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Plasma Surface Functionalized Polyetheretherketone for Enhanced Osseo-Integration at Bone-Implant Interface Ying Zhao,†,‡,§,# Hoi Man Wong,†,# So Ching Lui,† Eva Y. W. Chong,† Guosong Wu,§ Xiaoli Zhao,‡ Chong Wang,† Haobo Pan,‡ Kenneth M. C. Cheung,† Shuilin Wu,⊥ Paul K. Chu,*,§ and Kelvin W. K. Yeung*,†,∥ †
Department of Orthopaedics and Traumatology, The University of Hong Kong, Pokfulam, Hong Kong Center for Human Tissues and Organs Degeneration, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China § Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China ∥ Shenzhen Key Laboratory for Innovative Technology in Orthopaedic Trauma, The University of Hong Kong Shenzhen Hospital, 1 Haiyuan first Road, Futian District, Shenzhen 518053, China ⊥ Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Ministry-of-Education Key Laboratory for the Green Preparation and Application of Functional Materials, Hubei Province Key Laboratory of Industrial Biotechnology, Faculty of Materials Science and Engineering, Hubei University, Wuhan 430068, China ‡
S Supporting Information *
ABSTRACT: This study aims at improving osseo-integration at the bone-implant interface of polyetheretherketone (PEEK) by water (H2O) and ammonia (NH3) plasma immersion ion implantation (PIII). The pertinent surface characteristics including surface energy, roughness, morphology, and chemical composition are investigated systematically and the in vitro biological performance is evaluated by cell adhesion and proliferation, alkaline phosphatase (ALP) activity, real-time RT-PCR evaluation, and mineralization tests. In vivo osseo-integration is examined via implanting samples into the distal femur of the rats. The hydrophilicity, surface roughness, cell adhesion, and proliferation, ALP activity, and osteogenic differentiation after H2O PIII or NH3 PIII are improved significantly. Furthermore, substantially enhanced osseo-integration is achieved in vivo. Nonline-of-sight plasma surface functionalization, which is particularly suitable for biomedical implants with an irregular geometry, does not alter the bulk compressive yield strength and elastic modulus of the materials. Consequently, the favorable bulk attributes of PEEK are preserved while the surface biological properties are enhanced thus boding well for wider orthopedic application of the biopolymer. KEYWORDS: osseointegration, polyetheretherketone (PEEK), plasma immersion ion implantation, surface modification, interface implant interface.1 Polyetheretherketone (PEEK) has recently attracted much attention because their bulk mechanical properties are similar to those of human bones and the flexibility in processing allows easy manufacturing of different
1. INTRODUCTION Stainless steels, titanium-based alloys, and cobalt chromium alloy are widely used in bone screws, artificial hip joints, and spinal cages because of their favorable mechanical properties, corrosion resistance, and biocompatibility. However, the mismatch in the elastic moduli between human bones and metals often results in stress shielding that can lead to reduced new bone formation and mechanical stability of the bone© XXXX American Chemical Society
Received: November 11, 2015 Accepted: January 21, 2016
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DOI: 10.1021/acsami.5b10881 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
2.2.3. Surface Composition. The surface composition of the untreated control, as well as H2O PIII and NH3 PIII samples, was determined by X-ray photoelectron spectroscopy (XPS, Physical Electronics PHI 5802 system, Minnesota, USA). The C 1s and O 1s spectra were deconvoluted by Gaussian−Lorentzian peak fitting. 2.3. Mechanical Characterization. The compression test was conducted on the MTS 858.02 mini Bionix (USA) according to the ASTM standard D695-08. The loading speed was 1.3 mm/min and the experiment was conducted at ambient temperature. The yield strength and elastic modulus were determined from the load−displacement graph and the data represented averages of ten measurements ± SD. 2.4. In Vitro Studies. 2.4.1. Cell Culture. Mouse MC3T3-E1 preosteoblasts were employed to investigate the effects of PIII on the cell behavior. They were incubated in a complete Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, USA) supplemented with 10% (v/v) fetal bovine serum (French Origin, Biosera, UK), antibiotics (100 U/ml penicillin and 100 μg/mL of streptomycin (Invitrogen, USA)), and 2 mM L-glutamine (Invitrogen, USA) in a humidified atmosphere with 5% CO2 at 37 °C. Before culturing, the samples were sterilized with 70% alcohol for 40 min and rinsed with sterile phosphate buffered saline (PBS) thrice. The medium for cell culture was refreshed every 3 days. 2.4.2. Cell Adhesion. MC3T3-E1 preosteoblasts at a density of 10 000 cells per well were seeded evenly on the various plasma-treated and untreated control samples on 96-well plates. After 4 h, the samples were washed with PBS and fixed with 4% paraformaldehyde in PBS at 37 °C for 10 min. The cell nuclei were stained with Hoechst 33342 and the images were captured by florescence microcopy (Eclipse 80i, Nikon) with a UV-2A filter (excitation 330−380 nm, emission at 420 nm). The cells from five random areas were quantified using the Image-Pro Plus software (Media Cybernetics, USA). The quantities of cells attached to the untreated and PIII samples were derived based on the ratio of the image area to the PEEK surface area. 2.4.3. Cell Morphology. The morphology of the MC3T3-E1 preosteoblasts was observed by field-emission scanning electron microscopy (FESEM, Leo 1530 FEG, Oxford) after incubation for 4 h. The cells were first fixed by 10% neutral buffered formalin for 4 h at 4 °C and after rinsing with the cacodylate buffer containing 0.1 M sucrose to remove excess fixatives, they were dehydrated sequentially with a series of ethanol (30%, 50%, and 70% for 10 min; 90% and 100% for 15 min) in duplicate for each passage. A critical point dryer was used to ensure total removal of water from the samples and a layer of gold−palladium was sputter-coated onto the samples before examination by SEM. 2.4.4. Cell Viability. The MTT assay was employed to quantitatively assess the cytotoxicity of the PIII samples against murine cells. 150 μL of the cell suspension was seeded on each sample on the 96-well tissue culture plates at a density of 5 × 103 cells/well and cultured for 2, 4, and 7 days. At every prescribed time point, the specimens were gently rinsed twice with PBS and transferred to a new 96-well plate. After introduction of the MTT solution prepared by adding thiazolyl blue tetrazolium bromide (Sigma) powder to PBS, the specimens were incubated at 37 °C to form formazen, which was subsequently dissolved in the sodium dodecyl sulfate (SDS) solution. The absorbance at 570 nm was determined on a microplate reader (DTX 800 Series Multimode Detectors, Beckman Coulter, USA). 2.4.5. Alkaline Phosphatase (ALP) Activity. MC3T3-E1 preosteoblasts were seeded at a density of 25 000 cells per well on the various samples on 24-well plates. The medium was renewed every other day. After culturing for 4 and 7 days, the cells were washed with PBS three times and lysed with 0.1% Triton X-100 at 4 °C for 30 min. The cell lysates were centrifuged at 574 g at 4 °C for 10 min (2−5 Sartorius, Sigma, USA) and 10 μL of the supernatant from each sample was transferred to a 96-well tissue culture plate. The ALP activity was determined by a colorimetric assay using an ALP reagent containing pnitrophenyl phosphate (p-NPP) (Stanbio, USA) as the substrate. The absorbance was recorded by the multimode detector (Beckman Coulter DTX 880) at a wavelength of 405 nm. The ALP activity was normalized to the total protein level of the samples measured by the
biomedical products by injection molding, extrusion, and machining.2 Moreover, PEEK can be repeatedly sterilized by conventional autoclaving without materials degradation and the radiolucent nature of PEEK facilitates X-ray assessment at the surgical site.3 Recent in vitro and in vivo studies have also suggested that the materials are generally biocompatible.4−7 However, there are concerns on its inertness, hydrophobic nature, and ensuing limitation in bone fixation.8−11 There have been increasing efforts to improve the bone-implant interface by fabricating composites with hydroxyapatite or plasma spraying hydroxyapatite coatings onto the PEEK surface.12−14 Unfortunately, the inherent elastic modulus of PEEK is altered by fabricating composites with hydroxyapatite.12 Moreover, the hydroxyapatite coating on plasma sprayed PEEK may delaminate upon mechanical shear and bending.14 Among the various surface treatment methods, plasma surface functionalization by means of plasma immersion ion implantation (PIII) is a desirable alternative. The technique not only improves the surface properties without altering the bulk mechanical properties, but also produces a graded surface layer that does not delaminate as easily as coatings as a result of energetic ion bombardment and mixing.15−17 In addition, the nonline-of-sight nature of this technique enables easy conformal treatment of biomedical implants with a complex geometry. Previous studies18−20 have revealed that the proper plasma surface treatment can indeed enhance the cytocompatibility and calcium phosphate deposition on PEEK but there have been few studies on the effects of different PIII parameters and conditions on the biological and mechanical properties of PEEK. In this study, we perform a comprehensively study on the effects of water (H2O) and ammonia (NH3) PIII on PEEK by characterizing the surface structure, mechanical properties, in vitro cellular response, as well as in vivo bone formation.
2. MATERIALS AND METHODS 2.1. Sample Preparation. Medical grade PEEK (Ketron LSG, Quadrant EPP, USA) was used and disks with dimensions of Φ5 × 2 mm3 and Φ14 × 2 mm3 were prepared for surface characterization and in vitro studies. The rod samples for the mechanical tests were Φ3 × 9 mm3, and those used in the in vivo animal studies were Φ2 × 6 mm3. The samples were sequentially ground with 600, 1200, 2400, and 4000 grit silicon carbide paper, as well as 1 μm diamond paste to achieve a mirror finish. Afterward, the samples were first cleaned with pure acetone in an ultrasonic bath and then distilled water for 15 min at room temperature. The GPI-100 plasma immersion ion implanter (PIII) in the Plasma Laboratory of City University of Hong Kong is utilized in this study. The PEEK samples underwent PIII in the H2O or NH3 plasma under the following conditions: radio frequency of 50 Hz, pulse width of 30 μs, pulsed voltage of −10, −20, or −30 kV, and treatment time of 2 h. 2.2. Characterization. 2.2.1. Surface Energy. The contact angles were determined by the sessile drop method with six types of liquids including water, tricresyl phosphate, glycerol, formamide, ethylene glycol, and diiodomethane. A 10 μL droplet of the liquid was put on the sample surface and the readings were taken after 1 min on the contact angle goniometer (Rame-Hart, Mountain Lakes, NJ, USA). The surface energy was calculated according to the literature,21 and five samples of each type were measured to obtain average values. 2.2.2. Surface Roughness and Morphology. Atomic force microscopy (AFM, Auto Probe CP, Park Scientific Instruments) was performed to determine the surface roughness and topography on the microscale in the noncontact mode. The scanned area was 5 μm × 5 μm and the average root-mean-square (RMS) roughness ± standard deviation (SD) was determined from three measurements for each sample. B
DOI: 10.1021/acsami.5b10881 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces Table 1. Primer Pairs Used in Real-Time PCR Analysis gene
forward primer
reverse primer
Gapdh ALP Col1a1 Runx2 OPN
5′-ACCCAGAAGACTGTGGATGG-3′ 5′-CCAGCAGGTTTCTCTCTTGG-3′ 5′-GAGCGGAGAGTACTGGATCG-3′ 5′-CCCAGCCACCTTTACCTACA-3′ 5′-TCTGATGAGACCGTCACTGC-3′
5′-CACATTGGGGGTAGGAACAC-3′ 5′-GGGATGGAGGAGAGAAGGTC-3′ 5′-GTTCGGGCTGATGTACCAGT-3′ 5′-TATGGAGTGCTGCTGGTCTG-3′ 5′-AGGTCCTCATCTGTGGCATC-3′
2.5.3. Histological Analysis. The rats were sacrificed at 8 weeks postsurgery and the bone samples were harvested and fixed in 10% buffered formalin for 3 days. A standard tissue processing step was conducted to change the samples from the aqueous stage to organic stage. A dehydrating process was first performed using 70%, 95%, and 100% v/v ethanol for 3 days each. The samples were transferred to xylene as a transition between ethanol and methyl methacrylate for 3 days. Finally, the samples were embedded in methyl methacrylate (Technovit 9100 New, Heraeus Kulzer, Hanau, Germany) according to the manufacturer’s instructions. The embedded samples were cut and ground into sections with a thickness of 50−70 μm and stained with Giemsa (MERCK, Germany) stain. The morphological and histological analyses were performed on an optical microscope to evaluate the bone on-growth or integration with host tissues. The percentage of bone contact of both PIII and untreated samples were measured by using ImageJ after Giemsa staining. 2.5.4. Elastic Modulus of Newly Formed Bone. The elastic moduli of the newly formed bone on both the PIII and untreated samples 8 weeks after implantation were determined by nanoindentation (Nano Indenter G200) and the data represented averages of four measurements ± SD. To facilitate the indentation, the samples were mounted by methyl methacrylate fixation prior to the test. Then, the bone structure was exposed for testing. 2.6. Statistical Analysis. The in vitro experiments were independently performed at least in triplicates. The in vitro and in vivo data were analyzed by the one-way ANOVA and expressed as means ± standard deviations. A p value
