In Vitro and in Vivo Evaluation of Silicate-Coated

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In Vitro and in Vivo Evaluation of Silicate-coated Polyetheretherketone Fabricated by Electron Beam Evaporation Jin Wen, Tao Lu, Xiao Wang, Lianyi Xu, Qianju Wu, Hongya Pan, Donghui Wang, Xuanyong Liu, and Xinquan Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b10229 • Publication Date (Web): 28 Apr 2016 Downloaded from http://pubs.acs.org on April 29, 2016

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In Vitro and in Vivo Evaluation of Silicate-coated Polyetheretherketone

Fabricated

by

Electron

Beam

Evaporation Jin Wena, b, 1, Tao Luc, 1, Xiao Wang a, b, 1, Lianyi Xua, b, Qianju Wua, b, Hongya Panb, Donghui Wangc, Xuanyong Liuc, * and Xinquan Jianga, b, *

a

Department of Prosthodontics, Ninth People’s Hospital affiliated to Shanghai Jiao

Tong University, School of Medicine, 639 Zhizaoju Road, Shanghai 200011, China b

Oral Bioengineering Lab, Shanghai Research Institute of Stomatology, Ninth

People's Hospital Affiliated to Shanghai Jiao Tong University, School of Medicine, Shanghai Key Laboratory of Stomatology, 639 Zhizaoju Road, Shanghai 200011, China c

State Key Laboratory of High Performance Ceramics and Superfine Microstructure,

Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, China

Corresponding Authors:

Prof. Xinquan Jiang Department of Prosthodontics, Ninth People’s Hospital affiliated to Shanghai Jiao Tong University, School of Medicine, 639 Zhizaoju Road, Shanghai 200011, China E-mail: [email protected] Tel.: +86 21 63135412, Fax: +86 21 63136856 1

ACS Paragon Plus Environment

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Prof. Xuanyong Liu State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, China E-mail: [email protected] Tel.: +86 21 52412409, Fax: +86 21 52412409

1

These authors contributed equally to this work.

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ABSTRACT: Intrinsic bioinertness severely hampers the application of polyetheretherketone (PEEK), although in the field of dentistry it is considered to be an ideal titanium substitute implanting material. In this study, a bioactive silicate coating was successfully introduced onto PEEK surface by using electron beam evaporation (EBE) technology to improve its bioactivity and osseointegration of PEEK. Through controlling the duration of EBE, the incorporated amounts of Si could be exquisitely adjusted to obtain proper bio-functionality, as assessed by cell adhesion, proliferation, osteogenic gene expression and protein detection. In vivo, the samples were then tested in a femur implantation model to assay osseointegration effects in ovariectomized (OVX) rats. Remarkable enhancement of adhesion, spreading, osteogenesis and differentiation of bone marrow stem cells (rBMSCs-OVX) were noted on silicate-coated samples. In particular, the group that was processed for five minutes with EBE (EBE-5min) showed the most improvements in ALP activity and osteogenic-related gene expression compared to the remaining groups. Better osseointegration of the group that was processed for 8 minutes with EBE (EBE-8min) was observed in vivo, as indicated by micro-CT test, fluorescent labeling, and histological and histomorphometric analyses. Collectively, the outcomes of the above experiments demonstrated that the present work is a meaningful attempt to promote osseointegration under osteoporotic conditions with only Si element incorporated to PEEK surface by the application of EBE technique. To the best of our knowledge, this work is the first demonstration of tuning the surface properties of PEEK via the adoption of an EBE-fabricated silicate coating to address an osteoporotic problem

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both in vitro and in vivo.

KEYWORDS: PEEK; silicate; electron beam evaporation; stem cells; osseointegration; ovariectomy

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INTRODUCTION As medical technology advances, implant surgery is becoming increasingly common in the dental and orthopedic fields1-2. Although titanium and its alloys have been widely used as implant materials for several decades, there are still concerns over metal ions release, radiopacity and other inherent unsatisfactory properties3. Employing PEEK as a substitute for titanium and its alloys is advantageous owing to a set of characteristics4 including excellent mechanical properties, non-toxicity, good chemical and sterilization resistance, natural radiolucency and compatibility with magnetic resonance imaging (MRI)5. In the clinic, PEEK has already been widely used in the fabrication of interbody spinal fusion cages6. However, the adoption of PEEK as a dental implant is severely hampered by its inherent bioinertness7-8. Especially for the osteoporosis patients, limitations such as low bone mass and inferior quality imposed by a given patient’s bone and surrounding tissue, severely affect the osseointegration between bone and implants9. Moreover, applying additional therapy to manage the problem can lead to accumulating medical costs and decreased patient satisfaction. During the past few years, numerous strategies have been employed to modified bioinert PEEK10. For instance, bioceramics such as hydroxyapatite are used to optimize PEEK by enhancing its mechanical property and biological function11-12. However, the weak bonding of the coating to the substrate material, low mechanical strength and the fragility or degradation result in unsatisfactory osseointegration13-16. Therefore, proper surface modifications should be presented as an alternative possible

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solution. Coating techniques such as plasma spraying17, plasma immersion ion implantation18, plasma ion deposition19 and chemical methods like selective wet-chemistry20, chemical covalent bonding11 can construct bioactive surface and endow the implant surface with some different biological properties. All of these techniques aim at improving the bioactivity of PEEK to promote the level of direct bone attachment at the interface to get enough bonding strength or improve bone growth12，21-24. Among all, electron beam evaporation (EBE) is a feasible method in which an intense electron beam is generated from a filament and steered to strike source material in a vacuum environment. As the heat accumulates, atoms of source materials will be vaporized and deposited onto the substrate. Thus EBE could be an optimal way to modify the PEEK surface without unsatisfactory effects while doping some bioactive elements onto the surface. Osteoporosis is an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Many bioactive elements are important in the synthesis of the constituents of bone matrix, such as calcium, zinc, copper, boron and manganese). Silicon is an essential element to the development of healthy connective tissues and skeletal tissues and is concentrated at the mineralization front of growing bone25. Recently it is proven that silicon-containing bioceramics such as silicate can enhance bone regeneration26-28. Besides, studies have disclosed that CaSiO3 and some silicate bioceramics possess the osteogenic and angiogenic effects due to the existence of silicon29-30. Normally Si-doped coatings fabricated by plasma spraying, sol-gel and electrophoretic deposition techniques contain other bioactive elements which will

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interfere to address the direct mechanism of osteogenic behavior by sole silicon. EBE is very suitable to address the independent role in bone regeneration31 and this actually can help us investigate the exquisite role of silicon on osseointegration process. In this work, EBE was applied to fabricate silicate coatings onto PEEK samples. This is the first attempt to introduce a silicate coating onto PEEK using EBE technique32. Moreover, we manipulated the amount of silicon applied to the PEEK samples by conducting different durations of the coating process33. To evaluate its bioactivity, a series of tests were performed, including examining the osteogenic response in vitro by assessing initial cell adhesion, proliferation, and osteogenic gene mRNA and protein expression. All of the implants were inserted into femurs of ovariectomized rats for 8 weeks, after which they were to be examined by micro-CT, sequential fluorescent labeling and histomorphometric staining to evaluate the osseointegration effect in vivo.

EXPERIMENTAL SECTION Specimen fabrication and modification. PEEK samples that were machined into different dimensions were used in this study. Square samples (10×10×1 mm³ or 20×20×1 mm³) were fabricated for surface characterization, immersion tests and in vitro studies. Rods of Φ2 ×8 mm³ dimension were used in in vivo studies. All samples had one side polished and were ultrasonically cleaned in acetone, ethanol, and ultra-pure water to remove contamination prior to surface treatment. Calcium

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silicate (CaSiO3; Analytical reagent, China) powders and 5% wt polyvinyl alcohol (PVA) gel were mixed uniformly and then pressed into ceramic disks, after which they were sintered at 1000 ℃ in a furnace with heating rate of 5 ℃/min for 2 h to discharge PVA and condense the ceramic. For silicate coating, an electron beam evaporator (Pulsetech, China) was applied at 8.5 kV and 1.6 A to deposit silicate onto the PEEK samples, using the CaSiO3 disk as evaporant. The PEEK samples were divided into three different groups that were treated with different deposition times, including 2 minutes, 5 minutes, and 8 minutes, and were denoted as EBE-2min, EBE-5min, and EBE-8min，respectively.

Surface characterizations. The surface morphologies of the coatings were examined with field emission scanning electron microscopy (FESEM; HITACHI S-4800, Japan) at different magnifications. The surface chemical states and elemental compositions of the coatings were measured using X-ray photoelectron spectroscopy (XPS, Physical Electronic PHI 5802 equipped with a monochromatic Al Kα source).

Contact angle measurement. The surface wettability was detected by contact angle measurement (Automatic Contact Angle Meter Model SL200B, China) using deionized water droplets. Generally, a microliter syringe with a 2-µL droplet of water suspended from its tip was advanced toward the sample surface until the droplet made contact with the surface. Images were taken and a magnified image was used to measure the contact angle between the drop and the coating. The measurement was

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conducted five times for each group and the results are displayed as the mean ± standard deviation (SD).

Surface zeta-potential measurements. The surface zeta-potential was measured from the motion of ions in the diffusion layer using a Surpass electro kinetic analyzer (Anton Parr, Austria). Under conditions of pH-adjusted KCl solution (0.001 M), the electrolyte solution’s viscosity, vacuum permittivity and dielectric constant were obtained according to the Helmholtz-Smoluchowski equation, where dI/dP represents the slope of the streaming current versus the pressure difference. The zeta-potential was measured three times at each pH value.

Ion release. All four groups of PEEK samples were soaked in 10 mL ultrapure water at 37 ºC for 8 weeks and the leaching liquid was collected at the end of incubation. The concentration of the Si ions that were being released was analyzed by inductively coupled plasma atomic emission spectroscopy (ICP-AES, JY2000-2, France).

Isolation and culture of primary rBMSCs from OVX rats. All experimental protocols concerning animals were approved by the Animal Committee of the Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. Briefly, ovariectomy was performed on six-month-old female SD rats through two dorsal incisions that were made in each rat. Three months after the operation, the

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OVX rats were sacrificed, and the metaphysis from both ends of the femurs were cut off to enable the marrow to be flushed out. The cells were then cultured in Dulbecco’s modified Eagle medium (DMEM; GIBCO Laboratories, Grand Island, NY) in 10 cm diameter plates for nearly 2 weeks at 37℃ in a 5% CO2 incubator. It takes approximately 6-7 days to obtain primary rBMSCs-OVX. The cultured cells from passage 2 were used for further studies.

Cell adhesion. The rBMSCs-OVX were seeded onto the PEEK samples in a 24-well plate at a density of 5×104 cells per well. After incubating for 4 hours, all samples were removed to a new 24-well plate and fixed in 3% glutaraldehyde overnight. After gradient dehydration, the samples were submitted to a final dehydration step in varying concentrations of hexamethyldisilizane (HMDS) ethanol solution and then dried in a fume cupboard. Following this, all of the samples were sputter-coated with platinum to enable SEM (Hitachi S-3400, Japan) observation.

rBMSCs-OVX proliferation assay. To investigate the cell proliferation of rBMSCs-OVX on different PEEK samples (con/2min/5min/8min), a Cell Counting Kit-8 (CCK-8, Dojindo Laboratories Inc., Kumamoto, Japan) was used. Briefly, cells were seeded on PEEK samples (10×10×1 mm³) in 24-well plates at a density of 5×104 cells per well and cultured for 1, 4, and 7 days. After incubation for one hour, the DMEM-CCK8 solution was carefully moved to a 96-well plate, and absorbance was read at the wavelength of 450 nm by a microplate spectrophotometer (Benchmark

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Plus, Tacoma, Washington, USA). All experiments were performed in triplicate, and the results were analyzed by plotting cell growth curves according to the absorbance readings.

Alkaline phosphatase (ALP) staining and calcium deposition assay. To determine the early differentiation of rBMSCs-OVX on PEEK samples, the rBMSCs-OVX were seeded on PEEK samples in 24-well plates at a density of 5×104 cells per well and cultured for 7 days. On day 7, ALP staining was performed according to the manufacturer’s instructions (Beyotime, Jiangsu, China). Briefly, after fixation in 4% paraformaldehyde, the cells were incubated in a mixture of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate. Calcium deposition was observed by Alizarin Red S staining. Initially, 1×105 cells/well were seeded onto the PEEK samples in 6-well plates. After 14 days of incubation in DMEM, the cells were fixed and then stained using 0.1% Alizarin Red S solution for 30 minutes at 37℃.

RNA isolation and gene expression by quantitative real-time PCR analysis. Real-time PCR analysis was performed to investigate the relative gene expression levels of rBMSCs-OVX that were seeded onto PEEK samples. The rBMSCs-OVX was seeded onto PEEK samples (20×20×1 mm³) in 6-well plates at a density of 1×105 cells per well and incubated for 4 or 7 days. Total cellular RNA extraction was performed with TRIzol reagent (Invitrogen, Carlsbad, USA) according to the

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manufacturer’s instructions. Purified gene-specific primers were synthesized commercially (Shengong Co., Ltd. Shanghai, China), and the genes, accession numbers, primer sequences, and amplicon sizes are listed in Table 1. All mRNA values were compared to a single number value after normalization.

Immunofluorescence of osteocalcin. To detect osteocalcin (OCN) expression, 5×105 cells per well were seeded onto PEEK samples in 24-well plates and cultured for 4 days. Cells were fixed in 4% paraformaldehyde after washing with PBS and were then treated with Triton X-100 for permeabilization. Following this, the samples were incubated in specific primary antibodies against osteocalcin (Abcam, USA) overnight at 4℃. Finally, the samples were incubated with secondary antibody for 30 minutes at room temperature. After washing with PBS, all specimens were observed using a confocal laser-scanning microscope.

Surgical procedures. In this study, a rat femoral model was used, and the surgical procedures were conducted as described previously. The rats were anesthetized by intraperitoneal injection of ketamine. After their hind limbs were shaved, a 10 mm longitudinal incision was made across the knee joint along the lateral side of the extensor mechanism. Using a rotary drill, a cylindrical hole measuring approximately 2.2 mm in diameter was created along the long axis of the femur. Two implants were randomly and bilaterally placed into each rat. The soft tissues were sutured after relocation of the patella and reconstruction of the extensor mechanism. Sixteen rats

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were operated on in this study, and they were randomly divided into 4 groups.

Sequential fluorescent labeling. The process of new bone formation and mineralization was assessed by polychrome sequential fluorescent labeling method. At 2, 4 and 6 weeks after surgery, 30 mg/kg alizarin red S (Sigma, USA), 25 mg/kg tetracycline

hydrochloride

(Sigma)

and

20

mg/kg

calcein

(Sigma)

were

intraperitoneally administered in sequence.

Sample preparation. The 16 animals that underwent surgery were randomly allocated into 4 groups that were labeled as the PEEK, PEEK-2min, PEEK-5min and PEEK-8min groups. Each observation group included 4 rats, and both legs from each rat were assessed, for a total of 8 legs per group (n=8). All rats were sacrificed at 8 weeks after surgery34. Following this, 32 femurs were harvested and trimmed into smaller blocks. . Micro-CT assay. The presence of newly formed bone around the implants was detected by Micro-CT (GE explore Locus SP Micro-CT, USA). The parameters of scanning were set at 80 Kv and 80 Ua with an exposure time of 3000 ms and a resolution of 15 µm. Three-dimensional images were reconstructed using NRecon software (SkyScan, USA) and a CTvol program (SkyScan). The bone volume fraction (bone volume/total volume, BV/TV) and trabecular pattern factor (Tb.pf) were determined for newly grown bone tissues using DataViewer software (SkyScan) and a

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CTAn program (SkyScan).

Histomorphometric observation. After being measured by Micro-CT, the femur specimens of each group were dehydrated in a graded concentration of 75% to 100% alcohol and then embedded in polymethylmetacrylate (PMMA). After being embedded, all specimens were cut into 150-µm-thick sections using a Leica SP1600 saw microtome (Leica, Hamburg, Germany). All sections were ground and subsequently polished to a final thickness of approximately 40 µm, and their fluorescent labeling was observed using a confocal laser-scanning microscope (CLSM, Leica). The excitation/emission wavelengths of the included chelating fluorochromes were 543/617 nm, 405/580 nm and 488/517 nm for alizarin red S (red), tetracycline hydrochloride (yellow) and calcein (green), respectively.

Statistical analysis. All data were expressed as the mean ± standard deviation. Statistical analysis for the above assays was carried out via ANOVA and SNK post hoc based on a normal distribution and equal variance assumption test. All statistical analyses were conducted using SPSS v.10.1 software (SPSS Inc.). Values of *p < 0.05 and **p

In Vitro and in Vivo Evaluation of Silicate-Coated

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