Multifunctional Protein-Immobilized Plasma Polymer Films for

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Multifunctional Protein-Immobilized Plasma Polymer Films for Orthopedic Applications Callum A. C. Stewart,†,‡,§ Behnam Akhavan,*,†,‡,∥ Juichien Hung,‡ Shisan Bao,§,⊥ Jun-Hyeog Jang,#,¶ Steven G. Wise,‡,⊥,¶ and Marcela M. M. Bilek*,†,§,∥,○,¶ †

School of Physics, University of Sydney, Physics Road, Camperdown, NSW 2006, Australia Heart Research Institute, 7 Eliza Street, Newtown, New South Wales 2042, Australia § Charles Perkins Centre, University of Sydney, Camperdown NSW 2006, Australia ∥ School of Aerospace Mechanical and Mechatronic Engineering, University of Sydney, Camperdown, NSW 2006, Australia ⊥ Sydney Medical School, University of Sydney, Camperdown, NSW 2006, Australia # School of Medicine, Inha University, Incheon 400−712, Korea ○ Sydney Nano Institute, The University of Sydney, Camperdown, NSW 2006, Australia

ACS Biomater. Sci. Eng. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 11/04/18. For personal use only.

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ABSTRACT: Osseointegration is essential for ensuring optimal functioning and longevity of orthopedic implants. In a significant number of patients, the body does not fully integrate with the orthopedic implant, which opens the potential for the formation of bacterial biofilms and adverse foreign body reactions. Protein-functionalization of the implant surfaces can reduce this potential by stimulating rapid cell attachment or bone formation. Ideally, a multifunctional protein surface should simultaneously stimulate cell attachment and bone formation for optimal osseointegration. In this study, we utilized primary mouse osteoblasts to examine the osteogenic potential of a multifunctional fusion protein, combining the fibronectin (FN) attachment and osteocalcin (OCN) bone signaling sequences, compared against that of the individual proteins. These three biomolecules were immobilized on radical-functionalized plasma polymer films (rPPFs) that covalently bond proteins through interactions with embedded radicals that migrate to the surface. The fusion protein was also compared to a coimmobilized ratio of FN:OCN prepared through a two-step sequential exposure to OCN solution followed by FN solution. The preparation and characterization overhead for the two protein surfaces was substantial when compared to the fusion protein functionalization process. Significantly greater osteoblast attachment and spreading were observed for the FN, FN:OCN, and fusion protein surfaces compared to titanium (p < 0.05), while the calcium deposition after 17 days showed a significant increase (p < 0.01) on the fusion protein surface alone. The greater osseointegration potential of the fusion protein surface compared to the single and coimmobilized protein surfaces is attributed to the homogeneous distribution of the attachment and signaling sequences. Overall, the fusion protein-coated rPPFs produced easily functionalizable and highly osteogenic surfaces with the potential to greatly improve the tissue integration of orthopedic implants. KEYWORDS: osseointegration, surface, multifunctional biointerface, bone implants, osteoblasts

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results in the encapsulation of the implant in fibrous tissue2,7,8 that often inhibits its function. Surface functionalization with biologically active molecules has the potential to address both problems. Antimicrobial molecules can be included on the surface to prevent biofilm formation;9,10 while ECM and signaling biomolecules can be used to promote rapid colonization by bone-forming cells. Osteogenic proteins that promote cell attachment and bone formation resemble the natural physiological environment

INTRODUCTION

Orthopedic medical devices, such as artificial knees and hips, are in ever-increasing demand with millions of operations performed annually. However, many operations experience postoperative complications due to the formation of a biofilm1 or fibrotic encapsulation of the implant2 and require revision surgeries to correct.3 Biofilms occur when bacteria colonize a surface and produce a bacterial extracellular matrix (ECM) that protects the colony and makes it resistant to antibiotics.4−6 Fibrotic encapsulation occurs when the body identifies the implant as foreign and activates the immune system, triggering a negative foreign body reaction (FBR) that © XXXX American Chemical Society

Received: August 13, 2018 Accepted: October 19, 2018

A

DOI: 10.1021/acsbiomaterials.8b00954 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering more closely and stimulate native tissue to win the “race for the surface”,11,12 outcompeting bacteria. Osteogenic proteinfunctionalization, therefore, has the potential to prevent both biofilm and foreign body reactions as well as providing functional integration of the implant, known as osseointegration. Biomolecule functionalization of orthopedic surfaces is a strategy being explored to facilitate functional bone integration. Previous works have shown that surface-immobilized proteins are more effective for osseointegration compared to freesolution injections.13,14 Protein functionalization of orthopedic surfaces has been performed through physical adsorption, covalent attachment using chemical linkers, and physical covalent immobilization. Physical adsorption relies on physical forces such as electrostatic and hydrophobic interactions to temporarily attach proteins on the target surface. Adsorption methods are easy to perform, but the protein molecules experience competitive adsorption−desorption when placed in the biological environment in a process often referred to as the Vroman effect.15−17 Permanent surface-immobilization of proteins has been achieved using chemical linkers, such as polydopamine (DOPA)18 and silanes,19,20 covalently anchored to the surface. However, wet-chemistry methods applied for covalent protein functionalization are typically time-consuming21−23 and produce side-reactions that reduce the immobilization efficiency and limit reproducibility.24 Dry, plasmabased methods, such as plasma immersion ion implantation and plasma polymerization, have been applied to modify surfaces for covalent attachment of biomolecules through embedded unpaired electrons or specific chemical functional groups, respectively.25 Although the capital cost of a plasmabased technology is higher than that of a wet-chemistry approach, this cost can be quickly compensated by substantially lower variable costs due to minimal usage of chemicals and the relative simplicity of the process. The proteins and other biomolecules investigated for increasing osseointegration can be separated into two main classes: (i) adhesive extracellular matrix (ECM) proteins such as collagen26,27 and fibronectin;28 and (ii) direct signaling proteins, such as bone morphogenic protein-2 (BMP2).21,22,29,30 Both classes have demonstrated significant increases in osseointegration in vitro and in vivo.27,29,31 ECM proteins are involved in the recruitment and establishment of osteoblasts and stem cells at the surface of the implant. Fibronectin and collagen contain the canonical RGD cell binding motif that interacts with a range of cell-surface integrins, including α5β1, α2β1, and αvβ3, to adhere the cells to the protein-functionalized surface.32 In this case, bone formation at the ECM-functionalized surface occurs through natural mineralization processes and is typically increased due to the greater cell coverage rather than enhanced mineralization activity.33,34 The signaling proteins, for example BMP-2,35 and other signaling molecules, such as Simvastatin,36−38 on the other hand, increase the rate of bone formation by accelerating the differentiation and mineralization of the adherent cells through the upregulation of osteogenic metabolic products, e.g., osteocalcin (OCN/BGlaP), osteopontin (OPN), and alkaline phosphatase (ALP).39−41 The single protein approach to biomolecule functionalization has shown some success, but a deeper understanding of the osseointegration process reveals that a multifunctional protein-coated surface is required. The synergistic combination of biological functionalities would allow for increased cellular

attachment in conjunction with more rapid bone formation, leading to greater osseointegration. The required multifunctionalization can be achieved via two approaches; (i) the immobilization of multiple proteins on a single surface through multiple exposures and/or exposure to protein cocktail solutions, or (ii) the immobilization of synthetic multifunctional biomolecules, known as fusion proteins.32,42 Fusion proteins are created synthetically to incorporate multiple peptide sequences responsible for various cellular interactions into a single protein. A few fusion biomolecules have been utilized for orthopedic applications such as a branched antibacterial/attachment protein,43 a composite attachment protein,44,45 and an attachment/signaling protein (fibronectin/ BMP-2 fusion).46 Previous work has proven the capacity for both adsorption and chemical covalent immobilization of peptide cocktails.35,47 The adsorption of multiple proteins or peptides will suffer from competitive adsorption−desorption resulting in compromised biomolecule layer integrity15 and potential protein denaturation, triggering an exaggerated immune response that leads to fibrotic encapsulation.48,49 Chemical covalent immobilization prevents loss of biomolecule layer integrity, but the chemistry involved in linker-mediated protein immobilization is complex and difficult to replicate consistently due to side reactions,24 making immobilization of protein cocktails challenging. Recently, plasma deposited polymer films containing embedded radicals (rPPFs) with robustness and resistance to delamination as required for implantable orthopedic devices, have been developed for titanium and other carbide forming substrates.50,51 The carbon-based interlayers formed under energetic ion bombardment have been shown to allow singlestep, reagent-free immobilization of biomolecules through embedded radicals.52−54 The rPPFs routinely demonstrate functional lifetimes up to 4.5 months postdeposition due to the reservoir of radicals contained within the films,52 exceeding the reported radical quantities for conventional PPFs deposited in the absence of enhanced ion-bombardment by 1−2 orders of magnitude.53,55,56 The single-step covalent attachment of proteins to rPPFs allows for the reproducible production of multifunctional protein surfaces via the immobilization of multiple proteins or synthetic multifunctional fusion proteins. A promising multifunctional protein, comprised of the osteocalcin (OCN) 22−49 amino acid (AA) c-terminal sequence and the fibronectin (FN) 9,10 cell attachment domains (FN9−10),57 has been shown to increase cellular attachment and promote cell differentiation and mineralization (compared to FN) on an ion-implanted spin-coated polystyrene film.39 Here we examine the attachment and spreading, proliferation, and mineralization of primary mouse osteoblasts (OBs) on rPPFs functionalized with the FN−OCN fusion protein, a FN:OCN multiprotein counterpart, and the single component proteins to comparatively investigate the potential of these various approaches to osseointegration. Our findings open up the possibility of fabricating a new class of multifunctional interfaces with improved tissue-implant integration for orthopedic applications.

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METHODS

Deposition of Radical-Functionalized Plasma Polymer Films (rPPFs). Ti-6Al-4 V foils (70 μm thickness, Firmetal, China) were cleaned via a nitric acid procedure and inserted into a custom-built B

DOI: 10.1021/acsbiomaterials.8b00954 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering plasma deposition chamber, as previously described.56 The chamber was pumped down to below 5 × 10−5 Torr via a screw (Ebara PVD250) and a turbomolecular (Edwards NEXT400) pump. The Ti foils were plasma cleaned (Ar flow rate = 40 standard cubic centimeters per minute (sccm)) for 10 min (RF input power = 75 W, substrate bias voltage = −500 V, pressure ∼70 mTorr). A gaseous mixture of acetylene (5 sccm), N2 (10 sccm), and Ar (15 sccm) was added via a showerhead-like ring at the top of the chamber. The deposition pressure was raised to 110 mTorr before setting the RF input power to 50 W. The substrate holder was biased to −500 V (pulse width = 20 μs, frequency = 3 kHz). The RF power supply was 13.56 MHz Eni OEM-6, and substrate pulsed bias voltages were delivered by RUP 6 pulse generator (GBS-Electronik). The rPPF was deposited for 2 min, giving a thickness of approximately 32 nm as measured by spectroscopic ellipsometry. Gas flow rates were individually controlled by an Allicat Scientific mass flow controller. The rPPFs were stored in atmosphere for use between 5 and 7 days. Fusion Protein Synthesis. The FN−OCN fusion protein was produced as previously described by Jang et al.39,42 The synthesis was initiated via polymerase chain reaction (PCR) amplification of the OCN and FN sequences. The OCN forward and reverse primers were 5′-TAGGAGCCCTCACACTCCTC-3 and 5′-CTGGAGAGGAGCAGAACTGG-3′, respectively. A restriction site was generated using the forward primer 5′-AACAGATCTTACCTGTATCAATGGCTGGGA-3, for BglII site, and the reverse primer 5′AATGGTACCGACCGGGCCGTAGAAGCGCCG-3′, for the KpnI site. The FN sequence was generated with the forward and reverse primers, 5′-GGTACCGGTCTTGATTCCCCAACTGG-3′ and 5′AAGCTTTGGTTTGTCAATTTCTGTTCGG-3′, respectively. PCR was conducted over 30 cycles; annealing at 55 °C for 1 min; extension at 72 °C for 1 min, and denaturation at 94 °C for 1 min. The amplified PCR products were digested using BglII, KpnI and KpnI, HindIII then ligated into pBAD-HisB vector (Invitrogen, Carlsbad, CA), giving rise to a pBAD-HisB-FN−OCN. TOP10 cells were cultured at 37 °C overnight in LB medium containing ampicillin after transformation. The bacteria were harvested and pelleted by centrifugation at 6000 g for 10 min, lysed in NaCl−Tris−EDTA (STE) buffer, and sonicated. The soluble extract was centrifuged at 13 000g for 2 × 10 min. The supernatant containing the fusion protein was then purified by passing through a chromatography column containing a nickel−nitrilotriacetic acid resin (Invitrogen, Carlsbad, CA) to 90% purity by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Protein Immobilization of rPPF Surfaces. All protein stocks were handled under sterile conditions in a biological safety cabinet (BSC) to reduce potential contamination for enzyme-linked immunosorbent assays (ELISAs) and cellular assays. The fibronectin (FN) (purity >85%, F2006-Sigma-Aldrich), osteocalcin (OCN) (purity >94%, O5761 Sigma-Aldrich), and the FN−OCN fusion protein were diluted to the required concentrations in sterile PBS (Sigma-Aldrich) at physiological pH from premade protein stock solutions of100 μg/mL (1.7 × 10−6 M), for OCN, 100 μg/mL (2.3 × 10−7 M) or 500 μg/mL (1.1 × 10−6 M) for FN, and 1000 μg/mL (2.5 × 10−5 M) for fusion protein. The rPPF foils were sterilized using UV light. The protein solutions were placed on the surfaces of the foils at the desired concentrations and allowed to covalently bind overnight at 4 °C. The coimmobilized ratio of FN:OCN was deposited in a twostep exposure. The specified concentrations corresponding to the proportional fraction of the monolayer concentration for OCN were applied to the rPPF surfaces for 1 h at room temperature. The surfaces were gently rinsed once with PBS and exposed to a 15 μg/mL FN solution overnight at 4 °C. The protein-functionalized rPPF surfaces were used within 24 h. Surface Characterization. The surface chemistry, surface energy, and radical density of the rPPF surface were examined via X-ray Photoelectron Spectroscopy (XPS), water and diiodomethane contact angle measurements, and electron paramagnetic resonance (EPR) spectroscopy, repsectively. XPS was performed using a SPECS (FlexMode) spectrometer equipped with a monochromatic Al Kα (hν = 1486.7 eV) radiation source, a hemispherical analyzer

(PHOIBOS 150), and an MCD9 electron detector. The radiation source operated at 200 W (10 kV and 20 mA), and the electron takeoff angle was 90° relative to the sample surface. Measurements were performed at pressures below 5.0 × 10−8 mbar. The survey spectra were collected in an energy range of 0−1000 eV at a pass energy of 30 eV and a resolution of 0.5 eV. High-resolution (0.1 eV) C 1s spectra were collected at a pass energy of 20 eV. The CasaXPS software (version 2.3.18PR1.0) was used for spectral analysis. The surface energy of the rPPF was examined using a Krüss DS10 analyzer equipped with a CCD camera. The contact angles of water (polar) and diiodomethane (nonpolar) were measured following the sessile-drop method. The contact angles were extracted from the captured images using the Krüss drop shape analysis software (version 1.90.0.11) and averaged over multiple readings (n = 5). The total, dispersive, and polar surface energies were then calculated by the software using the Owens−Wendt−Rabel−Kaelble (OWRK) model. EPR was performed with a Bruker EMXplus Xband to evaluate the radical functionalization of the rPPF coating. Polystyrene films (7 cm × 7 cm) were coated with rPPF and rolled into borosilicate glass NMR tubes (Wilmad). The spectrometer was calibrated using a weak pitch sample (∼1 × 1013 spins/cm). The spectra were recorded with a central magnetic field of 3510 G, modulation amplitude of 3 G, microwave frequency of 9.8 GHz, and power of 0.025 W. The resonator detection cavity had a minimum signal-to-noise ratio is 400:1. The field modulation frequency was 1 × 105 Hz and the sampling time was 85 ms/G. Ten scans were averaged per sample. Enzyme-Linked Immunosorbent Assays. Enzyme-linked immunosorbent assays (ELISAs) were used to quantify the surface concentrations of the respective proteins. To demonstrate covalent immobilization, the substrates were washed in 1 mL of 5% sodium dodecyl sulfate (SDS) (w/v) for 15 min at 90 °C before undergoing quantification. These values were compared to non-SDS washed samples. A FN and OCN rabbit-antihuman primary (F3648, SigmaAldrich and Ab093876, Abcam) were used at 1:5000 and 1:2000 dilutions, respectively. A goat-antirabbit secondary antibody (ab6721, Abcam) was used at 1:20,000 and 1:10,000 dilutions, respectively. The ELISAs were exposed to 1-Step Ultra TMB - ELISA Substrate (cat. 34028B, Thermofisher Scientific) for 30 min before 0.2 M sulfuric acid was added to stop the reaction. The 150 μL aliquots were measured for absorbance signal at 450 nm. Primary Osteoblast Harvesting and Culturing. Primary osteoblasts were collected from 7-week old C57BL/6 mice as per the standard protocol.58 Experiments were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. All personnel involved in the animal procedures have completed an approved animal care and ethics course. Mice long bones were retrieved from sacrificed mice, cleaned, and the marrow flushed out with buffer. The bone shards were incubated in low glucose DMEM media containing 20% fetal bovine serum (FBS) and 1% Penicillin-streptomycin to allow for cell outgrowth before trypsinization and culturing. The cell cultures were maintained at 37 °C in 5% CO2 atmosphere with 50 μg/mL ascorbate added to the culture media to promote osteoblast differentiation. The osteoblast cultures were monitored through visual examination of the cellular morphology and staining with alizarin red. Any subcultures that indicated ingrowth of other cell types were discarded. Cellular Attachment Assays. Cellular attachment and spreading were examined after 1 h via fluorescent microscopy. Titanium and rPPF substrates were cut to 0.8 cm × 1 cm and sterilized under UV light before being transferred to an eight-well multichannel slide (Nunc154534, Lifetechnologies). The primary osteoblasts (OBs) were seeded onto bare Ti, rPPF, and protein functionalized surfaces at the seeding quantity of 10,000 cells. After 60 min, the seeding media was removed and the substrates fixed with 70% (v/w) ethanol. The cells were stained with actinRED555 (Thermofisher) and mounted with DAPI fluorescent mounting media (Agilent) under dark conditions. The samples were examined with a Zeiss Axio Imager.Z2 fluorescence microscope at the corresponding wavelengths. All surfaces were examined under 5× magnification (≈4 mm2) with a C

DOI: 10.1021/acsbiomaterials.8b00954 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Figure 1. Characterization of the rPPF surface. (a) The elemental composition was 71 ± 4% carbon, 20 ± 1% nitrogen, and 9 ± 1% oxygen as calculated from the XPS survey spectrum. (b) The high resolution C 1s spectrum was curve-fitted with components at binding energies (284.6 ± 0.5 eV (C−C/C−H), 286.5 ± 0.5 eV (C−O/C−N), 287.5 ± 0.5 eV (C = O/N−C = O), and 289 ± 0.5 eV (COOH). (c) A representative image of the water drop placed on the rPPF-coated Ti showed a water contact angle (WCA) of 59.4°. (d) A representative EPR spectrum of the rPPF was deposited on polystyrene. Signal broadness originates from a variety of carbon-centered radicals and/or anisotropic signal from surface radicals.

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minimum of nine images per surface. The images were separated into their component colors and analyzed with “ImageJ” software for cell size and quantity. The background signal was removed by applying the minimum area restrictions of 20 pixels and 50 pixels to the DAPI and actinRED555 images, respectively. Cellular Proliferation Assay. The 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) end point assay was used to measure the proliferation of osteoblasts. Osteoblasts were seeded onto the bare Ti, rPPF, and proteinfunctionalized surfaces in triplicate at 4000 cells/well and media replaced every 3 days. The OBs were examined at 3 and 14 days post seeding. Samples were incubated in 200 μL of 20% MTS in DMEM for 2 h under dark conditions. After the allotted time, a 100 μL aliquot was transferred to a 96 well plate and the absorbance measured at 490 nm. MTS was used for the primary osteoblasts due to the slow doubling time of the cells and the subsequent need to detect subtle proliferation differences. Osteoblast Mineralization. The mineralizing capacity of the different protein conditions was examined using a 40 mM alizarin red stain (ARS), which binds to calcium. The Ti, rPPF, and protein functionalized substrates were prepared as described and primary osteoblasts were seeded at 10,000 cells per well (confluence). The media was replaced every 3 days. At day 17 post seeding, the media was removed, and the respective samples were stained with freshly prepared ARS for 2 h. The excess ARS was removed, and the samples washed gently before being placed into individual Eppendorf tubes. 400 μL of 10% (v/v) acetic acid was added before vortexing and heating to 85 °C for 10 min. The samples were then centrifuged at 16 000g for 15 min, and 50 μL of ammonia hydroxide was added to each sample to neutralize the solution. The 200 μL aliquots were transferred to a 96 well plate, and the absorbance was read at 405 nm. Statistical Analysis. Statistical analysis of the cell studies was performed with Microsoft Excel using the two-tailed student’s t test assuming equal variance to calculate statistical significance. The calculated p values were determined significant if p ≤ 0.05 and highly significant if p ≤ 0.01.

RESULTS Characterization of rPPF Coating. The rPPFs were left to age for 5−7 days before protein functionalization was performed and have been previously characterized.59 The surface chemistry was analyzed using XPS, and the survey spectrum is shown in Figure 1.a. The atomic composition was 71 ± 4% carbon, 20 ± 1% nitrogen, and 9 ± 1% oxygen. The large concentration of carbon is typical of amorphous carbon deposited from monomers, such as acetylene, that have been highly cross-linked through ion bombardment. The nitrogen was incorporated into the gas mixture to promote sp2 bonding and relieve internal stresses.60,61 The presence of 9 ± 1% of oxygen suggests that the rPPF has undergone autoxidation reactions between carbon-centered radicals and atmospheric oxygen.62−64 The C 1s high-resolution peak was curve-fitted with binding energies corresponding to C−C/C−H (284.6 ± 0.5 eV), C−O/C−N (286.5 ± 0.5 eV), CO/N−CO (287.5 ± 0.5 eV), and COOH (289 ± 0.5 eV).56,65,66 The peak fitting further illustrates autoxidation with the presence of single and double bonded carbon−oxygen groups (blue and magenta curves in Figure 1.b, respectively) and the minor formation of carboxylic acid functionalities (cyan peak at 289 ± 0.5 eV). The organic polar groups formed as autoxidative products are known to be beneficial for cellular attachment and proliferation.67 Unlike for chemical-functionalized PPFs, the formation of autoxidative products has minimal effect on the protein immobilization potential of the rPPFs.51−54 The water contact angle (WCA) of the rPPF surface, measured 1 week after deposition, was approximately 59.4° (Figure 1.c) and the diiodomethane contact angle was 31.4°. The total surface energy was 56.2 mJ.m−2 with dispersive and polar components of 43.6 and 12.6 mJ.m−2, respectively.59 The hydrophilic nature of the rPPF is derived from the free energy of radicals and polar oxidative products formed by exposure to atmospheric oxygen.51,53,68 The WCA shows the surface is mildly hydrophilic (WCA < 90)69,70 and allows proteins to D

DOI: 10.1021/acsbiomaterials.8b00954 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 2. ELISA detection of fibronectin (FN), osteocalcin (OCN), and fusion (FN−OCN) proteins on radical-functionalized plasma polymer surfaces. The ELISA absorbance signals as a function of protein solution concentration are shown for fibronectin (a), osteocalcin (b), and the FN− OCN fusion protein (c). The signal saturates at around 15 μg/mL, indicating that a surface monolayer has been achieved. The post-SDS washing retention of the three proteins (d) shows the degree of covalent protein attachment. Significant quantities of the protein layer, ranging from 60− 80%, were detected on the rPPF after washing, while up to only 15% was retained on Ti, indicative of covalent protein bonding on rPPFs. ELISA signals for fibronectin and osteocalcin coimmobilized on the rPPFs (e) show that both proteins are covalently immobilized. The ratios shown indicate the proportion of a monolayer expected according to the titration curves shown in parts a and b for the solution concentrations used.

OCN) (40 kDa) proteins were immobilized on the surfaces of rPPFs through reactions with the surface-embedded radicals. The protein coverage and the degree of covalent bonding were investigated via protein-specific ELISA assays. A titration curve for each protein was constructed in the range of 0−30 μg/mL to identify the solution concentration required to immobilize a monolayer (Figure 2a−c). A concentration of 15 μg/mL was selected for the FN (3.4 × 10−8 M), OCN (2.5 × 10−6 M), and the fusion (3.8 × 10−7 M) proteins as the subsequent data points were equal within the uncertainty, indicating the formation of a monolayer. The degree of covalent immobilization of each protein was determined using SDS washing. SDS is a detergent that disrupts all physical interactions, leaving only covalent bonds intact.74−76 The SDS washing step demonstrated covalent immobilization of the three proteins on rPPFs and near complete removal of the noncovalently bound proteins on Ti

retain a native conformation and consequently their biological activity.71 The 59.4° WCA places the rPPF within the optimal wettability range for bone lineage cell attachment.72 The unpaired electrons representing radicals contained in the rPPF were detected via EPR (Figure 1d). The spectrum contains a symmetrical peak centered at a g-value of 2.003, indicating the presence of surface-embedded radicals. The broadness of the peaks indicates a variety of carbon-centered radicals and/or signal anisotropy due to the radicals appearing at the rPPF surface.53,68,73 The rPPFs have been shown to possess radical densities 1−2 orders of magnitude greater than conventional PPFs deposited in the absence of enhanced ionbombardment,53,55,56 and this enables the covalent immobilization of a monolayer of biomolecules directly to the surface.51,59 Protein Functionalization of rPPFs. Fibronectin (FN) (440 kDa), osteocalcin (OCN) (5.9 kDa), and fusion (FN− E

DOI: 10.1021/acsbiomaterials.8b00954 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering (Figure 2d). The greatest protein retention was found on the rPPF surfaces with 79 ± 7%, 62 ± 8%, and 73 ± 13% of the adsorbed FN, OCN, and fusion layers retained, respectively. In contrast, Ti retained only 15 ± 6%, 0 ± 5%, and 5 ± 7% of the adsorbed layers, respectively. The higher retention of proteins on rPPF surfaces after SDS washing agrees with previous publications.53,77 A sequential 2-step exposure was used to coimmobilize FN and OCN in various ratios. The solution concentrations used were determined according to the desired monolayer fraction as per Figures 2, parts a and b. A series of ratios were examined pre- and post- SDS washing with the FN and OCN ELISAs, as shown in Figure 2e. The 100% protein surfaces show similar post-SDS proportional retentions as in Figure 2d, indicative of covalent immobilization. The same conditions resulted in near complete removal of the corresponding protein layers on Ti surfaces. When coimmobilized, the signals of OCN and FN were decreased compared to their 100% controls, most noticeably for OCN prior to SDS washing. The transition from a 100% to 90% OCN surface with FN produced a signal reduction resembling SDS washing due to competitive adsorption−desorption of OCN. The OCN immobilization follows the expected trends, while the FN shows minimal variation across the given ratios. The 2-step coimmobilization approach accounts for the reduction in ELISA signal from competitive adsorption−desorption and the titration trend for the OCN, but not for the FN behavior, suggesting the FN could bond in a different orientation or conformation due to the change in surface properties after OCN immobilization. Ultimately, the ratio of 1:3 FN:OCN was selected for cellular studies as it provided an optimized signal from both ELISAs, while expressing a substantial difference in surface protein composition. Osteoblast Attachment. Osteoblast attachment and spreading was analyzed via fluorescence microscopy. The average cell numbers, shown in Figure 3.a, revealed that the FN−OCN fusion protein-coated surfaces had the highest cell average of 174 ± 35 cells/field of view, followed by the FN and FN:OCN coated surfaces, 157 ± 35 and 149 ± 38, respectively. All three demonstrate cell attachment significantly greater than the 73 ± 25 cells/field of view observed for the uncoated titanium. The OCN and rPPF surfaces had 78 ± 30 and 97 ± 25 cells/field of view, respectively, showing no significant increase over Ti. The average cell size of the OBs (Figure 3.b) on the FN, FN:OCN, and fusion protein-coated surfaces were 2664 ± 421, 2569 ± 529, and 1999 ± 278 μm2, respectively, showing a significant increase in spreading over cells on Ti alone (912 ± 203 μm2). The rPPF surfaces also induced a significant increase in cell spreading (2213 ± 331 μm2); while OCN (1214 ± 345 μm2) produced a slight, but not significant, increase in size compared to Ti. The cell population and spreading for each surface can be observed in the corresponding representative fluorescence images in Figure 3c. The increased cell number and spreading on the FN, FN:OCN, and fusion surfaces is attributed to the presence of the RGD binding sequence in the FN9−10 domains. The RGD sequence is one of the primary binding sequences in ECM proteins responsible for cellular attachment.43,44 Both multifunctionalized protein-coated surfaces demonstrated cell attachment and spreading on par with the FN-functionalized surface, indicating that the RGD sequence was well presented and accessible to the cells. OCN and rPPF surfaces provided

Figure 3. Average osteoblast cell quantity (a) and cell size (b) after 1 h incubation as determined by fluorescence cell staining with DAPI/ ActinRED. Uncertainties are determined from the standard deviation between samples (n = 9). (c) Representative 10× magnification fluorescence microscope images of osteoblast cell attachment on bare titanium, rPPF coatings, fibronectin-functionalized (FN), osteocalcinfunctionalized (OCN), FN−OCN fusion protein-functionalized, and FN:OCN ratio-functionalized surfaces. The cell viability was determined by staining with DAPI (blue, nuclei), and cell spreading was determined by staining with ActinRED (red, cytoskeletons).

equivalent cell attachment to Ti as they do not possess binding motifs. The cell spreading was significantly increased on the rPPF compared to Ti, possibly due to the presence of nitrogenand oxygen-containing carbon groups at the interface as observed from XPS data (Figure 1, parts a and b).78 An examination of the overall cellular affinity for the surfaces agrees with the expected trends: the RGD containing surfaces (the fusion, FN, and FN:OCN) are favored most for cell attachment and spreading, followed by the organic surfaces (rPPF and OCN), and the inorganic Ti substrate being least favored; i.e., RDG motif (FN, FN:OCN, fusion) > organic nonbinding (OCN, rPPF) > inorganic (Ti). Proliferation. Osteoblast (OB) proliferation on the protein-functionalized rPPFs were measured at day 3 and day 14 post seeding via MTS (Figure 4). On day 3, the Ti (0.87 ± 0.04), FN (1.00 ± 0.21), and FN:OCN (0.96 ± 0.18) surfaces demonstrated elevated absorbance compared to the rPPF (0.74 ± 0.01), OCN (0.74 ± 0.04), and fusion (0.78 ± 0.07) protein-functionalized surfaces. The day 14 MTS F

DOI: 10.1021/acsbiomaterials.8b00954 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Figure 4. MTS proliferation signal for osteoblasts on Ti, rPPF, fibronectin (FN), FN:OCN ratio, osteocalcin (OCN), and FN−OCN fusion protein-coated surfaces at day 3 and day 14 post seeding. Analysis of the growth ratios showed no difference between the surfaces. No significant difference in cell proliferation was observed between examined surfaces on day 3 with the exception of Ti compared to rPPF (p < 0.01, σ) and OCN (p < 0.05, ∗). On day 14, no protein-functionalized surfaces demonstrated significant differences compared to either Ti or rPPF.

Figure 5. Alizarin Red mineralization of primary osteoblasts on Ti, rPPF, fibronectin (FN), osteocalcin (OCN), FN:OCN ratio, and FN−OCN fusion protein-coated surfaces at day 17. The calcium deposition on the fusion-coated surfaces was significantly greater than that on rPPF and OCN surfaces (p < 0.05, ∗), as well as Ti (p < 0.01, #).

Overall, the fusion protein demonstrated the greatest mineralization potential in the short time frame investigated, indicating the significant benefits of its application in orthopedic implants.

absorbances demonstrate equal proliferation on the Ti (1.20 ± 0.04) and rPPF (1.19 ± 0.05), with the FN (1.32 ± 0.09) and FN:OCN (1.33 ± 0.13) surfaces remaining slightly elevated. The OCN (1.10 ± 0.14) and fusion (1.01 ± 0.11) proteinfunctionalized surfaces produced less proliferation signal relative to the rPPF and Ti. The osteoblast growth rates showed insignificant variations across all samples. The OB proliferation is correlated with the extent of cell attachment and spreading. We see the general trend of the FNcontaining surfaces producing more MTS signal than the OCN and rPPF based on the elevated cell numbers at the attachment stage (Figure 3a,b), as the proliferation rates are similar. Overall, the Ti, rPPF, and the protein-functionalized surfaces supported the attachment, spreading, and proliferation of osteoblasts. Mineralization. The mineralization potency of the multifunctional surfaces, as determined by calcium content, was probed at day 17 with Alizarin Red stain (ARS) (Figure 5). The fusion protein-coated surface produced a significant increase in mineralization compared to the rPPF and OCN coated surfaces (p < 0.05) and a highly significant increase when compared with Ti (p < 0.01). A visual examination of the surfaces on day 14 showed the formation of mineralized nodules on the OCN and fusion protein-coated surfaces, but were absent on the Ti, rPPF, and other protein-coated rPPF surfaces. The mineralization observed on the single protein-functionalized surfaces is derived from the increased cell quantity for the FN and the enhanced mineralization rate due to OCN signaling. The two multifunctional surfaces, FN:OCN and fusion, presented different mineralization behaviors related to the nature of the multifunctionalization. The FN:OCN ratio surfaces did not demonstrate an enhancement beyond the single protein surfaces, potentially due to the inhomogeneous presentation of the two proteins. In contrast, the fusion protein-coated surfaces exceeded the mineralization observed on the other surfaces as the protein is designed to uniformly present both signaling and attachment motifs by combining the adhesive FN9−10 domain with the signaling capability of the OCN (22−49 amino acid sequence) in a single molecule.39,42

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DISCUSSION The functionalization of orthopedic implants with biological cues for rapid osseointegration is a promising approach to reduce the postoperative complications associated with fibrotic encapsulation and biofilm formation.2,59 This biofunctionalization strategy requires the development of easily reproducible, multifunctional surfaces that encourage rapid cellular attachment and accelerate mineralization. Protein and biomolecule immobilization represents one direction for producing multifunctional orthopedic interfaces through the coimmobilization of multiple proteins. Alternatively, the benefits may be more practically achieved through the utilization of synthetic multifunctional biomolecules. In this study, we compared the osteogenic potential of a surface with coimmobilized fibronectin and osteocalcin (1:3 FN:OCN) with that of a multifunctional fusion protein surface. Both protein surfaces utilized radical-functionalized plasma polymer films (rPPFs) to achieve direct covalent bonding of the proteins to the Ti surfaces. Overall, the FN−OCN fusion protein surfaces are better candidates for biofunctionalization of orthopedic implants than the coimmobilized FN:OCN surfaces due to their simpler immobilization process, easier ELISA quantification, and greater osteogenic potency. The quantity of adsorbed proteins to the Ti and rPPF surfaces, via single or sequential exposures, is determined by a competitive adsorption−desorption equilibrium.16,79,80 Proteins adsorb to the exposed surface based on hydrophobic and electrostatic interactions, and become displaced by incoming proteins through transitional complexes with the rearrangement driven by solution partial pressure and surface affinity.15−17 The effective monolayer for surfaces occurs when the adsorption−desorption dynamic reaches equilibrium. The protein adsorption−desorption equilibrium on rPPFs is influenced by the probability of covalent bond formation between a surface migrating radical and the adsorbed proteins, thus preventing desorption. The competitive exchange of G

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Figure 6. Visual representation of protein equilibrium on Ti and rPPF surfaces. The proteins initially adsorb onto the Ti and rPPF surfaces. The adsorbed surface proteins will undergo competitive adsorption−desorption dynamics as the protein solution reaches equilibrium. The adsorbed proteins on rPPF surfaces form covalent bonds with the surface migrating radicals preventing desorption and leading to greater surface protein retention.

sequence presentation reduce the reliability of ELISA detection for the quantification of immobilized protein ratios. The examination of protein ratios is a time- and resourceintensive process. The quantification of multiprotein surfaces via ELISA requires the examination of each protein ratio with all the required ELISA systems to determine the concentrations of the individual proteins. Thus, the samples required increase exponentially as more different proteins are included onto the surface. In addition to the signal discrepancies from protein reorientation or refolding, the ELISA technique can also introduce effective area discrepancies related to the size differences between the proteins and the antibodies (Igs). The ELISA signal produced is considered representative of the protein quantity if the target protein is larger than the primary and secondary antibodies, e.g., fibronectin. However, smaller proteins and peptides can experience an ELISA detection limit based on steric hindrance due to the large footprint of the antibodies, which understates the surface protein quantity. In contrast, the multifunctionalization of an implant surface with fusion proteins reduces the number of samples required for characterization of an immobilization protocol and increases the ELISA accuracy. For example, the combination of the FN9−10 and OCN C-terminal sequences in a single protein allows for protein quantification via a single ELISA using an antibody targeting either of the sequences. The fusion protein in this study is reported to be approximately 40 kDa,39,42 which reduces the potential for misrepresentation via ELISA antibody steric hindrance. Overall, the utilization of the fusion protein allows for simpler and more accurate ELISA quantification. The most critical component of both multifunctionalization approaches is the presentation of the desired motifs to the biological environment. The primary osteoblasts demonstrated that both multifunctional surfaces presented the RGD sequence by the observed increase in cellular attachment and spreading (Figure 3). However, a significant increase in mineralization compared to the bare titanium was observed only on the fusion protein surfaces (Figure 5). This suggests

proteins will continue but with a reducing effective surface area, as depicted in Figure 6. This mechanism accounts for the observed increase in protein specific signal from the rPPFs compared to the Ti, and the significantly different protein retention values (Figure 2.d).81 The differences in mobilities and binding affinities between proteins make it difficult to translate the single protein titration results into a cocktail solution yielding the desired surface coverage ratios post immobilization.16 Thus, the coimmobilization of fibronectin and osteocalcin was performed by sequential protein solution exposures to allow for greater control of the competitive adsorption−desorption dynamics and surface composition. In principle, the covalently bound proportions of OCN would reduce the effective area, limiting the available sites for FN immobilization at the surface. The rPPF surfaces must have sufficient protein exposure time to ensure the optimal covalent attachment. The osteocalcin, immobilized in various ratios, reproduced a concentration behavior consistent with the titration curve, as expected for the first incubated protein solution. During the subsequent incubation, the FN bonded to the available surface and displaced the noncovalently bonded OCN.15−17 This mechanism explains why the ELISA signal was equivalent to that obtained post-SDS washing in the initial OCN retention experiments (Figure 2e). The observed fibronectin signals plateau across all protein concentration ratios, both pre- and post SDS washing. This behavior contradicts the trend observed from the FN titration curve (Figure 2a). The uniform signal strength suggests a change in FN adsorption dynamics that could invoke a protein reorientation or refolding in response to the partially OCN-functionalized rPPF surfaces. A reorientation of the bipolypeptide FN structure (2 × 220 kDa polypeptide units)82 could potentially double the availability of the primary antibody targeted genetic sequence presented in solution. The ELISA signal would then increase by a fraction equivalent to the proportion of reoriented FN present (Figure 2e). Such unexpected increases in target H

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that the OCN signaling sequence in the fusion protein is freely available to the biological environment, whereas, the OCN proteins on the FN:OCN surfaces are inefficiently presented, whether through obstruction by the FN or below a critical concentration. Similar observations of the effectiveness of coimmobilized vs fusion protein surfaces were noted in other biomolecule functionalization work,44,45 where a composite attachment fusion peptide outperformed the 50:50 mixture of the component peptides on the surface. The compromised performance was attributed to the inhomogeneity of the surface peptide layer.44,45 The FN:OCN ratio selected in our study is a close approximation to the 50:50 surface ratio but with a more inhomogeneous surface coverage due to the significant size difference between the FN (440 kDa) and the OCN (5.9 kDa) proteins. In contrast, the FN−OCN fusion protein surfaces presented the required densities of both the RGD cell binding sequence and the OCN signaling sequence homogeneously across the surface, resulting in significantly greater osteogenic potency. Overall, the multifunctional fusion protein coated rPPF surfaces presented the most significant potential for osseointegrating orthopedic implants based on the simplicity of the protein functionalization, the ease of quantification by ELISA, and the homogeneous availability of cell adhesive RGD and mineralization signaling OCN sequences.

The authors declare no competing financial interest.

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CONCLUSIONS The demand for orthopedic implants is increasing annually, and despite best efforts, biofilm formation and fibrotic encapsulation still represent a significant cause of revision surgeries. In this work, we investigated the osteogenic potential of an FN−OCN fusion protein in comparison with a 1:3 fibronectin (FN):osteocalcin (OCN) coimmobilized protein surface and the individual proteins alone immobilized on radical functionalized plasma polymer films (rPPFs). The preparation and characterization overhead for the ratio surface was substantial when compared to the fusion protein functionalization process. The FN, fusion protein, and FN:OCN ratio protein surfaces all demonstrated significant increases in cellular attachment compared to the bare titanium and rPPF surfaces, but only the fusion protein surfaces demonstrated significant increases of mineralization over titanium alone. The significantly increased osteogenic potential of the fusion protein is believed to be derived from the homogeneous availability of both the RGD attachment and OCN signaling sequences.44−46 Our findings indicated that the FN−OCN fusion-protein coated rPPF surfaces hold significant potential for improving the osseointegration of implantable orthopedic devices.

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AUTHOR INFORMATION

Corresponding Authors

*(M.M.M.B.) Telephone: +61293516079. Fax: +61 2 93517726. E-mail: [email protected]. *(B.A.) E-mail: [email protected]. ORCID

Behnam Akhavan: 0000-0002-1599-658X Steven G. Wise: 0000-0001-7964-819X Marcela M. M. Bilek: 0000-0003-3363-2664 Author Contributions ¶

Equal senior authors I

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DOI: 10.1021/acsbiomaterials.8b00954 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX