Effect of Microarc Oxidation-Treated Ti6Al4V Scaffold Following Low

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Bio-interactions and Biocompatibility

Effect of Micro-arc Oxidation-treated Ti6Al4V Scaffold Following Lowintensity Pulsed Ultrasound Stimulation on Osteogenic Cells in Vitro Jie Chen, Jiongjiong Li, Fu Hu, Qin Zou, QuanJing Mei, Shujun Li, Yu-Lin Hao, WenTao Hou, Jidong Li, Yubao Li, and Yi Zuo ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01000 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 6, 2019

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Effect of Micro-arc Oxidation-treated Ti6Al4V Scaffold Following Low-intensity Pulsed Ultrasound Stimulation on Osteogenic Cells in Vitro Jie Chen1, Jiongjiong Li1, Fu Hu1, Qin Zou1, Quanjing Mei1, Shujun Li2, Yulin Hao2, Wentao Hou2, Jidong Li1, Yubao Li1, Yi Zuo*1

1Research

Center for Nano Biomaterials, and Analytical & Testing Center, Sichuan University,

Chengdu, 610064, People’s Republic of China 2Shenyang

National Laboratory for Materials Science, Institute of Metal Research, Chinese

Academy of Sciences, 72 Wenhua Road, Shenyang, 110016, People’s Republic of China

*Corresponding authors: [email protected] (Yi Zuo)

Tel.: +28 85418178; Fax: +28 85417273.

Mailing address: Analytical & Testing Center, Sichuan University, Chengdu 610064, People’s Republic of China

Disclosure of conflict of interest: None

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KEYWORDS: titanium alloy scaffold; EBM–MAO strategy; affinity surface; stereoscopic cytoskeleton structure; LIPUS stimulation; cell mineralization

ABSTRACT: The porous Ti6Al4V alloy has emerged to solve the biomechanical mismatch between implant and bone as its tunable mechanical properties. Cell-surface interaction is related to numerous factors –the surface’s chemical composition, morphological structure, and external effect. The microarc oxidation (MAO) method was employed in this study to improve the surface properties of scaffolds produced by Electron Beam Melting (EBM), as well as low intensity pulse ultrasound (LIPUS) provide physical stimulation for cells in vitro. Although MAO-treated and untreated scaffolds shared the same three-dimensional (3D) structures, the former recreated a more affinity surface than the latter in the 3D room, which could stimulate cell adhesion, proliferation and differentiation. Therefore, MG63 cells were represented with a stereoscopic cytoskeleton structure on the MAO-treated scaffold as numerous cellular filopodia/lamellipodia with rich extracellular matrix secretion, while flat and sheet-like cells were observed on the untreated scaffold. The expression of ALP, OCN, BMP2, Bmpr1a and Runx2 were up-regulated by the MAO-treated scaffold, in addition, LIPUS stimulation effectively promoted cell proliferation and osteogenesis differentiation. In the future, the EBM–MAO strategy can be applied to prepare 3D hierarchical macro-/micro-structure titanium implants for bone grafts, and LIPUS stimulation can be used as a therapeutic method simultaneously.


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1 Introduction The quantity and quality of bone formed at the implant–skeletal interface play a decisive role when determining implant success.1 The titanium alloy is currently promoted in clinical settings, as a widely accepted bonding mechanism known as a titanium film (e.g. Na2TiO3) forms on the surface of the titanium alloy, further resulting in a carbonated apatite coating, which provides a biocompatible interface with peri-implant tissues.2 However, abrasion and corrosion of implant continue to be a major problem in many long-term clinical applications.3,4 According to the available statistics, 25% of hip replacement procedures are revisions for previous implant failure and 10% of cases of dental implants fail within a 5-year period.5,6 Furthermore, the allergic reactions of toxic Al and V elements can further lead to neurological disorders and to the deterioration of Alzheimer’s disease in some studies,7,8 as well as to an accumulation of particulates around the material.9 Electron beam melting is one of the most striking additive manufacturing techniques, which allow us to manufacture porous architecture flexibly with customized shapes and appropriate mechanical strength at an economic cost.10 With tunable mechanical properties and optimized cellular structures, porous titanium alloys are expected to exhibit mechanical properties that more closely resemble bones than compact titanium materials.11 Previous researches have shown that porous Ti6Al4V alloy rods prepared by the EBM technique can be used to treat early-stage talus necrosis when implanted into the tail of sheep.12,13 The elastic modulus of the scaffold is about 2.2GPa, depending on different cellular sizes, which is suitable for bone regeneration.14 Various treatments employing surface modification have been proposed and explored, with the view of further improving the surface properties of titanium alloy, such as plasma spraying,15 sandblasting,16 acid etching,17 alkaline treatment,18 laser surface treatment,19,20 and surface



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mechanical attrition treatment.21,22 Beyond these methods, micro-arc oxidation has largely attracted increased attention because of its great superiority. The MAO process, also called plasma electrolytic oxidation (PEO), is a convenient and effective technique that can form micro-nano porous and firmly adherent ceramic coatings in situ, which could modify the surface topography and chemistry of titanium alloy.23 Moreover, the MAO treatment is favorable for products and scaffolds that feature curved surfaces and sophisticated shapes. Therefore, the combined EBM–MAO strategy shows promising potential when constructing three-dimensional (3D)-hierarchical macro-/microstructure/topography titanium implants for bone healing. In vitro studies offer valuable insights into biological responses of implants. Cell–surface interactions highly depend on the chemical element, crystallinity, roughness, porosity, and surface macro- and microstructure.24 Among these, surface chemistry and morphology are two of the most important properties that determine the biological performance of biomedical implants.25,26 MAO provided a more suitable environment for cell generation, bonding, and differentiation in orthopedic and dental implants, and the cells exhibited notable osteogenic gene expression.27-29 However, most of the experiments stemmed from plate samples that were obtained during MAO treatment.30 Recently, Xiu, P and Zhou reported that MAO treatment could promote the surface bioactivity of Ti and Ti alloy scaffolds that enhanced the bone in-growth in vivo.31,32 To our knowledge, there is the absence of researches addressing the influence of the stereo-topography of macro- and microstructures of titanium scaffolds on osteogenic cells’ adhesive interactions and proliferation in vitro. Meanwhile, a large number of experiments have demonstrated that low-intensity pulsed ultrasound (LIPUS) had promoted effect in vitro migration of seed cell.33 Transmission at a certain intensity in the organism, LIPUS causes changes in the structure or function of cells. With a positive impact in adjusting stimulus, LIPUS produces a variety of biological effects such as mechanical, thermal and


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physicochemical influences on cells. Furthermore, it strengthens the cytoplasmic movement due to the cell membrane potential and intracellular enzyme activity change.34,35 If exposure to LIPUS, cells are stimulated to release alkaline phosphatase (ALP) into the extracellular space. Binding with adenosine triphosphate (ATP), proteins on –cell membrane can regulate the release of Ca2+,36 which is critical for bone formation. So LIPUS effect have been introduced to this study, hoping that cell proliferation and secretion could be promoted deep inside the scaffolds. This study aims to investigate the initial cell–surface interactions of the cells on titanium scaffolds and to compare the biological effects of a MAO-treated Ti6Al4V scaffold with those of an untreated scaffold in vitro, both of which are EBM-printed. Especially, the influence of LIPUS stimulation on cellular activities for titanium alloy scaffolds was studied by examining the preliminary attachment, proliferation, morphology, and viability of human osteosarcoma cells (MG63). From a cellular point of view, actin cytoskeletons and microtubules are particularly essential for cell adhesion and motility. The influences of material surface and stimulated condition on the distribution patterns and cytoskeleton organization of osteoblast-like cells were thus investigated in detail using fluorescent staining. From the biomolecular point of view, the mineralization capacity of the scaffolds was explored through the expression of related proteins. 2 Materials and methods 2.1 Scaffold fabrication Ti6Al4V scaffolds were fabricated by utilizing the EBM system based on a well-defined CAD program containing 3D mesh models, which can be adjusted to create different spacing and meshstrut diameters. The MAO process was then performed in an aqueous electrolyte prepared by



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dissolving 0.2 M sodium acetate trihydrate (CH3COONa), 0.015 M disodium glycerophosphate (C3H7Na2O6P·5H2O), and 0.01 M ethylene-diaminetetraacetic acid disodium salt (C10H14N2Na2O8·2H2O), with the Ti6Al4V mesh structure as an anode. The electrolyte concentration remained constant and the temperature of the electrolytes was maintained in the range of 35 °C ± 10 °C through a combined stirring and cooling system. The oxide layers were formed at a fixed current density of 2 A/dm2 until the DC voltage was raised to 400V. After the MAO process, the scaffolds were successively cleaned ultrasonically in acetone, ethanol, and deionized water. 2.2 Surface characterization Scanning electron microscopy (SEM; JSM-6500LV, JEOL, Japan) coupled with EDX (51-XMX1136, OXFORD, USA) at 20 KV was employed to examine the surface morphology and to detect the contents of the elements. After the MAO-treated scaffold broken by quenching into liquid nitrogen, the interior surface morphology has been observed with SEM and the elements of coating has been analyzed with EDX. The dried samples were sputter coated with gold prior to the SEM examination. Scaffolds were tested by X-ray diffractometer (XRD; DX-2500, FANGYUAN instrument co. LTD., Dandong, China), and its analysis conditions were Copper target ( = 0.15418 nm), 40 kV tube voltage, 25 mA tube current, 0.03/min canning speed and scanning range in 15 ~ 90. Chemical bonding energy on the surface of the scaffold was tested by X-ray photoelectron spectrometer (XPS； XSAM800 ， KRATOS ， UK) ， and the data were analyzed by XPS Peak Fit 4.1 software. Roughness measuring instrument (TR100A, Time technology co. LTD., Beijing, China) measured the surface roughness of the sample (n=5). The contact angles of the samples were measured by static contact angle meter (JY-82A, DINGSHENG testing machine checkout equipment Co., Ltd., Hebei, China). The static liquid drop method was used in the experiment and the liquid was distilled water.


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2.3 Extract assay of Al and V ions To study if the MAO surface modification could affect the release of Al and V ions from the EBMprinted Ti6Al4V scaffold, the concentrations of Al and V ions in the extract solutions were measured by using of Inductive Coupled Plasma Emission Spectrometer (ICP; VG PQExCell, TJA, US). The extract solution has been prepared according to ISO 10993-10:1995. In details, 2g samples were placed in container, then 10mL ultra-pure water was added and placed at 37℃ for 3 days and 7 days (n=5). The extract solutions were collection and the concentrations of Al and V elements were measured. 2.4 Cell culture Human osteosarcoma (MG63) cells were cultured in F-12 Nutrient Mixture medium (Gibco®; Thermo Fisher Scientific, Waltham, MA, USA) containing 10% (v/v) newborn bovine serum (Gibco®; Thermo Fisher Scientific) and 1% (v/v) penicillin/streptomycin antibiotic solution (MP Biomedicals, LLC, Santa Ana, CA, USA). Prior to cell seeding, the samples were sterilized in an autoclave. After sterilization, the cells were seeded onto the specimens at a density of 2×104 cells/mL using 24-well tissue culture plates as the holders. The plates were incubated in a humidified atmosphere of 5% CO2 and 95% air at 37°C, and the culture medium was changed every 2 days. 2.5 Low-intensity pulsed ultrasound stimulation Customized low-intensity pulse ultrasonic (LIPUS) instrument (RONGHAI Ultrasonic Medical Engineering Research Center Ltd., Chongqing, China) includes a controller, which owns the functions of parameter display, parameter setting, function control, transducer drive and 6 transducers. The transducer with a surface area of 6 cm2 emits the modulated sine wave of which the frequency is



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1.5 MHz, strength is 100mW/cm2, pulse repetition frequency is 1KHz, and duty ratio (the duty ratio is the output and the stopping time of the transducer in 1 second) is 20％. From the day of seeding, LIPUS was set for 20 min daily via a 2mm gel to stimulate the bottom of the cell culture plates at 37℃. 2.6 Cell viability assay Cell viability on the scaffolds was evaluated using the Live/Dead® reagent Viability/Cytotoxicity Assay Kit (Molecular Probes, Eugene, OR, USA). It provides a two-color fluorescence cell viability assay that is based on the simultaneous determination of living and dead cells with calcein AM and ethidium homodimer-1 (EthD-1), respectively, which measure recognized parameters of cell viability  intracellular esterase activity and plasma membrane integrity. Calcein AM is well retained within living cells, producing an intense uniform green fluorescence in these cells (ex / em ~ 495 nm / ~515 nm), while EthD-1 enters cells with damaged membranes, producing a bright red fluorescence in dead cells (ex / em ~ 495 nm / ~ 635 nm). EthD-1 is excluded by the intact plasma membrane of living cells. After initial cell seeding for 4 days and 7 days, respectively, the samples were washed twice with PBS and incubated with Live/Dead solution (4μM calcein AM and 4μM ethidium homodimer) at room temperature in the dark. After dye for 30 minutes, the samples were subsequently analyzed and photographed with an inverted microscope (TE2000-U; Nikon, Tokyo, Japan). The viable cells (in green) and nonviable cells (in red) could be easily distinguished under the fluorescence microscope. 2.7 Cell morphology After 4 days and 7 days, the cell/scaffold constructs were retrieved and processed to assess cell


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attachment and morphology using SEM (JSM-6500LV, JEOL, Japan) and confocal laser scanning microscopy (CLSM; A1R MP+, Nikon, Japan). 2.7.1 Scanning electron microscope (SEM) Briefly, cell/scaffold constructs were removed from the culture well, washed in PBS, fixed in 2.5% glutaraldehyde, and dehydrated in a graded ethanol series of 30%, 50%, 75%, 80%, 95%, and 100%; this was followed by critical point drying. Specimens were sputter coated with gold and the morphology of the MG63 cells was investigated with backscattered and secondary electrons via SEM under 20 kV. 2.7.2 Confocal laser scanning microscopy (CLSM) Cell attachment onto scaffold surfaces was mainly visualized with mitochondrion, actin and nuclei staining by CLSM. Briefly, cell/scaffold constructs were fixed in 3.7% (w/v) formaldehyde solution, permeabilized with 0.2% (v/v) Triton X-100/phosphate buffered saline (PBS; 00-3002, Invitrogen, USA), and successively stained with working concentrations of approximately 0.25μM MITOTRACKER® Green FM probes (Thermo Fisher Scientific, USA), 0.15μM Alexa Fluor® 532 phalloidin (Thermo Fisher Scientific, USA), and 5μg/mL blue fluorescent Hoechst 33342 (Thermo Fisher Scientific, USA) in 1% (w/v) bovine serum albumin (BSA; Gibco, Thermo Fisher Scientific, USA)/PBS. The cell/scaffold constructs were then washed with PBS in order to remove excess stains. Viewing cellular ultrastructure was performed using CLSM. The fluorescent Hoechst 33342 dyes are cell-permeable reagents that can stain the nucleus bright blue (ex / em ~ 350 nm / ~ 461 nm). The cell-permeant Mito Tracker® Green FM probes, which contain a mildly thiol-reactive chloromethyl moiety, can accumulate in active mitochondria after passively diffusing across the plasma membrane (ex / em ~ 490 nm / ~ 516 nm). The Alexa Fluor ® 532 phalloidin, isolated from the deadly Amanita phalloides mushroom, can be used interchangeably in



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most applications and bind competitively to the same sites in F-actin (ex / em ~ 531 nm / ~ 554 nm). All of the dyeing methods were based on the manufacturer’s instructions. 2.8 Cell proliferation The proliferation rate of MG-63 cells co-cultured on the scaffolds was determined by the highly water-soluble tetrazolium salt WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4disulfo-phenyl)-2H-tetrazolium, monosodium salt] (CCK-8; Sigma-Aldrich Co., St. Louis, MO, USA), which was reduced by dehydrogenases in cells to give a yellow-colored product (formazan). After MG-63 cells cultured on the scaffolds for 1, 4, 7 and 11 day(s), the samples were rinsed three times with sterile PBS and incubated with CCK-8 solution for 2 hours to generate formazan. Subsequently, the optical density of the solution was evaluated through a multi-label counter (WallacVictor3 1420; PerkinElmer Co., Waltham, MA, USA) at a wavelength of 450 nm. 2.9 Quantitative real-time polymerase chain reaction (qPCR) In order to determine the expression of osteogenic-related genes in MG63 cells, cells were seeded at 2×104 cells/mL on the samples. Total RNA of cells was extracted by using TRIZOL reagent (Thermo Fisher Scientific, US) after incubated for 7 days and 11 days respectively in 5% CO2 at 37°C. At the same time, the quantity of RNA was detected by OD value, and the integrity of RNA was determined by 1% agarose gel electrophoresis. The first-strand cDNA was synthesized by placing the reaction fluid (10µL 2× RT Mix, 1 µL GENESEED® Enzyme Mix, 2 µL Reverse Transcription Primer, 1 µg Total RNA and 20 µL RNase free ddH2O) for 10 min at 25 °C, 15 min at 42 °C and 5 min at 85 °C, respectively. The qPCR reaction system was prepared with 10 µL GENESEED® qPCR SYBR® Green Master Mix, 0.5 µL Forward primer, 0.5 µL Reverse primer, 0.4 µL 50x ROX Reference Dye


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2, template DNA and 20 µL sterilized distilled water. The PCR initiated denaturation at 95 °C for 5 min. Then 40 cyclic reactions were carried out, each of which was composed of denaturation at 95 °C for 10 s and acquisition signals at 60 °C for 34 s. Eventually, the dissolution was reacted at 95 °C for 15 s, 60 °C for 60 s and 95 °C for 10 s. The primer sequences used in this study were listed as follows: Bmp2-F2: 5'- CACAGGGACACACCAACCAT-3' Bmp2-R2: 5'- TTACCGTCGTGGCCAAAAGT-3' Product length 163 Runx2-F1: 5'- TTCAGACCCCAGGCAGTTC-3' Runx2-R1: 5'- GTGTGGTAGTGAGTGGTGG-3' Product length 152 Bmpr1a-F2: 5'- TTTATGGCACGCAAGGCAAG-3' Bmpr1a-R2: 5'- CCCAGGTCAGCAATACAGCA-3' Product length 100 2.10 Enzyme-linked immunosorbent assay (ELISA) The alkaline phosphatase (ALP) and osteocalcin (OCN) were quantified by Human Elisa Kit (ELISA, MLBIO technology Co. Ltd., Yuanjiang road, Minhang District, Shanghai) according to manufacturer’s recommendations. Cultured 7 days and 11 days respectively, the cells were disrupted with 0.2% (v/v) Triton X-100/phosphate buffered saline (PBS) after the culture medium was rejected. Moreover, the lysate of the cells was collected. Then, 50 µL of standard and 40 µL of samples were added to each well. Next, 10 µL of sample diluents were added into each sample well. Following this, 50μL enzymes labeling reagent was added into each hole after incubation for 0.5 h at 37 ℃ . Afterwards, the 100μL colorant was added. Last but not least, the reaction was terminated by adding



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50 µL stop solution after avoiding the light for 10 min. The absorbance at 450 nm was measured by enzyme standard instrument, and the concentration was obtained by drawing a standard curve. 2.11 Statistical analysis The data were obtained from five samples for the statistical analysis. All results are presented as the mean ± standard deviation. One-way analysis of variance (ANOVA) tests, together with Tukey’s test, was conducted using the GraphPad InStat statistics program (GraphPad Software, La Jolla, CA, USA) to examine the date using the following significance levels: p

Effect of Microarc Oxidation-Treated Ti6Al4V Scaffold Following Low

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