraction spectra of substrates were performed using a PANalytical Philips X’pert MRD system 2 diffractometer with Cu K alpha radiation


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wavelength (λ = 0.15418 nm), generated at a voltage of 45 kV and a filament emission of 40 mA. Theta angle (2θ) was collected from 2θ = 30-80, with a step size of 0.02°, and the analysis was performed by Origin and Jade software. Further SEM and EDS analysis was performed using a JEOL 7000F Analytical SEM operating at 10 kV. EDS data was collected and analyzed with Thermo Electron EDS X-ray Microanalysis System with a 90-second live-time. Electron Backscatter Diffraction (EBSD) was performed using a Thermo Scios2 Dual-Beam SEM/FIB operated at 30 kV and 13 nA with the sample at a 70-degree tilt. EBSD patterns and data were captured and processed using the EDAX TEAM EBSD Analysis System with a Hikari Super EBSD detector. Kikuchi patterns were collected and indexed using either a 0.20 or 0.60 um step size. Surface energy of irradiated samples was evaluated by contact angle testing with deionized water

through

a

Ramé-Hart

Goniometer

Model

500-Advanced

contact

angle

goniometer/tensiometer with DROPimage Advanced Software. The sessile method of contact angle analysis was employed, using 3 μL of deionized water drops, with 6 drops per sample to measure the contact angle on each sample.

2.3. In vitro biological evaluation of Ti6Al4V samples modified by DIS. Cell biocompatibility experiments were performed by MTT and Alamarblue assays of Human Aortic Smooth Muscle cells (HAMSCs, Thermofisher Scientific Cat # C00725PAC)) at 24. At the same time, cell adhesion state was analyzed by cell morphology evaluation using cell cytoskeleton and nuclei staining as well as SEM. To that end, HASMCs cells were grown in Medium 231 (ThermoFisher Scientific Cat # M231500) supplemented with Smooth Muscle growth supplemented SMGS (S-008-5) and completed with 10% fetal bovine serum (FBS Thermofisher Scientific) at 37°C with 95% humidity and 5% CO2 gas exchange until reached 80% of


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confluence. Then, HASMCs were harvested by adding 0.05% trypsin (Thermofisher Scientific) at 37°C and centrifuge to obtain the cell pellet. Titanium samples were previously sterilized by autoclave (in water steam at a pressure of 2300 mbar at 121 °C) and placed in 24 well tissue culture plates (Corning Costar). HASMCs suspension was seeded at a cell density of 5 x 105 cells/mL and after 24 hours, cell viability was determined by the yellow tetrazolium MTT (Sigma) method. The stop solution consisted of 10 % sodium dodecyl sulphate (Fischer, USA) and 50 % isopropanol. Production of formazan was measured using a microplate reader (Benchmark, Biorad) at 570 nm. Finally, results were expressed as the cell viability percentage using cells growing on the TCP as control surface. At the same time point, cell metabolic activity was evaluated using Alamarblue assay (ThermoFisher DAL1100) assay to confirmed cell viability results following the manufactured instructions. In short, HASMCs metabolic activity was measured reading fluorescence levels at 530/590 nm (Exc/Emi) using a microplate reader (BioTek Synergy HT). Untreated Ti6A14V (CPTI), SLA type surface and TCP were used as controls. Regarding the cell morphology, titanium samples were seeded at a cell density of 10,000 cells/cm2. After 24h of incubation, samples were fixed using 4% paraformaldehyde (PFA) for 20 mins and then washed three times with PBS. Subsequently, samples were permeabilized using 0.1 % Triton X100, washed with PBS followed by 30 mins of Texas Red®-X phalloidin (Thermo Fisher) at room temperature in dark conditions. As a contrast cell nucleus were stained using Hoechst (Invitrogen, Molecular Probes ®) and finally samples were evaluated using confocal microscope Leica (LSM 800) using for actin fibers TRICT filter (λex/λem=550/600 nm) and DAPI filter for Hoechst (λex/λem = 380/455 nm). To further analyze cell morphology, HASMCs were then fixed with 2% glutaraldehyde, 12 h, at 4°C. Cell dehydration was achieved with increasing concentrations of


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ethanol (30, 50, 70, 80, 90 and 100%) in phosphate buffered saline (PBS). Finally, the samples were subjected to critical point drying and coated with gold to be observed by SEM (Hitachi 4800).

2.4. Statistical analysis All experiments were carried out in triplicates using three samples per condition (n=3). Data were expressed as mean ± standard deviation. One-way ANOVA followed by Tukey post treatment were applied to evaluate data for significant differences (P < 0.05) using Origin software.

3. RESULTS AND DISCUSSION

3.1. Normal incidence irradiation synthesis of Ti6Al4V

Figure 1 summarizes the SEM images of a variety of Ti-based surfaces studied in this work. The microstructure of un-irradiated Ti6Al4V polished and using an etching treatment (SLA-type surface) revealed a conventional α+β mill-annealed alloy consisting of α phase (hcp), equiaxial grains and Widmanstätten plates, dispersed in an untransformed β matrix (bcc). In contrast, after DIS exposure at a moderate fluence of 2.5 x 1017 cm-2, this surface morphology changes as a consequence of both heating and milling at the α+β thermodynamically stable region, and further slow cooling, allowing β-α transformation. This combination of phases and constituents results in an excellent balance between mechanical strength, toughness, ductility and fatigue resistance while reaching surface nanopatterning in an ultrashallow region of the surface below a few microns.36 This is an advantaged of surface-terminated synthesis approaches that enables modification of the surface without any change to the underlying bulk structure and properties.


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Figure 1. Surface characteristics of Ti6Al4V samples before (Polished Ti and Acid-etched Ti) and after DIS processing (S1 to S4) at normal and off-normal incidence angle. S1 corresponds to Ti6Al4V sample after irradiation with normal incidence angle (0°) and S2, S3, and S4, Ti6Al4V samples irradiated using off-normal incidence angles of 30°, 60° and 80° degrees, respectively.

The β phase (bcc) grains of the Ti6Al4V have higher resistance to DIS surface modification indicated by a reduced amount of surface nanostructuring at lower incidence angles (e.g. angles near normal incidence with respect to the surface). This distinction in surface modification is reduced at higher incidence angles as it is seen in S4_80° (Fig.1) and higher fluences as shown in Figure 2. Although these SEM images did not emphasize this β phase behavior, other experiments using DIS revealed clearly this surface modification which are summarized in Figure 2.


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Figure 2. SEM microphotographs of nanopatterned Ti alloy surfaces after surface modification using low (1.0 x1017 cm-2), medium (5.0 x1017 cm-2) and high fluence (1x1018 cm-2) of DIS process.

Samples were irradiated using argon ions, and off-normal incidence angle (60 degrees). Low fluences revealed small features, similar to cones, with random orientation and dispersed within Ti surface. However, nanofeatures become bigger in size, with more sharp cones and nanoplatelets or nano-walls more compacted at higher fluences. These images confirmed the diminished effect of grain orientation on nanofeatures shapes and direction when the irradiation is performed through oblique incidence angles (60 and 80 degrees) and higher fluence. Figure SI_1 of Supporting Information includes in more low-magnification SEM images using different fluences and the grain boundaries affection due to ion beam exposure in DIS.

A detailed evaluation of surface structural modification and nanostructuring due to DIS as a function of incident angle of Ti6Al4V samples is summarized in Figure 3.


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Figure 3. SEM images showing control surfaces (polished Ti and SLA type surface) and the evolution of surface nanopatterning of Ti6Al4V samples for different incidence angles under 1 keV Ar+ irradiation and 2.5 x 1017 cm-2 fluence. The bottom row consists of the higher magnification image of the region inside the marked black-dotted squares, indicated in the corresponding upper lower-magnification SEM images. S1_0° sample resulted in non-directional


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sub-micron structures between 0.05-0.1 m. S2_30° resulted in 10-20 nm thin nanowalls with 250500 nm length, S3_60° resulted in ripple structures of 20-nm width and 50-nm wavelength and S3_80° resulted in 50-100 nm wide and 0.5-1.5 m long pillar structures. Red and black arrows highlight the ion-beam direction and nanopatterning formation, respectively.

For ion-incident angles between 0°-30° it appears that the high plastic deformation resistance phase is correlated with resistance to DIS nanopatterning except at higher oblique angles (e.g. 6080°) in which both phases are modified as one can observe in Figure 7 about EBSD in SEM 2 image. Intrinsic to the DIS modification is its ability to only modify the first few 100’s of nm as shown in Figure SI_2 and SI_3 of Supporting Information and therefore not affect the optimized mechanical properties described above.37 These figures showed the projected range of ion damage is about 3-4 nm with a spread of ~2 nm, and under these conditions the damage is localized to be lower than

ab_2018_00879q Designing nanostructured Ti6Al4V bioactive

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