Osteoconductive Enhancement of Polyether Ether Ketone: A Mild

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Osteoconductive Enhancement of Polyether Ether Ketone: A Mild Covalent Surface Modification Approach Andrew Kassick, Saigopalakrishna Saileelaprasad Yerneni, Eric Gottlieb, Francis Cartieri, Yushuan Peng, Gordon Mao, Alexander Kharlamov, Mark C Miller, Chen Xu, Michael Oh, Tomasz Kowalewski, Boyle Cheng, Phil Gordon Campbell, and Saadyah E. Averick ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00274 • Publication Date (Web): 29 Aug 2018 Downloaded from http://pubs.acs.org on September 4, 2018

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ACS Applied Bio Materials

Osteoconductive Enhancement of Polyether Ether Ketone: A Mild Covalent Surface Modification Approach Andrew J. Kassick,a,b Saigopalakrishna S. Yerneni,c Eric Gottlieb,d Francis Cartieri,e Yushuan Peng,c Gordon Mao,b Alexander Kharlamov,f Mark C. Miller,f,h Chen Xu,b Michael Oh,b Tomasz Kowalewski,d Boyle Cheng,b Phil G. Campbell,g and Saadyah Avericka,b* a

Neuroscience Disruptive Research Lab, Allegheny Health Network Research Institute, Allegheny General Hospital,

Pittsburgh, PA 15212, USA. b

Neuroscience Institute, Allegheny Health Network, Allegheny General Hospital, Pittsburgh, PA 15212, USA.

c

Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15217, USA.

d

Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15217, USA.

e

Department of Surgery Allegheny Health Network, West Penn Hospital, Pittsburgh, Pennsylvania 15212, USA.

f

Department of Orthopedic Surgery, Allegheny General Hospital, Pittsburgh, Pennsylvania 15212, USA.

g

Department of Biomedical Engineering and Engineering Research Accelerator, Carnegie Mellon University,

Pittsburgh, PA 15217, USA. h

Departments of Mechanical Engineering and Materials Science & Bioengineering, University of Pittsburgh,

Pittsburgh, PA 15213, USA.

ABSTRACT Polyether ether ketone (PEEK, 1) is an important material for the fabrication of implants employed in spinal fusion surgery. While its radiolucency and favorable elastic modulus have made PEEK an attractive choice for interbody fusion devices, its poor osseointegrative properties prevent the formation of a strong union between implant and surrounding bone structures and remain a major liability. Recent advancements in PEEK surface technology have resulted in improved osseointegration; however, the identification of an ideal implant material has proven challenging. In this manuscript, we describe our preliminary investigation into the realm of 1 ACS Paragon Plus Environment

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PEEK-based fusion devices that has culminated in the discovery of a mild, solution-based process for the preparation of covalently surface modified PEEK biomaterials that display enhanced osteoconductive properties.

Surface modification occurred under mild reaction

conditions via the acid-mediated addition of various commercially available hydrophilic oxyamine and hydrazine nucleophiles to the diaryl ketone moiety of PEEK. The resulting modified surfaces have been confirmed by contact angle measurements and X-ray photoelectron spectroscopy (XPS).

Subsequent in vitro studies demonstrated the enhanced capability of

several modified PEEK variants to promote osteogenic differentiation and mineralized calcium deposition relative to unmodified PEEK surfaces. Keywords: PEEK, Osteoconductivity, Surface Modification, Bone Ingrowth, Polymer Biomaterial

INTRODUCTION Spinal arthrodesis, more commonly known as spinal fusion, is a valuable surgical procedure for the treatment of conservatively unmanageable pain resulting from various medical conditions of the lower back and neck including degenerative disc disease, spinal stenosis, degenerative spondylolisthesis, and trauma.1-5 The process involves the removal of a damaged intervertebral disc and subsequent installation of a fixative device (e.g. interbody spacer, cage, etc.) to promote the union of vertebrae through osseointegration of new bone tissue with the implant surface. The resulting restriction of vertebral motion serves to alleviate the pressure on surrounding nerves thus reducing pain and enhancing the overall spinal functionality of the patient. Modern fusion devices are most commonly fabricated from titanium metal alloys or the synthetic thermoplastic polymer, polyether ether ketone (PEEK, 1). Historically, titanium-based

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devices have demonstrated a superior level of osseointegration and resultant bone fusion relative to other implant materials; an advantage that has been attributed to the hydrophilic nature of the metal surface which provides a favorable contact area for osteoblast adhesion, growth, and differentiation.6,7

While titanium-based appliances are currently among the most widely

employed implants for spinal fusion procedures, they still present several limitations. The characteristic radiopacity exhibited by titanium devices can lead to challenges in the postoperative imaging of implant sites which is crucial to evaluating the fusion process. Titanium-based implants also possess a much higher elasticity modulus relative to bone which has been speculated to result in subsidence and an increased probability of device destabilization and failure.8,9 PEEK, on the other hand, exhibits an elasticity modulus which more closely resembles that of natural bone while also being radiolucent, thus overcoming the principal liabilities associated with titanium-based fusion devices.8,

10-12

However, due to its intrinsic

hydrophobicity, PEEK is not conducive to osteogenic differentiation and does not readily form a strong fusion with surrounding bone structures. These poor osseointegrative properties lead to longer required times for spinal fusion and thus a greater risk of device failure. An ideal fusion implant material would be capable of successfully promoting osseointegration while also matching the elastic modulus of bone and retaining radiotransparency. Currently, the primary mechanisms for increasing PEEK’s ability to promote osteoblast growth require extensive surface modification. Recent advances in PEEK surface technology involve the incorporation of pores and channels into the polymer via acid-mediated etching or leaching techniques that provide a dramatically increased surface area toward creating a more interlocking interface between existing bone and the implant.13-19 These porous PEEK fusion devices have demonstrated a marked enhancement in the level of cell adhesion, proliferation, and



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mineralization relative to the corresponding smooth PEEK appliances although the preparation of their porous topographies typically requires harsh chemicals along with high temperatures and pressures.13,15,18 Coating the surface of PEEK with a layer of metal such as titanium or other inorganic minerals such as hydroxyapatite to improve its overall hydrophilicity has also been investigated.19-23 The major liability to such a strategy is that spray coatings and composites have been shown to delaminate, potentially leading to inflammation or infection at the fusion site and subsequent device failure.24-26 Additionally, coating all surfaces of a PEEK-based medical device with separate materials, especially titanium, is not operationally simple and can lead to a significant added cost to the manufacture of the device while also resulting in low throughput due to complex device geometries.27 Direct chemical modification of the polymer backbone of PEEK has also been explored.28-32 Of the examples that have been reported, most rely on harsh chemical treatment or multiple synthetic steps to achieve hydrophilic surface functionalization and enhanced osseointegration. Thus, a need exists for improved osteoconductive hydrophilic polymer surfaces with a production procedure that is facile, economical, and scalable. To that end, we sought to develop a mild, solution-based process to enhance the overall hydrophilicity of PEEK. Our approach to arrive at the corresponding hydrophilic or aqua PEEK biomaterials was predicated on the covalent surface modification strategy illustrated in Figure 1. Installation of the desired polarity would be achieved via reaction of the diaryl ketone functionality present in the polymeric scaffold of PEEK (1) with variously substituted oxyamine and hydrazine nucleophiles (2a-d) bearing a hydrophilic substituent capable of forming favorable interactions with cell membranes to promote adhesion, growth, and osteogenic differentiation. The feasibility of the required bond formation has been documented in the literature as hydroxylamine can be reacted with semicrystalline PEEK films to introduce


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unsubstituted oxime functional groups on the surface of the polymer.33 Oxyamine and hydrazine nucleophiles were selected for investigation as the carbon-nitrogen double bonds present in the resultant oxime and hydrazone products (3a-d) are both stable in a physiological environment and can be formed under mild reaction conditions that should not impact the mechanical integrity of PEEK.

Being a covalent, solution-based process, this approach would hold a distinct

advantage over currently employed coating processes by allowing for uniform reaction with all exposed surfaces of a PEEK implant regardless of size or geometric complexity and without risk of future delamination. Moreover, surface functionalization of PEEK could be achieved in a single chemical step from a variety of commercially available starting materials. It was therefore speculated that this strategy would provide a novel and operationally simple approach for improved PEEK surface hydrophilicity. Herein we report our results for the preparation and characterization of covalently surface-modified aqua PEEK biomaterials that demonstrate enhanced osteoconductive properties compared to unmodified PEEK.

Figure 1. Covalent surface modification of PEEK with oxyamine and hydrazine nucleophiles

EXPERIMENTAL SECTION Materials. Amorphous PEEK film was obtained from McMaster-Carr (Elmhurst, IL). Girard’s Reagent T and 4 M hydrogen chloride in dioxane were purchased from Sigma-Aldrich (St. Louis, MO).

Girard’s Reagent D was purchased from TCI America (Portland, OR).



Oxyamino

P-15

peptide

was

sourced

from

GenScript

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(Piscataway,

NJ).

2-

(Aminooxy)ethanamine dihydrochloride was purchased from Ark Pharm Inc (Libertyville, IL). 4, 4’-Dimethylbenzophenone was obtained from ACROS Organics (Morris Plains, NJ). Methanol and ethanol were purchased from Sigma Aldrich (St. Louis, MO) and pharmcoAAPER (Shelbyville, KY), respectively. Water was purified via a Millipore Synergy water purification system. All reagents and solvents were used as received unless otherwise noted. General Procedure for the Preparation of Surface-Modified PEEK films An amorphous PEEK disc (0.26 mm thick, ~60 mg) was washed with 95:5 MeOH/H2O (immersed for 30 min then dried at ambient temperature) and added to a glass vial with a magnetic stir bar. The disc was then treated with a 25 mg/mL solution of an oxyamine or hydrazine nucleophile in MeOH. The vessel was sealed and the reaction was heated at 45 ˚C for 24 h. The reaction was cooled to ambient temperature and the PEEK disc was rinsed with MeOH, ultra-pure Milli-Q water, and 70% EtOH. PEEK samples were then allowed to dry at ambient temperature. Static Water Contact Angles Contact angles (θ) were obtained with a VCA Optima contact angle measuring instrument (AST Products, Inc.) with a drop size of 1.0 µL of deionized water. PEEK Tensile Strength Testing Dogbone-shaped samples were placed in a reaction vial, submerged in 5 mL of MeOH or ultra-pure Milli-Q water, and treated with 50 µL of 4 M HCl in dioxane. The vessel was sealed and the reaction was heated at 45 ˚C for 24 h. The reaction was cooled to ambient temperature


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and the PEEK dogbones were rinsed with MeOH, ultra-pure Milli-Q water, and 70% EtOH. The samples were allowed to dry at ambient temperature for 24 h. The samples were then mounted in a biaxial load frame (Bionix 858, MTS, Eden Prairie, MN) for axial tension testing. The cross-sectional dimensions and length of the central section of uniform width were measured with calipers (accuracy 0.05 mm). Specimens were nominally 4 mm wide, 0.14 mm thick and 12.25 mm long in the constant length section between the tabs of each specimen. The measured averages were 4.005 × 0.143 × 12.258 mm. Each specimen was aligned with the vertical axis of the load frame and rigidly clamped on the dogbone tabs using knurled vice grip fixtures. Using an extension rate of 0.05 mm/s, the machine applied a load to the specimen until the load reached an essentially steady state while the specimen continued to elongate. (Figure S1. Supporting Information) X-ray Photoelectron Spectroscopy (XPS) X-ray photoelectron spectroscopy (XPS) was conducted on a Thermo Fisher ESCALAB 250 Xi spectrometer with an Al Kα source and a 0.9 mm spot size with charge compensation. Spectra were recorded under normal emission conditions (take-off angle of 90° from the plane of the surface). Cell Culture C2C12 cells (ATCC®CRL-1772™) were grown in Dulbecco’s modified eagle media (DMEM; ThermoFisher Scientific, Waltham, MA) supplemented with 10% (v/v) fetal bovine serum (FBS; ThermoFisher Scientific, Waltham, MA) and 1% (v/v) penicillin-streptomycin (PS; ThermoFisher Scientific, Waltham, MA). MC3T3-E1 (subclone-4; ATCC®CRL-2593™) cells were grown in ascorbic acid-free α-Modified Eagle Media (α-MEM; ThermoFisher Scientific, Waltham, MA) supplemented with 10% FBS (v/v) and 1% PS (v/v) . 7 ACS Paragon Plus Environment


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Alkaline Phosphatase (ALP) Assay ALP assay was performed and quantified as previously described.29 Briefly, C2C12 cells were seeded on PEEK discs (treated and untreated groups) in 12-well plates and incubated in the presence/absence of 100 ng/ml recombinant human bone morphogenetic protein–2 (BMP2; INFUSE®, Medtronic, Minneapolis, MN) in triplicates for 72 hours. Post osteogenic differentiation, the discs were washed in PBS and fixed for 20 min in 10% neutral-buffered formalin at ambient temperature. Alkaline phosphatase activity was detected using ALP detection kit (Sigma Aldrich, St. Louis, MO) according to the manufacturer’s instructions. Briefly, ALP cell monolayers were incubated with the ALP stain overnight at ambient temperature in the dark followed by three 5 minute rinses with distilled water to wash-off excess stain. For quantification, three representative bright-field images were acquired on the PEEK disc surface or tissue culture (TC) plate well using 5X objective of Carl Zeiss epifluorescence microscope. ALP-stained images were converted to CMYK format as this color format is representative of the reflected light colors as opposed to emitted light colors (RGB). Since cyan and magenta form the color blue, these channels were added together and inverted to get intensity values in both the channels. The average pixel intensity was determined using the image histogram tool in Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA). Mineralization assay PEEK discs (treated and untreated groups) were seeded in triplicates with MC3T3-E1 (subclone-4) cells at a density of 2.5 × 103 cells/cm2 in a 12-well plate and allowed to reach confluency. To induce osteogenic differentiation, 50 µg/mL ascorbic acid and 100 mM βglycerophosphate with and without 100 ng/mL BMP2 were supplemented in the growth media. Cells were allowed to differentiate for 21 days with media change every 72 hours. Calcium 8 ACS Paragon Plus Environment

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deposits were stained using alizarin red staining solution (MilliporeSigma, St. Louis, MO) according to manufacturer’s instructions. Briefly, cells were washed in PBS and fixed in 10% neutral-buffered formalin (Sigma Aldrich, St. Louis, MO) for 15 minutes at ambient temperature. Excess fixative was washed-off thoroughly with distilled water and alizarin red staining solution was added for 30 min at ambient temperature. Excess stain was washed off with distilled water overnight and discs were imaged. For quantification, alizarin red deposits were solubilized with 10% acetic acid and absorbance was measured at 405 nm using TECAN spectrophotometer (Tecan Group Ltd, Männedorf, Switzerland). Relative absorbance values were plotted post background subtraction to evaluate the extent of mineralization. Statistical Analysis For statistical analysis, all data was subjected to Analysis of Variance (ANOVA) followed by Tukey’s Post Hoc test for multiple comparisons between each treatment group and the controls using Systat Software. Statistical significance was defined at p≤0.05. RESULTS AND DISCUSSION Model Studies To establish optimal reaction conditions for the hydrophilic surface enhancement of PEEK, model studies were initiated employing the substituted hydrazine nucleophile, Girard’s reagent T (2c), and 4, 4’-dimethylbenzophenone (4) as a PEEK monomer surrogate (Table 1). Given our interest in a mild reagent system to effect surface modification without impacting the mechanical integrity of the polymer, we first attempted to achieve the desired reactivity under neutral reaction conditions. Benzophenone 4 was treated with a solution of Girard’s T (2c) in MeOH (25 mg/mL) and maintained at ambient temperature. After 2 h, LC-MS analysis showed no



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desired product formation, only unreacted starting material (entry a). A similar lack of reactivity was observed after 24 h (entry b). However, the same reaction conditions in the presence of 4 M HCl in dioxane afforded the desired hydrazone adduct 6c in 63% conversion (entry c), demonstrating the necessity of acid in the reaction. This observation is not surprising given that ketones typically require Brønsted or Lewis acid activation in order to undergo nucleophilic addition due to the steric hindrance encountered around the reacting carbon center. Moreover, the ketone moiety in PEEK possesses an added level of chemical stability as a result of resonance stabilization provided by the electron donating substituents at the 4’ positions of the flanking aryl rings making activation of the carbonyl a prerequisite for functionalization. Conveniently, this acid requirement can also be satisfied when the nucleophilic reacting partner is a hydrochloride salt (entry e) resulting in a more facile procedure for achieving the desired bond construction.


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Table 1. Optimization of Nucleophilic Addition to 4, 4’-Dimethylbenzophenone

O

2a or 2c

4

N

a R=

h

Cl

O N

N H

6a, 6c

a

NH2

O

R

c

°

Conversions are based on TIC data from LC-MS analysis of crude reaction mixtures

The role of temperature in the nucleophilic addition of oxyamines and hydrazines to 4, 4dimethylbenzophenone (4) was also investigated (Table 1). Keeping with our desire for mild reaction conditions, we next studied the reaction at a moderately elevated temperature (45 °C). LC-MS analysis after 2 h showed that the increased temperature made little difference in reactivity for the addition of Girard’s reagent T (64% conversion). However, a notable reactivity difference was realized with the oxyamino salt, 2-(aminooxy)ethanamine dihydrochloride (2a). Reaction of benzophenone 4 and oxyamine 2a in MeOH solvent for 2 h resulted in conversions of 37% and 58% at ambient temperature and 45 °C, respectively. Ultimately, both oxyamine and hydrazine addition reactions were judged to be complete after 24 h at 45 °C. Based on these



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results, we adopted an acidic reagent system under moderately elevated temperature (45 °C) as optimized reaction conditions for further investigation. Surface Modification of PEEK Having arrived at a mild procedure to affect the synthesis of substituted oxime and hydrazone adducts from the corresponding PEEK monomer surrogate 4, attention was focused on the application of our optimized reaction conditions to the surface modification of PEEK. A series of commercially available oxyamine and hydrazine nucleophiles were selected to probe the feasibility of the proposed strategy (Figure 1 and Schemes S1-S4).

A typical surface

modification experiment entailed the treatment of a PEEK disc (0.26 mm thick) with an oxyamine or hydrazine hydrochloride salt in methanol, water, or a suitable mixture of MeOH and H2O and heating the resulting mixture for 24 h at 45 °C in a sealed vial. In the case where the chosen nucleophile was a neutral species, 2 equivalents of 4 M HCl in dioxane were added to the reaction mixture. (See Supporting Information for full experimental details). The modified PEEK samples were then characterized via contact angle measurements, tensile strength testing, and X-ray photoelectron spectroscopy (XPS). Contact Angle Measurements Our modified PEEK surface technology was initially assessed by measuring the static water contact angles (θ) for PEEK films 3a-d as a means to verify surface modification and estimate hydrophilicity (Figure 2A). Unmodified PEEK (1) displayed a contact angle of 91° indicative of its hydrophobic nature (entry a). Samples with θ >91° were viewed as being more hydrophobic than PEEK, while samples with θ

Osteoconductive Enhancement of Polyether Ether Ketone: A Mild

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