Surface chemistry and osteoblast-like cell response on a titanium surface modified by a focused Nd:YAG laser

    Surface chemistry and osteoblast-like cell response on a titanium surface modified by a focused Nd:YAG laser Naofumi Ohtsu, Taro Kozuka, Misao Yamane, Hirofumi Arai PII: DOI: Reference:

S0257-8972(16)31018-0 doi:10.1016/j.surfcoat.2016.10.024 SCT 21669

To appear in:

Surface & Coatings Technology

Received date: Revised date: Accepted date:

1 June 2016 7 October 2016 9 October 2016

Please cite this article as: Naofumi Ohtsu, Taro Kozuka, Misao Yamane, Hirofumi Arai, Surface chemistry and osteoblast-like cell response on a titanium surface modified by a focused Nd:YAG laser, Surface & Coatings Technology (2016), doi:10.1016/j.surfcoat.2016.10.024

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ACCEPTED MANUSCRIPT Surface chemistry and osteoblast-like cell response on a titanium

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surface modified by a focused Nd:YAG laser

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Instrumental Analysis Center, Kitami Institute of Technology, 165 Koen-cho, Kitami,

Hokkaido 090-8507, Japan Department of Biotechnology and Environmental Chemistry, Kitami Institute of Technology, 165 Koen-cho, Kitami, Hokkaido 090-8507, Japan

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Naofumi Ohtsu1,*, Taro Kozuka1, Misao Yamane1, Hirofumi Arai2

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*Corresponding author: Naofumi Ohtsu, Instrumental Analysis Center, Kitami Institute of Technology, 165 Koen-cho, Kitami, Hokkaido 090-8507, Japan

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E-mail: [email protected]. Tel&Fax: +81-157-26-9563

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ACCEPTED MANUSCRIPT Abstract

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The present paper demonstrates that the cellular response on a Ti surface could be enhanced

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through surface treatment by an accurately focused beam of a low-power pulsed Nd:YAG

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laser. Focusing the beam leads to the generation of a laser-induced plasma on the target surface, which makes it possible to enhance the effect of laser irradiation. A chemically stable

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and thin layer of titanium dioxide (TiO2) was formed on the laser-treated substrate, while

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changes in the surface microstructure were hardly observed. This result indicates that the oxide formation was developed through a reaction to the plasma instead of an energy transfer

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from the laser to the surface. The resulting TiO2 layer suppressed the adsorption of the

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contaminant carbon on the Ti surface, which made it possible to maintain hydrophilicity, even after sterilization. In fact, the cellular adhesiveness and cell proliferation on the Ti

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surface were notably improved by the laser treatment despite the low power of 5 and 25 mJ·pulse-1. On the other hand, differentiation of the osteoblast-like cells was not facilitated by the laser treatment.

Key Words: titanium implant, focused Nd:YAG laser, cellular response, surface chemistry

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ACCEPTED MANUSCRIPT 1. Introduction

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Titanium implants have become major devices in orthopedics and dentistry in recent years to

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replace failed or damaged human hard tissues such as hip joints and tooth roots. While the

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biocompatibility and mechanical properties of Ti materials are sufficient in most medical settings, some medical doctors believe that their bio-functions should be further improved. A

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Ti implant used as a substitute for hard tissue should be able to support loads in daily

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activities, and strong bonding between the implant and human bone is required for successful surgical operation [1-2]. However, metallic materials are known as bio-inert substances, and

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thus, bone formation around the metallic surface is generally inactive. They must spend a

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long time to acquire sufficient bonding strength, especially when implanted in an aged patient. Based on these limitations, researchers consider the formation of a bioactive surface the most

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important criterion for producing an ideal implant, and they have devoted much effort to develop new surface-modification processes [3-17]. Bone formation in the human body is achieved by three types of bone cells: osteoblast, osteocyte, and osteoclasts. Forming, maintaining, and remodeling the bone are achieved by the collaboration of these cells. Accordingly, enhancement of cellular viability leads to the acceleration of bone formation. When a medical device is surgically implanted, proteins in human tissues are immediately adsorbed on its surface. Thereafter, the bone cells attach themselves to the proteins, and the cells develop through their proliferation and 3

ACCEPTED MANUSCRIPT differentiation. Bone formation around a material surface is achieved as a result of these

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cellular developments. In the sequential process, protein adsorption is the first event

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occurring around the surface; therefore, controlling the surface chemistry and morphology

improving the bone formation performance [18-20].

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that dominate the protein adsorption behavior is considered to be a major strategy for

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Many methods designed to control the surface properties of a material can modify an

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entire surface, but they cannot be applied to a restricted area. In general, biomedical materials are small pieces with complex shapes, and surface treatments that can effective modify only

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small selected areas are considered innovative techniques. For example, surface processing

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by laser irradiation has excellent flexibility and can treat only the desired part of a surface [21-26]. Laser irradiation of a metallic surface induces several different phenomena such as

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the ablation of the naturally formed oxide and melting of the metal owing to the energy transfer from the laser beam, resulting in changing surface chemistry and increased surface roughness. Lawrence et al. treated a Ti-alloy surface by using a pulsed Nd:YAG laser with a power of 400 W; they demonstrated that the adhesiveness and proliferation of osteoblast cells were enhanced owing to an increase in surface roughness and improved wettability [21]. Hao et al. employed a continuous-wave CO2 laser with a power of 3 kW for laser-surface processing; they revealed that improvement of the surface wettability resulted in an increase in the amount of adsorbed fibronectin, an essential protein for cellular attachment [22]. 4

ACCEPTED MANUSCRIPT However, the laser beams employed in these studies were emitted from relatively

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high-powered laser instruments because the beam had to be “defocused” before irradiating

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the surface to increase the irradiated area. A high-power laser is an expensive instrument with

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a large body, which is a disadvantage in simplicity and flexibility.

It is well known that a plasma consisting of atmospheric gases and solid constituents is

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induced on a solid surface when it is irradiated by a focused laser beam [27]. Such

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laser-induced plasma is expected to aggressively react with the surface, and the resulting layer is different from that formed with a defocused laser. So far, several researchers

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employed a focused laser for forming micro-texture on metallic substrate [28-29]. On the

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other hand, our group has been devoted to developing a new surface process using focused laser irradiation for modifying surface property [30-31]. We previously reported that the

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cellular adhesiveness on a Ti substrate was improved by using a “focused” Nd:YAG laser beam accompanied by a nitrogen gas blow, despite the relatively low power compared with that of a standard laser [31]. The treated surface comprised a primary TiN layer with thickness of approximately 1100 nm, together with a thin overlying oxide layer with thickness of 30 nm. We deduced that the improvement in cellular adhesiveness was due to the thin oxide layer. Based on these results, the cellular attachment on a Ti substrate can presumably be improved by laser processing in air with a low-power laser if the beam is accurately focused. In the present study, therefore, a Ti substrate was treated by focused 5

ACCEPTED MANUSCRIPT Nd:YAG laser irradiation in air. Its surface characteristics such as chemical composition,

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wettability, and surface morphology were then examined. After that, the cellular behavior on

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the treated surface, such as adhesiveness, proliferation, differentiation, and calcification, were

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evaluated using osteoblast-like cells. Together with these results, we investigated the effect of

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focused laser irradiation on the surface characteristics and cellular response.

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2. Experimental procedure

2.1. Surface treatment using a focused pulsed Nd:YAG laser

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A Ti plate (10 mm × 10 mm × 1 mm) and a Ti disk (ϕ 15 mm × 1 mm) with a purity of

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99.5% (Furuuchi Chem. Co., Japan) were used as substrates for cellular response tests and surface analyses, respectively. The substrates were chemically polished using a colloidal

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silica suspension with an average particle size of 40 nm, after which they were ultrasonically washed in ethanol. The substrates were placed on a stepping-motor-driven xy-axis stage (ALD-4011-G1M-R, Chuo Precision Industrial, Japan), and the stage was scanned at a speed of 1 mm s-1. A Q-switched Nd:YAG laser (Minilite II, Continuum Inc., USA), emitting at a wavelength of 532 nm (SHG mode) with a pulse width of 4 ± 1 ns ,was used for the treatment. The laser energy per pulse, determined using a laser power meter, was fixed at 5 or 25 mJ [29]. The laser was repeatedly and automatically triggered at a rate of 8 Hz. The laser beam was focused with a convex lens before it was incident on the Ti substrate in air. The 6

ACCEPTED MANUSCRIPT theoretical spot size of the focused laser beam was about 100 m in diameter, and the

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corresponding energy density was calculated to be about 300 J·cm-2. Under these

surface when a laser beam was irradiated.

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2.2. Characterization of laser-treated Ti surfaces

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experimental conditions, generation of a plasma with blue color was observed on a substrate

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Images of the laser-treated Ti surfaces were obtained with a laser microscope (VK-9500, Keyence, Japan). Changes in the chemical states and compositions of the surfaces in the

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depth direction were analyzed by X-ray photoelectron spectroscopy (XPS; PHI 5000

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VersaProbe, Ulvac-Phi, Japan), using monochromatic Al K radiation (h= 1486.6 eV). The photoelectron take-off angle was set to 45°, and the pass energy was set to 23.5 and 58.7 eV

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for narrow scan and depth analysis, respectively. An Ar ion gun with an acceleration voltage of 3 kV was used to obtain elemental depth profiles. The etching rate, estimated from a SiO2 layer, was approximately 12.5 nm min-1. The contact angle of distilled water was measured using a contact angle meter (DM-CE1, Kyowa Interface Science, Japan).

2.3. Evaluation of cellular response MC3T3-E1 cells (RIKEN BioResource Center, Japan), an osteoblast-like cell line, were cultured in a α-modified minimum essential medium (α-MEM; Gibco-BRL, USA) containing 7

ACCEPTED MANUSCRIPT 10% fetal bovine serum (FBS; JR Scientific, USA) and 1% of an antibiotic–antimycotic

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solution (100 U mL-1 penicillin, 100 µg mL-1 streptomycin, and 0.25 µg mL-1 amphotericin

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B; Gibco-BRL, USA) at 37 °C under a humidified atmosphere of 5% CO2.

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The Ti disk was sterilized with the 70% ethanol solution. Each sterilized Ti disk was placed in a single well of a 24-well polystyrene (PS) cell-culture plate. The MC3T3-E1 cells

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were seeded within each well at a concentration of 1 × 104 cells mL-1 and then incubated for 4

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or 72 h. Morphological analysis of the cells was then performed using scanning electron microscopy (SEM; JCM-5000 NeoScope, JEOL, Japan). To prepare specimens for SEM

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analysis, the cells were fixed with 3% glutaraldehyde and dehydrated by sequential soaking

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in 30%, 50%, 70%, 90%, 95%, and 100% ethanol, for 15 min in each solution. The number of cells attached during incubation was determined using a WST-8 assay (Cell Counting Kit-8,

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Dogin, Japan).

After incubation for 72 h, the medium was replaced with -MEM containing 10% FBS, 1% antibiotics, 2 mmol L-1 -glycerophosphate (Merck KGaA, Germany), and 50 g mL-1 L-ascorbic

acid (Wako Pure Chemical, Japan) to induce the differentiation of MC3T3-E1

cells. The differentiation-inducing medium was changed every 3 days. The alkaline phosphatase (ALP) activity of the differentiation-induced cells was assayed using a LabAssayTM ALP kit (Wako Pure Chemical, Japan) after the cells were retrieved from the Ti substrates. The levels of ALP activity were normalized to the number of cells counted by the 8

ACCEPTED MANUSCRIPT aforementioned procedure. The calcification of the differentiation-induced cells on each Ti

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substrate was evaluated by the alizarin red S staining method [32]. In brief, the cells were

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fixed with 4% formalin and then stained with a 1% alizarin red S solution (pH 4.2) at room

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temperature for 30 min.

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2.4. Statistical analysis

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The data for the cell numbers and the ALP activity are in the format mean ± standard deviation (S.D.), where mean represents the mean value of n measurements and S.D.

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represents the standard deviation. All experiments were performed with n = 3, which means

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that they were repeated three different times to confirm the reproducibility. The experimental data were analyzed with analysis of variance (ANOVA) followed by a post-hock Tukey–

3. Results

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Kramer test to identify the levels of significance (p < 0.01) between the groups.

3.1. Surface characteristics of a laser-treated surface Microscopic images of the untreated and laser-treated surfaces (at laser power levels of 5 and 25 mJ·pulse-1) are compared in Fig. 1. A striped pattern was observed on the laser-treated surface regardless of the laser power during irradiation. The width of the stripe was ~500 μm, which is notably larger than the diameter of the laser beam, ~100 μm. Despite similar 9

ACCEPTED MANUSCRIPT appearances of the surfaces, the arithmetic average roughness (Ra) of the surface treated at 25

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mJ·pulse-1, calculated from the image, was approximately three times larger than that of the

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surface treated at 5 mJ·pulse-1. On the other hand, the Ra value of the surface treated at 5

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mJ·pulse-1 was similar to that of an untreated surface. SEM images of Ti surfaces treated using a focused laser with a power of 5 and 25 mJ·pulse-1 are shown in Fig. 2. A relatively flat

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irradiated by different levels of laser power.

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morphology that included small cracks was observed on both surfaces despite being

To confirm the change in the surface oxide layer, XPS O 1s and Ti 2p spectra were

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obtained (Fig. 3). The shapes of the Ti 2p3/2 and 2p1/2 spectra are symmetric, regardless of the

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laser power. The binding energy of the Ti 2p3/2 peak was 458.7 eV, which is consistent with that of stoichiometric titanium dioxide (TiO2). On the other hand, the untreated Ti surface

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exhibited a small peak corresponding to elemental Ti at a binding energy of 453.8 eV. The O 1s spectra mainly comprise the spectra of metallic oxide states, whereas the spectrum of an untreated surface includes a small tail corresponding to hydroxide or surface-adsorbed water (or both hydroxide and surface-adsorbed water). The atomic ratio of O to Ti ([O]/[Ti]) was calculated from the spectral intensities of Ti 2p and O 1s for further analysis. The [O]/[Ti] ratios from the laser-treated surfaces were approximately 2, which corresponds to the ratio for stoichiometric TiO2, while the ratio of an untreated Ti surface was over 3. In addition, the thicknesses of the surface oxide were estimated from elemental depth profiles by XPS. The 10

ACCEPTED MANUSCRIPT layer thickness on the specimen treated at 25 mJ·pulse-1 was ~100 nm, which is twice the

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thickness of the specimen treated at 5 mJ·pulse-1.

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3.2. Cellular attachment and proliferation on the laser-treated surface

The number of MC3T3-E1 cells attached on the Ti surface was counted after they were

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cultivated for 4 and 72 h (Fig. 4). The number of cells after 4 h reflects the cellular

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adhesiveness on the surface, and the number of cells after 72 h was used to analyze the proliferation behavior. The cell count for the intact PS surface of the culture plate was used as

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a control, and it is also shown in Fig. 4. The quantitative results of the cell count were

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normalized to the initial number of seeded cells. After 4 h of cultivation, statistical analysis showed significant differences between an untreated surface and the laser-treated surfaces,

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although the differences did not depend on the laser power used for irradiation. Similarly, after cultivating the cells for 72 h, statistically significant differences in cell counts were only found between an untreated surface and the laser-treated surfaces, where the cell counts on the laser-treated surfaces were similar to that on the intact PS surface. Fig. 5 presents SEM images showing the cell morphology. While there was no visible difference in cell size, cells on the untreated substrate appeared less spread out; the pseudopods of these cells, which were related to cell viability, were more retracted. It appears that laser treatment enhanced the cell viability, regardless of the laser power. 11

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3.3. Differentiation behavior of osteoblast-like cells on the laser-treated surface

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The ALP activities of the attached MC3T3-E1 cells cultivated for 7, 14, and 21 days after the

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differentiation induction are shown in Fig. 6. The course of the increase in ALP activity was similar on all of the specimens: the activity increased for the first 14 days but leveled off

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thereafter. In addition, no statistically significant differences in the activity were found among

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the three specimens at all times. The cells on each substrate were stained using alizarin red S 35 days after the differentiation induction (Fig. 7). The stained cells on the intact PS surface

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are also shown as a control. The red deposits in the images correspond to the stained calcified

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part. The small red deposits scattered in the images were observed in all substrates, whereas

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hardly any difference in the deposited amount was observed among the three Ti surfaces.

4. Discussion

On the Ti surfaces treated with a focused pulsed Nd:YAG laser, a striped pattern was formed regardless of the laser power, and the width of the striped pattern was considerably larger than the diameter of laser beam used. It is believed that the pattern was formed by the reaction of the Ti surface with the laser-induced plasma. The Ra value on the surface treated at 25 mJ·pulse-1 was larger than those of the untreated surface and the surface treated at 5 mJ·pulse-1, whereas drastic changes in surface microstructure due to melting and 12

ACCEPTED MANUSCRIPT resolidification were not observed on the treated surfaces. It can be said that any predominant

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effect on the surface would have been derived from the plasma induced by the focused pulsed

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laser. Compared with a surface irradiated by a defocused high-power laser, the energy

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transfer from the laser to the Ti surface was considerably low in our study. As a result, drastic changes in the surface microstructure were hardly observed. It was inferred that the use of the

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laser at 25 mJ·pulse-1 could have generated a dense plasma when compared to its usage at 5

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mJ·pulse-1, thereby facilitating the ablation of the Ti surface and the formation of an oxide layer. The difference in the Ra values for different values of laser power originated from the

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depth of the striped pattern. Moreover, the chemical state and composition of the laser-treated

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surface almost agreed with those of stoichiometric TiO2 regardless of the laser power because the topmost layer was formed through the reaction of the Ti surface with the laser-induced

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plasma.

Enhancement of the cellular attachment and cell proliferation was clearly observed on the laser-treated surfaces. It was reported that the formation of a stable TiO2 layer on a Ti substrate through anodic oxidation suppressed the adsorption of surface contaminants, leading to improved surface hydrophilicity, which enhances cellular adhesiveness [33]. We believe that the TiO2 layer formed by the focused laser probably played a role similar to that of the anodic oxide layer. To confirm the hypothesis, the water contact angle (Fig. 8(a)) and the atomic ratio of C to Ti ([C]/[Ti]) adsorbed on the topmost surface (Fig. 8(b)) were 13

ACCEPTED MANUSCRIPT evaluated using a contact angle meter and XPS, respectively. Here, all the data were acquired

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from the specimen surface after it was sterilized with the 70% ethanol solution. As expected,

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the water contact angle on the laser-treated surface was notably lower than that on the

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untreated surface, and the [C]/[Ti] ratios were consistent with the measured contact angles. It was thus concluded that formation of a stable TiO2 layer through the reaction with the

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laser-induced plasma could suppress the adsorption of the contaminant carbon, thereby

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enhancing cellular adhesiveness and cell proliferation. On the other hand, differences in the adhesiveness and cell proliferation with varying laser power were hardly observed in our

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study, which means that the increase in surface roughness by laser processing did not affect

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the cellular response in our case. In general, increasing the surface roughness is advantageous for cellular adhesiveness; for instance, Deligianni et al. reported that the cell attachment and

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cell proliferation on a Ti alloy were increased with a sub-micrometer increase in surface roughness [20]. However, the roughness in our study was over a micrometer in size. This mismatch in surface roughness was probably associated with our unexpected results. In the bone-forming process, osteoblasts are finally differentiated into osteocytes by embedding in the calcified deposits and extra-cellular matrix. In general, the ALP activity and calcified deposits are used as the early and late markers, respectively, in the forming process. Enhancements of the ALP activity and calcification derived from the laser treatment were hardly observed in this study. It is generally accepted that the differentiation of cells is 14

ACCEPTED MANUSCRIPT induced by an external stimulus such as an addition of chemical substance. However, the only

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benefit shown by the laser-treated surface was the protein adsorption behavior related to the

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cellular adhesiveness and cell proliferation. Thus, we consider that this result was consistent

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with the general expectation.

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5. Conclusion

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A titanium surface was modified to improve the cellular response through irradiation by a focused pulsed Nd:YAG laser at relatively low power levels of 5 and 25 mJ·pulse-1. With

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these laser settings, notable changes in the surface microstructure due to melting and

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resolidification were hardly observed. The predominant modifier of the Ti surface was the laser-induced plasma, which resulted in the formation of a thin TiO2 layer on the surface. The

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water contact angle on the sterilized laser-treated substrate was low because the adsorption of the contaminant carbon was suppressed. The cellular adhesiveness and cell proliferation on the Ti surface were notably improved because a hydrophilic surface was formed after the treatment with the focused pulsed laser. It is generally accepted that the enhancements in cellular adhesiveness and cell proliferation follow the facilitation of bone-formation around a surface. On the other hand, differentiation of osteoblast-like cells was not facilitated on the laser-treated surface. In conclusion, surface modification using a focused pulsed laser with relatively low power is a promising strategy for improving bone-formation on a Ti implant. 15

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Acknowledgement

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The authors gratefully acknowledge Mr. Hirano, a graduate student in our laboratory, for his

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support in cell experiments. This work was supported by the Grants-in-aid for Scientific Research (C) (No. 15K06452) from the Ministry of Education, Science, Sports, and Culture

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(MEXT) of Japan.

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micro-texturing of Ti-6Al-4V substrates for improved cell integration, Appl. Surf. Sci. 253 (2007) 7738-7743.

[29] K.T. Voisey, C.A. Scotchford, L. Martin, H.S. Gill, Effect of Q-switched Laser Surface Texturing of Titanium on Osteoblast Cell Response, Phys. Procedia. 56 (2014) 1126-113.

[30] N. Ohtsu, W. Saito, M. Yamane, Thickness of titanium nitride layers formed by focused low-power pulsed Nd:YAG laser irradiation in nitrogen atmospheres, Surf. Coat. Technol. 22

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244 (2014) 57-62.

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[31] N. Ohtsu, W. Saito, M. Yamane, Selectable surface nitridation of titanium using focused

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pulsed Nd:YAG laser irradiation with nitrogen gas blow, Surf. Coat. Technol. 52 (2014)

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[32] K. Oya, Y. Tanaka, H. Saito, K. Kurashima, K. Nogi, H. Tsutsumi, Y. Tsutsumi, H. Doi, N. Nomura, T. Hanawa, Calcification by MC3T3-E1 cells on RGD peptide immobilized on

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titanium through electrodeposited PEG, Biomaterials 30 (2009) 1281-1286.

[33] N. Ohtsu, T. Kozuka, M. Hirano, H. Arai. Electrolyte effects on the surface chemistry

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and cellular response of anodized titanium, Appl. Surf. Sci. 249 (2015) 911-915.

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Fig. 1. Surface images obtained by a laser microscope: (a) untreated Ti surface; Ti surface

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treated by a focused laser with power of (b) 5 mJ·pulse-1 and (c) 25 mJ·pulse-1.

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Fig. 2. SEM images of Ti surfaces treated by a focused laser at different power levels: (a) 5 mJ·pulse-1; (b) 25 mJ·pulse-1.

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Fig. 3. XPS (a) Ti 2p and (b) O 1s spectra obtained from the topmost surface of an untreated

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substrate and substrates treated by a focused laser at 5 and 25 mJ·pulse-1. Fig. 4. Cell count on each specimen: PS, intact polystyrene; Ti, untreated Ti; 5mJ, Ti treated

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by a laser at 5 mJ·pulse-1; 25mJ, Ti treated a by laser at 25 mJ·pulse-1. The cell count was

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normalized to the initial number of seeded cells, after they were cultivated for (a) 4 h and (b) 72 h. Each value represents the mean ± S.D., while the asterisks indicate a statistically

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significant difference (**p < 0.01). The data were statistically analyzed using ANOVA with a post-hoc Tukey–Kramer test. Fig. 5. SEM images of MC3T3-E1 cells attached on (a) an untreated Ti surface and Ti surfaces treated by a focused laser at (b) 5 mJ·pulse-1 and (c) 25 mJ·pulse-1. Fig. 6. ALP activity of MC3T3-E1 cells on each Ti substrate at 7, 14, and 12 days after the induction of differentiation: PS, intact polystyrene; Ti, untreated Ti; 5mJ, Ti treated by a laser at 5 mJ·pulse-1; 25mJ, Ti treated by a laser at 25 mJ·pulse-1. Each value represents the mean ± S.D., while the asterisks indicate a statistically significant difference (**p < 0.01). 24

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Fig. 8 (a) Water contact angle and (b) [C]/[Ti] ratio on each Ti substrate after sterilization in a 70% ethanol solution: Ti, untreated Ti; 5mJ, Ti treated by a laser at 5 mJ·pulse-1; 25mJ, Ti

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> Cellular adhesiveness on a Ti surface was enhanced by a focused laser treatment > Focusing the beam leads to the a laser-induced plasma on the target surface > A thin layer of TiO2 was formed on the laser-treated substrate > The resulting TiO2 layer suppressed the adsorption of the contaminant carbon > Differentiation of the osteoblast-like cells was not facilitated by the treatment

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