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Enhanced osseointegration of hierarchical micro/nano-topographic titanium fabricated by micro-arc oxidation and electrochemical treatment Guanglong Li, Huiliang Cao, Wenjie Zhang, Xun Ding, Guangzheng Yang, Yuqin Qiao, Xuanyong Liu, and Xinquan Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b10633 • Publication Date (Web): 20 Jan 2016 Downloaded from http://pubs.acs.org on January 24, 2016

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ACS Applied Materials & Interfaces

Enhanced osseointegration of hierarchical micro/nano-topographic titanium fabricated by micro-arc oxidation and electrochemical treatment

Guanglong Lia,1,Huiliang Caob,1,Wenjie Zhanga,1, Xun Dinga, Guangzheng Yanga, Yuqin Qiaob, Xuanyong Liub,**, and Xinquan Jianga,*

a

Department of Prosthodontics, Oral Bioengineering and regenerative medicine Lab, Ninth

People’s Hospital Affiliated to Shanghai Jiao Tong University, School of Medicine, 639 Zhizaoju Road, Shanghai 200011, China b

State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai

Institute of Ceramics, Chinese Academy of Sciences, 1295 Ding-xi Road, Shanghai 200050, China 1

These authors contributed equally

* Corresponding author. Department of Prosthodontics, College of Stomatology, Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, 639Zhizaoju Road, Shanghai 200011, PR China. **Corresponding author. State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, PR China. Tel./fax: +86 21 52412409. E-mail addresses: [email protected] (X. Jiang), [email protected](X. Liu).

Keywords:

Micro-arc

oxidation;

Micro/nano-topography;

Osseointegration; Surface modification.

ACS Paragon Plus Environment

Dental

implant;


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ABSTRACT

Rapid osseointegration is recognized as a critical factor in determining the success rate of orthopedic and dental implants. Micro-arc oxidation (MAO) fabricated titanium oxide coatings with a porous topography have been proved to be a potent approach to enhance osteogenic capacity. hierarchical

coatings

with

similar

Now we report two kinds of new micro-morphologies

but

different

nano-topographies (i.e., MAO and MAO-AK coatings) and both coatings significantly promote cell attachment and osteogenic differentiation through mediating the integrin β1 signaling pathway. In this study, titanium with a unique hierarchical micro/nano-morphology surface was fabricated by a novel duplex coating process; i.e. firstly a titanium oxide layer was coated by MAO, and then the coating was electrochemically reduced in alkaline solution (MAO-AK). A series of in vitro stem cell differentiation and in vivo osseointegration experiments were carried out to evaluate the osteogenic capacity of the resulting coatings.

In vitro, the initial

adhesion of the canine BMSCs seeded on the MAO and MAO-AK coatings was significantly enhanced, and cell proliferation was promoted.

In addition, the

expression levels of osteogenesis-related genes, osteorix, ALP, OPN, and OCN, in the canine BMSCs were all up-regulated after incubation on these coatings, especially on the MAO-AK coating. And the in vitro ALP activity and mineralization capacity of canine BMSC cultured on the MAO-AK group were better than that on the MAO group.

Furthermore, six weeks after inserting the titanium implants into canine

femurs, both the bone formation speed and the bone-implant contact ratio of the MAO-AK group were significantly higher than those of the MAO group. All these results suggest that this duplex coating process is promising for engineering titanium surfaces to promote osseointegration for dental and orthopedic applications.


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1. INTRODUCTION

Titanium (Ti) and its alloys are extensively used as well-established biomaterials in dental and orthopedic therapies, due to their excellent mechanical strength, biocompatibility and anti-corrosion property.1-3 However, the surface of Ti-based materials is bio-inert,4-5which results in a limited potential for inducing new bone tissue formation and an inability to achieve ideal osseointegration. The surface of an implant plays a critical role in determining the rate as well as the quality and quantity of new bone formation around the implant.6-7 Therefore, the improvement of the Ti -based implants osseointegration has become a topic of great interest in related fields.

To stimulate desired biological responses, various methods have been used to modify titanium implant surfaces, such as sand blasting, etching, chemical methods, sol-gel treatments and ion beam-assisted deposition.8-11 Among those, micro-arc oxidation is a relatively simple and reliable technique used to create functional coatings on titanium substrates. Micro-arc oxidation (MAO), also called plasma electrolytic oxidation, is an effective and convenient surface modification technique that can produce porous, relatively rough, and firmly adherent titanium oxide films on the surface of Ti alloys.12-13 During the micro-arc oxidation process, the aqueous electrolyte ingredients can be incorporated into the oxide layers by discharges; classical electrolytic solution contains calcium acetate and glycerophosphate salt.14-15 After treatment, there is not only the formation of porous oxide layer structures on the Ti alloy surfaces but also the incorporation of abundant calcium and phosphorus chemical elements, which are significantly conducive for induce osteogensis and promote the biocompatibility of the Ti-based implants with the surrounding bone tissue.

Despite the wide application of the MAO technique for Ti-based implant surface



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modification, most studies have demonstrated that the traditionally fabricated porous structures were simple textures consisting of micro-scale pores.13,

16-17

As is well

known, osteogenic cell biological behavior is largely regulated by micro- and nano-scale topography. Many reports have shown that both micro-scale and nano-scale structures can facilitate osteogenic cell activity and increase bone formation around implants.18-19 In addition, the synergetic effects of the dual-length scale (i.e., hybrid micro/nano-scale) on BMSC and osteoblast activity have been corroborated by some studies.20-21 Overall, titanium with micro- and nano-scale morphological features elicits intensive osteogenic behavior and responses, including enhanced protein adsorption, cell adhesion, cell proliferation, alkaline phosphatase activity and up-regulated expression of bone-related genes, thus improving the rate and quality of osseointegration. To fabricate hierarchical structures on titanium surface after traditional MAO treatment, additional techniques, such as chemical treatment and hydrothermal treatment (HT), are typically employed.22-24 Huang et al. developed a MAO-HT-fabricated coating on titanium substrates; they found the hierarchical surface enhanced both fibronectin adsorption and initial MG-63 cell attachment.25 Zhou et al. reported a hierarchical structure produced by patterning Sr-doped hydroxyapatite nanorods on a micro-arc oxidized TiO2 layer; they found that the inter-rod spacing promoted apatite formation in simulated body fluid (SBF) and played an important role in mediating cell adhesion.26 Although these findings suggest titanium implants treated with both the MAO technique and a subsequent chemical treatment have great potential for a successful osseointegration with host bone, few reports provide in vivo data for the evaluation of integration effects between bone and these implants. Moreover, the reported paradigms toward micro/nano-topography of titanium are more complex for real applications. It generally starts with the development of micro-scale topography on titanium substrate (by using MAO technique), followed by the formation of nanostructure using chemical treatment, hydrothermal-treatment and so on.

Different from those reports aforementioned, we fabricated a novel porous TiO2 layer


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on Ti using an innovative MAO technique. By regulating the electrical parameters, the hierarchical micro/nano-topography created on titanium substrate is achieved by one step MAO treatment, named MAO group, which exhibits a porous surface with micro-scale pits and nano-petal-like structures. In addition, we took the samples treated with the new MAO technique into alkaline solution to execute the secondary electrochemical process as previously mentioned. Interestingly, the samples treated with the two-step process (MAO-AK group) changed the sharp edges (petal-like nanostructure) into granular-nanostructures while maintaining a micro-topography similar to that of the MAO group. Accordingly, in this study, the effects of these micro/nano-structural titanium surfaces on osteoblastic differentiation of BMSCs and osseointegration of titanium implants were studied in vitro and in vivo, respectively.

2. MATERIALS AND METHODS

2.1 Fabrication and Characterization of MAO and MAO-AK Coatings To evaluate cell behaviors in vitro, commercial TC4 titanium alloy was cut into squares (10×10×1mm or 20×20×1mm). In the animal experiments, medical titanium (Ti6Al4V) rods with an external diameter of 3.0mm and a length of 10.0mm were employed. The samples were ultrasonically cleaned in acetone, acid pickled, and ultrasonically cleaned with ethanol.

Then, the samples were micro-arc oxidized in an electrolyte solution composed of 4.5 g/L glycerophosphate disodium salt pentahydrate (C3H7Na2O6P•5H2O, Kelong, Shanghai, China) and 4.0 g/L sodium metasilicate nonahydrate (Na2SiO3•9H2O, Sinopharm, Shanghai, China) to fabricate a porous surface layer; the MAO group was composed of these samples. Micro-arc oxidized samples further electrochemically treated in a sodium hydrate solution (NaOH, 0.01M), were placed in a negative potential (0.5 A/cm2) for 30 min; the counter electrode was graphite. The MAO-AK



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group was composed of these samples, which exhibited the new coating after the twostep process (Figure 1).

The surface morphology of the coatings was observed using scanning electron microscopy (SEM, JEOL JAM-6700F, Japan). Coating chemical composition was analyzed using energy-dispersive X-ray spectrometry (EDS, EPMA, JAX-8100, Japan).16 The Titanium (Ti) chemical states were determined using X-ray photoelectron spectroscopy (XPS) (Physical electronics PHI 5802). Sample surface components were detected using a thin-film x-ray diffractometer (D/max 2500PC, Rigaku, Tokyo, Japan) with a Cu Kα (k = 1.5406 Å) X-ray source. Coating surface wettability was assessed using a contact angle instrument (SL20 0B, Solon, China) according to a method published in the literature.27

2.2 Culture of Canine BMSC Bone marrow stem cells (BMSCs) were isolated and cultured as previously described.28 In brief, under general anesthesia with ketamine (10 mg/kg) and xylazine (4 mg/kg), approximately 4 ml of autologous bone marrow was harvested from the iliac crest via needle aspiration. Phosphate-buffered saline (PBS) (10ml) was mixed with the bone marrow, and the mixture was then centrifuged at 1000rpm for 15 min. Primary cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (GibcoBRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) at 37°C in5% CO2. After 5 days of culture, non-adherent cells were removed, and fresh medium was added. The remaining adherent cells were mainly mesenchymal stromal cells. The cells were subcultured when they reached 80-90% confluence. The following in vitro experiments were carried out with cells from passages 2-4.

2.3 Cell Viability Assay To assess cell viability on the three different substrates, the Live-Dead Cell Staining Kit (BioVision) was applied.29 According to the standard protocol, live cells were


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easily

stained

by

Live-Dye,

a

cell-permeable

green

fluorescent

dye(Ex/Em=488/518nm). Propidium iodide (PI), a non-cell permeable red fluorescent dye (Ex/Em =488/615nm), was used to stain dead cells. In brief, 5.0×104 cells per ml were seeded on the substrates. After one day of culture, the staining solution was added to the complete culture medium and incubated with the cells for 15 min at 37°C. The cells were observed immediately using a fluorescence microscope. Healthy cells were stained only by the cell-permeable Live-Dye, fluorescing green. Dead cells could be stained by both the cell-permeable Live-Dye and the non-cell permeable PI (red), which caused an overlay of green and red that appeared yellow-red. The percentage of viable cells was calculated, and the experiments were performed in triplicate.

2.4 Cell Proliferation Activity Assay The MTT cell metabolism assay (Sigma) was applied to estimate canine BMSC proliferation activity on the three different substrates.6, 30 Primarily, 2.0×104 cells per mL were plated onto each specimen in a 24-well plate. After 1, 4 and 7 days of culture, the targeted wells were mixed with the MTT solution, and then incubated for 4 hours to form formazan. The formazan was dissolved in dimethyl sulfoxide and measured at 490 nm by an ELX Ultra Microplate Reader (BioTek, Winooski, VT). The experiment was performed in triplicate.

2.5 Cell Adhesion Assay The deposition of serum proteins plays a critical role in cell adhesion. It is recognized that fibronectin (FN), as an important component of the extra cellular matrix (ECM), is one of the main types of serum protein that support cell adhesion.31For instance, in this study, after two hours of incubation in the DMEM containing 10% fetal bovine serum (FBS), each sample was immunofluorescently stained for fibronectin. The titanium plates were co-incubated with rabbit anti-bovine fibronectin polyclonal antibody (dilution, 1:80; Millipore, Bedford, MA, USA) at 4°C overnight. After being washed three times with phosphate-buffered saline, all samples were incubated with



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DyLight 549-conjugated anti-rabbitIgG for one hour at 37°C.32 Digital images were acquired using a confocal laser scanning microscope (Leica, Solmes, Germany) and processed by ImageJ software (National Institutes of Health, Bethesda, MD, USA) to calculate the fluorescent area. When canine BMSCs had been seeded on the samples for four hours, the expression of integrin β1, a key cell-adhesion-related protein, was measured using its specific primary antibody (Abcam, Cambridge, MA) to detect the ability of the cells to adhere onto different surfaces. Cell nuclei were counterstained with DAPI (Invitrogen).The experiment was performed in triplicate. Integrin β1 expression was quantified via real-time PCR and western blotting. At the gene level, total RNA of the different samples were collected at 4 and 12 hours and then measured using real-time PCR; the primers used are shown in Table 1. At the protein level, canine BMSCs were incubated on titanium plates with different coatings for 12 hours. Total cell protein was collected using a protein extraction regent (Kangchen, China) with phenylmethanesulfonyl fluoride (PMSF). The protein concentration was measured via a Bio-Rad protein assay kit. Equal amounts of protein samples were separated

by

SDS

polyacrylamide

gel

electrophoresis

(PAGE)

and

then

electro-transferred to a polyvinylidene difluoride membrane (PVDF, Pall, USA).The membranes were blocked and incubated with integrin β1 primary antibody (Santa Cruz, USA, dilution, 1:500) and GAPDH primary antibody (Sigma, USA, dilution, 1:1000). The membranes were visualized using Kodak XO mat film (Rochester, USA), and the protein gray values were measured by Image J software.

2.6 Reverse Transcription and Real-time PCR Assay Cells were cultured on the substrates in complete Dulbecco's Modified Eagle Medium, and the total RNA was extracted with Trizol reagent (Invitrogen) after 14 days. The cDNA were generated using a PrimeScript 1stStrand cDNA Synthesis kit (Takara, Japan). The expression of osteogenic-related genes osterix, OCN, OPN, and alkaline phosphates (ALP) were measured using the Bio-Rad Quantitative Real-Time PCR system (qRT-PCR; Bio-Rad. MyiQTM, USA).33The housekeeping gene GAPDH was used for normalization and the Ti alloy group was used as a control. The specific


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primer sequences used in this study are listed in Table 1.

2.7 Alkaline Phosphatase Activity Canine BMSCs were seeded on the three different samples at a density of 2.0×104 cells/well. After being cultured for 7 days, cells on the substrates were fixed and stained by an alkaline phosphatase kit (Beyotime, China).34For the quantitative ALP assay, the cell lysates were incubated with p-nitrophenyl phosphate (p-NPP) (Sigma) at 37°C for 30 min. ALP activity was tested by detecting the optical density (OD) values at 405 nm and total protein content was assessed using a Bio-Rad protein assay kit (Bio-Rad, USA) with OD measured at 630 nm and normalized with a series of BSA (Sigma) standards. The ALP activity finally was presented as OD values per milligram of total proteins at 405nm. The ALP quantitative assay was also performed at the same time point (7days) in accordance with the ALP staining; the measurements were conducted in triplicate.

2.8 Calcium Deposition Assay After 21 days of incubation in DMEM without any osteoinductive factors, BMSCs were fixed in cold 70% ethanol and stained with 40mM alizarin red S.35 The staining results were observed under an optical microscope. For the quantitative assay, the stained cells were desorbed using 10% cetylpyridinium chloride (Sigma) and the OD values were detected at 590nm. Calcium deposition results were normalized to the total protein content and expressed as OD values at 590nm per milligram of total cellular protein.

2.9 In Vivo Osseointegration Evaluation 2.9.1 Surgical Procedures Twelve-month-old male beagle dogs (n=12) were utilized in this study. All animal procedures were reviewed and approved by the Animal Care and Experiment Committee of the Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University. The surgical procedures were performed under sterile conditions as previously



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described.36 After anesthesia via an injection of ketamine (10mg/kg), the mid-shaft of the left femur was carefully exposed through skin incision and muscle blunt dissection. Three holes (Φ=3.2 mm) were drilled into the shaft of canine femur; the space between each hole was approximately 10 mm. Three types of implants were inserted into the three holes randomly, and the wound was conventionally sutured.

2.9.2 Sequential Fluorescent Labeling For the purpose of evaluating new bone formation and mineralization, polychrome sequential fluorescent labeling was used as previously reported.36 At 2 and 4 weeks after the operation, 30 mg/kg of alizarin red S (Sigma, USA) and 20 mg/kg of calcein (Sigma), respectively, were administered intraperitoneally.

2.9.3 Radiographic Observation At 6 weeks post operation, all dogs were sacrificed, and the left femurs with the three groups of implants were harvested and fixed in 10% buffered formaldehyde. X-ray images were obtained to assess the quality of new bone formation and the degree of the mineralization around implants.37 All the radiographs were captured by a dental X-ray machine (Trophy, France) from distance of 7 cm (230V, 8 mA) and an exposure time of 0.28s.

2.9.4 Histological Analysis After the radiographs were obtained, all the femurs were trimmed into smaller cylindrical samples. The specimens were dehydrated with a graded series of alcohol from 75% to 100% ethanol and were embedded in polymethylmethacrylate (PMMA). The embedded specimens were cut into 150µm thick sections using a Leica SP1600 saw microtome (Leica, Hamburg, Germany). These sections were subsequently ground and polished to a final thickness of approximately 40µm. The polychrome fluorescent labeling of the sections was observed using confocal laser scanning microscopy (CLSM, Olympus). The excitation/emission wavelengths used to visualize the chelating fluorochromes were 543/600-640 nm and 488/500-550 nm for


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alizarin red S (red) and calcein (green), respectively.

After fluorescence microscopy, the same sections were counter-stained with van Gieson’s picrofuchsin to observe the mineralized bone tissue.32 The images were captured by a fluorescence microscope (Olympus, Japan) and analyzed using Image-ProPlus software. The percentages of bone-to-implant contact (BIC) and new bone area were evaluated on 3 to 4 sections of each implant for the histometric analysis.

2.10 Statistical Analysis Statistical comparisons were assessed via one-way ANOVA and SNK post-hoc based on the normal distribution and equal variance assumption tests. The data are presented as the mean± standard deviation. All statistical analysis was performed using an SAS 8.2 statistical software package (Cary, USA). Differences were considered statistically significant at p< 0.05 and p

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