Effect of plasma‐nitrided titanium surfaces on the differentiation of pre‐osteoblastic cells

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Effect of plasma‐nitrided titanium surfaces on the differentiation of pre‐

osteoblastic cells

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INTRODUCTION

Commercially pure titanium (CP‐Ti) and alloys derived from this metal have been used to manufacture bone and dental implants for over 40 years, mainly due to their

biocompat-ibility.1‒3 In general, titanium implants with modified

sur-faces, mainly concerning roughness, display positive effects on cellular adhesion and proliferation, leading to bone cell

and osteoblast osseointegration.4,5 _{This is due to nanoscale}

roughness, which mimics a bone surface, favoring the inte-gration process and stimulating the secretion and

differentia-tion of bone growth factors.6,7

These surfaces can be produced through a variety of

tech-niques, including plasma nitriding.8‒10 This method is not

costly, does not damage the environment, leads to excellent mechanical properties, chemical stability, and biocompat-ibility with the titanium surface, with prodigious cellular

response results.11‒14 _{As the nitrided titanium (TiN) layer}

presents a negative charge that favors increasing calcium ion deposition (positive charges), it is favorable for bone‐coating

applications.15 _{In addition, plasma TiN surfaces reduce}

bac-teria adhesion due to biofilm formation, reducing the risk of

infection and/or bone implant rejection.16

The chemical and physical properties of TiN surfaces are necessary to promote the initial protein adsorption and

cell adhesion processes.17‒19 _{Osteoblasts interact with the}

substrate through integrin binding.20 _{Integrins are dimeric}

transmembrane receptors formed by an α and a β unit and

M A I N T E X T A R T I C L E

Effect of plasma‐nitrided titanium surfaces on the differentiation

of pre‐osteoblastic cells

Carlos Eduardo B. Moura

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Moacir F. Queiroz Neto

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Janine Karla F. S. Braz

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Michelle de Aires

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Nainsandra B. Silva Farias

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Carlos Augusto G. Barboza

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Geraldo B. Cavalcanti Júnior

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Hugo Alexandre O. Rocha

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Clodomiro Alves Junior

Department of Animal Sciences, Federal Rural University of Semiarid Region (UFERSA), Mossoró, Brazil Correspondence

Carlos Moura, Department of Animal Sciences, Federal Rural University of Semiarid Region (UFERSA), Av. Francisco Mota, 572 Mossoró, Rio Grande do Norte 59.625‐900, Brazil.

Email: carlos.moura@ufersa.edu.br Funding information

Conselho Nacional de Desenvolvimento Científico e Tecnológico, Grant/Award Number: 479951/2008-0; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Abstract

A titanium surface nitrided by plasma contains nitrogen ions that guarantee resist-ance to corrosion and biocompatibility. Despite this, no descriptions concerning the influence of the expression of cell adhesion proteins and their influence on osteo-genic cell differentiation are available. Thus, the present study aimed to assess the response of murine pre‐osteoblastic cells (MC3T3‐E1) cultured on nitrided titanium surfaces. Pre‐osteoblastic cells were grown on polished titanium discs, used as con-trols, and on previously characterized plasma‐nitrided titanium discs. Cells from both groups were submitted to the MTT cell viability test. The expressions of α5, α2, and β1 integrin were assessed by flow cytometry and immunofluorescence, while osteo-calcin expression was assessed by flow cytometry. The nitrided surface presented higher α2 and β1 integrin expressions, as well as osteocalcin expression, when com-pared to the polished surface, with no alterations in cell viability. These findings seem to suggest that the plasma nitriding treatment produces a titanium surface with the potential for effective in vitro osseointegration.

K E Y W O R D S

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orga-nization.21‒24 _{According to Rosa et al,}25 _{a surface displaying}

titanium nanometer roughness produced by H2SO4/H2O2

sig-nificantly increased α2 and β1 integrin expression, indicating an osteoinductive effect on mesenchymal stem cells (MSC). Therefore, the integrin/extracellular matrix interaction

acti-vates cell migration, morphogenesis, and differentiation.26

Despite these claims, the literature does not describe the in-fluence of integrin expression nor, consequently, the differen-tiation of murine pre‐osteoblastic cells (MC3T3‐E1) grown on a titanium surface treated by plasma nitriding. Thus, this study aimed to assess the response of murine pre‐osteoblastic cells (MC3T3‐E1) cultures on nitrided titanium surfaces.

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MATERIALS AND METHODS

2.1

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Preparation and characterization of

titanium surfaces

CP‐Ti (grade II) discs with pre‐established dimensions (di-ameter: 9 mm; thickness: 3 mm) were produced, treated, and characterized in previous projects carried out by our group at the Plasma Processing Laboratory (LabPlasma) at the

Federal University of Rio Grande do Norte, Brazil.16 _Two

surfaces were evaluated: one surface only polished (PS) and one surface polished and later submitted to plasma treatment to obtain a nitrided surface (NS). All disks were previously sanded with a silicon carbide sandpaper 80 to 2000 MESH) and the polishing was performed using a solution composed of silica colloidal and hydrogen peroxide for 30 minutes. For NS preparation, previously polished disks were conditioned in a vacuum plasma reactor to receive the plasma treatment under the following conditions: nitrogen atmosphere at 8

sccm of hydrogen and 2 sccm of nitrogen (80% H2 + 20%

N2), plasma pressure of 2.2 mbar, and constant temperature

of 500°C for 1 hour.16

Surfaces were previously developed and characterized

by Aires et al.16 _{The surface wettability (θ = 70.20°, PS;}

θ = 45.00°, NS) was determined by the sessile drop method

by mean water contact angle. Surface nanotopography and roughness were observed using a Shimadzu SPM 9600 atomic force microscope (AFM Shimadzu Corporation, Tokyo, Japan). The surface roughness profile comprised av-erage surface roughness (Ra = 0.51 nm, PS; Ra = 21.00 nm, NS), mean maximum profile height (Rz = 10.67 nm, PS; Rz = 269.00 nm, NS), maximum profile peak height (Rp = 8.81 nm, PS; Rp = 186.00 nm, NS), and mean spacing between the peaks (Rp/Rz = 0.83 nm, PS; Rp/Rz = 0.69 nm, NS). Surface morphology was assessed by scanning electron microscopy (SEM‐SSX 550 Superscan, Shimadzu Corp.). TiN thin films’ structural properties were determined based on X‐ray dif-fraction (XRD, Shimadzu‐6000). Due to low thickness of the thin films, GIXRD (grazing incidence X‐ray diffraction)

measurements were obtained, at a flat and fixed incidence angle 2θ sweep detection in the diffractometer.

2.2

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Osteoblastic cell culture

Mouse pre‐osteoclast MC3T3‐E1 subclone 14 cell lines (ATCC‐American Type Culture Collection) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 μg streptomycin. Cells were incubated at 37°C in

humidified atmosphere containing 5% CO2, and the culture

medium was changed every 72 hours.

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Cell viability

Cells were seeded on polished and nitrided titanium discs in 24‐well plates (Techno Plastic Products—TPP, Trasadingen,

Switzerland) at a density of 5.0 × 104 _{cells/surface. The same}

cell density was cultivated in six wells without any discs (polystyrene surface), as a positive control for cell growth. After 24 hours of incubation, an MTT (Invitrogen, Waltham, MA, USA) solution prepared with DMEM medium was added into each well and incubated for 4 hours. The reac-tion product was dissolved in 1 mL of absolute ethanol and a 200‐μL aliquot of the solution was then transferred to 96‐ well plates and absorbances were determined by spectropho-tometry (MKX 200, μQuant, BioTek, Winooski, Vermont) using a Multiskan Ascent microplate reader (Epoch BioTek) at 570 nm. The sample absorbances determined in the MTT assay were used to determine the percentage of viable cells based on a standard curve obtained by culturing three dif-ferent concentrations of MC3T3‐E1 cells in triplicate in a 24‐well plate.

2.4

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Integrin expression

Quantitative integrin expression was assessed by flow

cy-tometry. To this end, 5 × 103 _{cells were grown on the PS,}

NS, and polystyrene surfaces (without Ti disks) of a 24‐well plate for 24 hours using DMEM supplemented with SFB and antibiotics. After the established time, the cells were washed with PBS and trypsinized. They were then incubated at 4°C with 200 μg/mL of each antibody against alpha2 (Santa Cruz Biotechnology, Dallas, TX, USA, Sc‐13546), alpha5 (Santa Cruz Biotechnology, Sc‐13547), and beta1 (Santa Cruz Biotechnology, Sc‐71392) for 60 minutes. After this time and following three washes in PBS, they were incubated for 45 minutes with a FITC‐conjugated anti‐mouse second-ary antibody at a 1:100 dilution (Santa Cruz Biotechnology, Sc‐2010). The samples were subsequently fixed in 0.5% par-aformaldehyde until analysis in the cytometer (FACScanto, BD Biosciences, San Jose, CA, USA). A total of 50 000 events were acquired for each tested surface and for the

controls. The WinMDIversion 2.8 software (a flow cytom-etry application) was used to analyze the mean fluorescence intensity (MFI) provided by the apparatus for each analyzed sample.

For qualitative integrin MC3T3‐E1 cell expression

eval-uations, experiments were performed with 5 × 103 _cells

cul-tured on the assessed surfaces (PS and NS) for 24 hours using DMEM supplemented with SFB and antibiotics. The cells were then fixed in methanol at 4°C for 6 minutes, washed in PBS and distilled water, and then incubated with the primary antibodies against alpha2, alpha5, and beta1 integrins, 2 mg/ mL at 4°C for 60 minutes. Subsequently, they were washed again and incubated for 45 minutes with 2 mg/mL FITC‐ conjugated anti‐mouse secondary antibodies. The surfaces were washed and the cell nuclei were labeled with 0.5 µg/mL DAPI (4′‐6‐diamidino‐2‐phenylindole, Molecular Probes, Eugene, OR, USA) for 15 minutes. After washing, the sam-ples were analyzed by fluorescence microscopy (Zeiss Axio Examiner LSM 710, Oberkochen, Germany). Images were overlain using the Adobe Photoshop 7 software.

2.5

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Osteocalcin expression

Pre‐osteoblastic cells were cultured for seven days on nitrided titanium surfaces and culture plates (positive control) using α‐MEM supplemented with 10% FBS, 50 μg/mL ascorbic acid, and 10 mM β‐glycerophosphate as the culture medium. Cells were trypsinized and incubated for 60 minutes at 4°C with 2 mg/mL murine anti‐osteocalcin antibodies obtained from goat (sc‐18322, Santa Cruz Biotechnology). The iso-tyoic monoclonal antibody was used as a negative control for each test. After three washes in PBS, the cells were incubated with 0.4 mg/mL of the Texas‐Red fluorochrome conjugate antibody (sc‐2783, Santa Cruz Biotechnology) for 30 min-utes at 4°C. Fifty‐thousand events were acquired per tested surface and controls in the flow cytometer (FACScanto BD Biosciences). The WinMDI software version 2.8 (Windows Multiple Document Interface) was used for the data analysis and the results were expressed as MFI.

2.6

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Statistical analyses

The experiments were performed in triplicate using five dif-ferent samples for each surface. Statistical data were sub-mitted to an ANOVA test, followed by the Tukey–Kramer Multiple Comparisons Test using the GraphPad Instat Software, version 3.5 for Windows.

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RESULTS

3.1

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Surface characterization

The SEM images showed finely dispersed particles uni-formly distributed in the Ti matrix (Figure 1). These nanopar-ticles were TiN confirmed by the TiN (1 1 1) and TiN (2 0 0) peaks observed in the indicated X‐ray diffraction analysis (Figure 2). The surface nanotopography was characterized by AFM. The micrographs displayed in Figure 3 indicate the varying topological surfaces before and after treatment. The pre‐treatment surface displayed a relatively smooth and flat surface topology. In contrast, the nitrited Ti surface presented a rough morphology.

3.2

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Cell viability

MC3T3‐E1 cells grown on polystyrene displayed higher vi-ability than those grown on the titanium surfaces (Figure 4). However, the percentage of viable cells was significantly higher on the NS when compared to the PS (P < 0.01).

3.3

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Integrin expression

Histograms generated by flow cytometry revealed an in-crease in α2 and β1 integrin expressions on the NS in rela-tion to the PS, whereas α5 integrin expression did not change significantly (Figure 5). The MFI confirmed the previous histogram, where a significant increase in α2 and β1 integrin expressions on the NS relative to PS (P < 0.001) and no sig-nificant difference for α5 integrin expression were observed.

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3.4

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Immunofluorescence

Differences in cell morphology can be observed in the im-ages obtained for each different surface (Figure 6). Pre‐os-teogenic cells cultured on the PS display a rounded, isolated, or small cluster morphology with irregular immunoreactiv-ity dispersed throughout the membrane. On the contrary, cells grown on the NS present a polygonal morphology with several filopodia, clustered in larger groups, with greater immunoreactivity in the cellular periphery and filopodia.

3.5

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Osteocalcin expression

The osteocalcin expressions’ flow cytometry analyses in pre‐ osteoblasts grown on Ti (NS and PS) surfaces and on the poly-styrene (control) culture plate are displayed in Figure 7. After 7 days of culture under osteogenic differentiation conditions, flow cytometry analyses indicate that osteocalcin expression in pre‐osteoblasts grown on the nitrided titanium surface was higher in comparison to the polished surface (P < 0.05).

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DISCUSSION

Rough nanoscale surfaces mimic the cellular microenviron-ment and may alter cytoskeletal formation, modifying intra-cellular signaling and increasing adhesion, proliferation, and

the production of transcription factors in these cells.5,6,27,28

Thus, indications that they act positively on cellular re-sponses, and, consequently, on better bone integration, are noted.

The surfaces present physical properties, such as hydro-philicity, which act in synergy with the roughness, improv-ing the quality of the cellular response to the biomaterial. This occurs due to the increase in the initial protein deposi-tion and the expression of pro‐osteogenic genes, which are responsible for the potential of cell adhesion and

differen-tiation into cells expressing the osteoblast phenotype.29‒31

Plasma nitriding promoted an increase in the roughness pa-rameters, as well as in surface hydrophilicity (reduced the wetting contact angle), capable of influencing pre‐osteoblast responses. Therefore, the plasma nitriding process altered the roughness and hydrophilicity properties of titanium, thereby

generating a surface with potential for osseointegration.16

Klein et al32 _{confirm that the morphological differences}

between cells cultured on rough and smooth titanium sur-faces exist, with more differentiated morphology noted on rough surfaces. The presence of a greater amount of filopo-dia scattered in several directions is characteristic of this dif-ferentiated morphology, as well as the presence of a greater

amount of focal contacts,5,32 _{similar to what is observed on}

nanomeric titanium surfaces. These morphologically differ-entiated cells express a greater amount of proteins related to osteoblastic differentiation, indicating a more differentiated

phenotype and better osseointegration.6

FIGURE 2 X‐ray diffractogram in GIXRD of the nitrided surface

The research reported herein has demonstrated that the nitrided titanium surface stimulated a significant increase in

α2 and β1 integrin expression. According to Moussa et al,33

α2 and β1 integrin expression on titanium surfaces coated with nitrate oxide plays a crucial role in the mechanism of increased osteoblast (HOS) adhesion when compared to the standard titanium surface. The different arrangements of the αβ units induce cellular responses, which are responsible

for osteoblast growth on titanium surfaces.34 _{Research has}

demonstrated the role of these receptors in the osteogenic differentiation of MSC, involving FAK autophosphorylation

and increased ERK 1/2 levels, which promote the activa-tion of osteogenic transcripactiva-tion factors, such as Runx2 and Osterix, which ultimately activate the genes that encode bone

matrix proteins.35,36

The α2 integrin is related to differentiation signaling, as its expression increases with increasing cell differentiation levels. Therefore, the NS induced higher α2 expression, confirmed by cytometry, indicating the presence of cells presenting a more differentiated phenotype when compared to polished titanium. In addition, the increase in β1 inte-grin expression is indicative of possible improvements in cell surface response. This integrin plays an important role

in cell adhesion to rough surfaces20 _{and is also related to}

the cytokine release that leads to cellular differentiation.32

Galli et al37 _{also demonstrated that FAK phosphorylation}

is prevented with integrin β1 blockade, resulting in cell signaling deficiencies, thus hindering differentiation. The α2β1 combination plays an important role in osteoblast be-havior in relation to the implant surface, and the greater the complexity of this surface, the greater the influence of

these integrins.20

Integrin α5 has also been reported as playing an im-portant role in osteoblast function. This integrin binds ex-clusively to the β1 subunit, forming an α5β1 heterodimer. This molecule binds to the RGD domain of fibronectin and activates several signaling pathways, including FAK/ERK and FAK/PI‐3‐kinase, while also regulating the survival, proliferation, and mineralization of the extracellular

ma-trix, especially in long‐term cell cultures.38‒40 _Keselowsky

et al39 _{produced acidic surfaces obtained by plasma}

blast-ing capable of inducblast-ing greater α5 integrin expression and function, regardless of roughness. The authors argue that the expression and role of this integrin are influenced by the surface chemistry, independent of microstructure. In the present study, the nitriding applied to the surface increased roughness and promoted a chemical change through the pre-cipitation of TiN on the surface, where this treatment was able to modulate the differential expression of α2, α5, and β1 integrins, which play critical roles in osteoblast func-tion regulafunc-tion. These structural microstructure and surface chemistry‐dependent changes differentially alter the affinity of specific integrins, resulting in changes in integrin‐bind-ing profiles. In this regard, knowledge of this mechanism could be explored in the treatment of biomaterial surfaces in order to control integrin‐binding specificity to induce de-sired cellular activities.41

According to Gittens et al,6 _{titanium surfaces with}

nano-structural modifications may increase osteocalcin, which is an osteoblast differentiation marker. A study carried out by Zhang

et al42 _{on hydrogen peroxide‐modified hybrid titanium surfaces}

indicated a high expression of osteocalcin in rat bone marrow

FIGURE 3 Atomic force microscope images of the (A) polished and (B) nitrided surfaces [Colour figure can be viewed at wileyonlinelibrary.com]

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FIGURE 4 Percentage of viable MC3T3‐E1 cells cultured for 24 hours on different surfaces. Significant difference

for a_{Polystyrene × NS P < 0.05;} b_{NS × PS}

P < 0.01 and c_{Polystyrene × PS P < 0.001}

FIGURE 5 α2 (A), β1 (B), and α5 (C) integrin expression in MC3T3‐E1 cells on the PS and NP titanium surfaces and the culture plate (control) after 24 hours of incubation. CN = isotypic control, CP = MC3T3‐E1 cells cultivated on Polystyrene, N = MC3T3‐E1 cells cultivated on the NS and PS = MC3T3‐E1 cells on the PS. D—Mean Fluorescence Intensity (MFI) for the beta1, alpha2, and alpha5 integrins in MC3T3‐E1 cells cultivated for 24 hours on the NS and PS. Upper and lower case letters indicate significant differences between surfaces (P < 0.001)

MSC, thus indicating a beneficial effect of the surface as a pro-moter of osteogenic differentiation. Our experiments corrobo-rate these results as osteocalcin expression in pre‐osteoblasts grown on NSs was significantly increased. As suggested by

Biggs et al,43 _{this increase can be regulated by the focal}

adhe-sion kinase pathway. In fact, the β‐catenin/integrin kinase path-way is indicated as an important signal mediator from surface topographic changes to promote osteogenic cellular

differen-tiation.44 _{In addition to this mechanism, it is possible that the}

expression of the α2β1 integrin heterodimer was necessary and

acted on osteocalcin expression, as observed by Wang et al.45

Taken together, the results reported herein suggest that plasma nitriding is an effective treatment in the develop-ment of a titanium surface more favorable for osseointe-gration. Further pre‐clinical tests are required to prove the efficacy of this method, especially to demonstrate both local and systemic responses to the nitrided titanium surface.

FIGURE 6 Immunofluorescence images of alpha2 (A, D), beta1 (B, E), and alpha5 (CF) integrins in MC3T3‐E1 cells cultured on the NS (A, B and C) and the PS (D, E and F). On the PS, isolated or small‐rounded cells presenting irregular immunoreactivity and dispersed in the cytoplasm (E, →). On the NS, polygonal cells with several filopodia, displaying greater immunoreactivity in the cellular periphery and in filopodia (A, →). DAPI‐ stained nuclei [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 7 Osteocalcin expression in MC3T3‐E1 cells on the PS and NP titanium surfaces and the culture plate (C) after 7 days of incubation (**P < 0.01)

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CONCLUSIONS

Titanium disks treated under a nitrogen atmosphere with surfaces presenting nanoscale roughness and more hy-drophilic than the untreated surface demonstrated a posi-tive influence on the biological activity of pre‐osteoblast MC3T3E1 cells. These treated surface properties were able to increase the expression of important proteins concerning cell adhesion, proliferation, and differentiation. The evi-dence from this study points toward the idea that plasma nitriding is an effective treatment to produce a surface that favors early osseointegration cellular events.

ACKNOWLEDGMENTS

The authors are grateful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ‐ Brasil (CAPES) ‐ Finance Code 001 and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support (pro-cess number 479951/2008 0).

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest with the contents of this article.

ORCID

Carlos Eduardo B. Moura https://orcid.

org/0000-0002-7960-5373

Janine Karla F. S. Braz https://orcid.

org/0000-0002-9570-6465

Michelle de Aires https://orcid.

org/0000-0002-4383-5804

Nainsandra B. Silva Farias https://orcid.

org/0000-0003-0828-109X

Carlos Augusto G. Barboza https://orcid.

org/0000-0003-1979-9919

Hugo Alexandre O. Rocha https://orcid.

org/0000-0003-2252-1221

Clodomiro Alves Junior https://orcid.

org/0000-0002-5547-5922

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How to cite this article: Moura CEB, Queiroz Neto MF, Braz JKFS, et al. Effect of plasma‐nitrided titanium surfaces on the differentiation of pre‐

osteoblastic cells. Artif Organs. 2019;00:1–9. https://

doi.org/10.1111/aor.13438

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