Biomimetic apatite coating on yttria-stabilized tetragonal zirconia utilizing femtosecond laser surface processing

    Biomimetic apatite coating on yttria-stabilized tetragonal zirconia utilizing femtosecond laser surface processing Ayako Oyane, Masayuki Kakehata, Ikuko Sakamaki, Alexander Pyatenko, Hidehiko Yashiro, Atsuo Ito, Kenji Torizuka PII: DOI: Reference:

S0257-8972(16)30201-8 doi: 10.1016/j.surfcoat.2016.03.075 SCT 21048

To appear in:

Surface & Coatings Technology

Received date: Revised date: Accepted date:

31 October 2015 23 February 2016 25 March 2016

Please cite this article as: Ayako Oyane, Masayuki Kakehata, Ikuko Sakamaki, Alexander Pyatenko, Hidehiko Yashiro, Atsuo Ito, Kenji Torizuka, Biomimetic apatite coating on yttria-stabilized tetragonal zirconia utilizing femtosecond laser surface processing, Surface & Coatings Technology (2016), doi: 10.1016/j.surfcoat.2016.03.075

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Biomimetic apatite coating on yttria-stabilized tetragonal zirconia utilizing femtosecond laser surface processing

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Ayako Oyane*1, Masayuki Kakehata2, Ikuko Sakamaki1, Alexander

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Pyatenko1, Hidehiko Yashiro2, Atsuo Ito3, and Kenji Torizuka2

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Nanomaterials Research Institute, National Institute of Advanced Industrial Science and Technology

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(AIST), Central 5-41, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan

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Electronics and Photonics Research Institute, National Institute of Advanced Industrial Science and

Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan

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Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST),

Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan

* Corresponding author: Ayako Oyane, Ph.D., Nanomaterials Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 5-41, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan. Tel:+81-29-861-4693, Fax:+81-29-861-3005 E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract (up to 300 words) Yttria-stabilized tetragonal zirconia polycrystals (Y-TZP) have been used in orthopedic and dental implants because of their excellent physicochemical properties. In this study, we

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developed an apatite coating technique with low thermal effects for Y-TZP ceramics based on a biomimetic process using a supersaturated calcium phosphate solution (CP solution) as a

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coating medium. To achieve this, we performed femtosecond laser processing with low thermal effects as a surface pre-modification tool for Y-TZP ceramics. By changing the laser

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scanning mode, we fabricated two different submicro-/micro-structures on the Y-TZP sample. Both laser-treated samples showed increased water wettability due to ablation plasma and

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partly formed an apatite layer on their surfaces in the CP solution within 7 days. To further enhance the apatite-forming ability, we applied an alternate dipping process to the

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laser-treated sample in order to precoat the sample with apatite precursors. The laser-treated

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and precursor-precoated sample successfully formed an apatite layer on the entire surface in

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the CP solution within a shorter time period (24 hours). The thus-coated apatite layer adhered to the sample so strongly that the layer remained on the sample even after the tape-detaching test. This strong adhesion may be attributed to the mechanical interlocking effects due to

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laser-induced surface roughening. Our proposed apatite-coating technique using the laser- and precursor-assisted biomimetic process would be useful for the creation of apatite-coated Y-TZP ceramics for orthopedic and dental applications.

Key words: femtosecond laser, ablation, apatite, coating, zirconia, biomimetic process

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ACCEPTED MANUSCRIPT 1. Introduction Yttria-stabilized tetragonal zirconia polycrystals (Y-TZP) are fine engineering ceramics with the advantages of high chemical durability, mechanical strength, fracture toughness,

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abrasion resistance, and esthetic appearance [1-3]. In addition, zirconia ceramics could show unique electrical and optical properties [4, 5]. Owing to these advantageous characteristics,

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zirconia ceramics have been applied as not only industrial materials but also biomaterials such as hip joint balls, knee joint components, and aesthetic crowns [6-8]. However, despite the

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increasing importance in clinical applications, zirconia ceramics are intrinsically bioinert and generally cannot form a direct bond with the surrounding living bone tissue. Therefore,

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zirconia ceramics with bone-bonding ability have been sought for particular implant applications that require integration with the bone tissue.

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The use of apatite coating is a well-established method for impairing bone-bonding ability

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to base artificial materials. This is because apatite, a calcium phosphate (CaP) compound

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found in human bone mineral, exhibits direct bone-bonding ability in vivo [9]. Among the various apatite coating techniques, low temperature biomimetic processes [10-14] based on pseudo-biomineralization reactions in supersaturated CaP solutions are particularly suitable

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for coating Y-TZP ceramics compared with conventional high temperature processes such as plasma spraying [15] and sputtering [16]. This is because the high temperature processes can induce partial thermal decomposition of apatite [15] and the transformation from the tetragonal phase to the monoclinic phase in Y-TZP, possibly leading to mechanical deterioration of Y-TZP ceramics [17,18]. Since the early 2000’s, a variety of biomimetic apatite coating techniques have been developed for stabilized zirconia ceramics [10-14]. For example, Uchida et al. proposed acid and alkaline treatment methods to induce apatite formation on zirconia/alumina nanocomposite ceramics in a simulated body fluid (SBF) with ion concentrations approximating those in the body fluid [11]. Takemoto et al. applied heat and CaP precoating 3

ACCEPTED MANUSCRIPT treatments to acid-treated zirconia/alumina nanocomposite ceramics to induce apatite formation in SBF [12]. Zain et al. employed polydopamine precoating to induce apatite formation on yttria-stabilized zirconia ceramics in 1.5SBF with ion concentrations 1.5 times

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higher than those of SBF [13]. Klopčič et al. modified the composition of the supersaturated CaP solution to obtain rapid apatite coating on zirconia ceramics [14]. On the other hand, we

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developed a plasma- and precursor-assisted biomimetic process for the formation of apatite coating on polymeric materials [19-22]. In this process, a polymeric material is first treated

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with oxygen gas plasma, and then precoated with apatite precursors to induce apatite formation on the surface in supersaturated CaP solutions. It was revealed that the

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plasma-induced surface wetting [19] and roughening [20-22] are the major factors affecting the apatite-forming ability of the polymeric material and the adhesion of the coating to the

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material surface. While such a gas plasma treatment is effective in increasing the surface

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wettability of zirconia ceramics, it is generally unable to change the surface morphology

materials.

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owing to the higher physicochemical stability of zirconia compared with that of polymeric

Recently, femtosecond laser processing with the high peak power and low thermal effects

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[23] has shown to be effective in producing micro-structures on Y-TZP ceramics via laser ablation [24, 25]. More recently, we fabricated a periodic grating submicro-structure in a checkered micro-pattern on a Y-TZP surface by femtosecond laser processing without a noticeable increase in the monoclinic phase content at the surface (~1 vol%) [26]. In this study, we applied this femtosecond laser processing to Y-TZP ceramics as a surface pre-modification tool in the precursor-assisted biomimetic apatite coating process. Our hypothesis was that the femtosecond laser processing should be effective in producing Y-TZP surfaces with increased surface roughness and wettability via ablation plasma, and this should enable the formation of strongly bonded apatite coatings on Y-TZP ceramics.

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ACCEPTED MANUSCRIPT We fabricated two different submicro-/micro-structures on the Y-TZP sample by changing the scanning mode of the femtosecond laser processing. The laser-treated samples and the samples that were further subjected to the alternate dipping process treatment in Ca and P

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solutions (CaP dipping process) were immersed in a supersaturated CaP solution (so-called CP solution [27]) to enable the formation of an apatite layer on the surfaces (Figure 1). These

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samples were compared to the Y-TZP sample that was subjected to the conventional oxygen gas plasma treatment followed by the CaP dipping process. The surface coverage and

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adhesion to the sample surface of the obtained apatite coatings were then characterized and

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discussed in terms of surface structures and wetting properties of the samples.

2. Material and methods

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2.1. Sample preparation

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Raw Y-TZP powders doped with 3 mol% yttria (TZ-3YB-E) were obtained from Tosoh

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Corp., Japan. Square Y-TZP plates with a thickness of 1 mm and lateral dimensions of 10 mm × 10 mm were prepared by sintering the raw Y-TZP powders at 1350 ºC followed by square-cutting and wet-polishing to mirrored surfaces (Ra < 0.05 m). The Y-TZP plates were

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ultrasonically washed three times with acetone and then dried in air. The resulting plates are hereafter referred to as the untreated Y-TZP samples.

2.2. Femtosecond laser processing for surface pre-modification For the femtosecond laser processing, we employed a laboratory-made Ti:sapphire chirped-pulse amplification system that generates linearly polarized 810-nm-centered 80-fs full width at half maximum (FWHM) pulses at a repetition rate of 570 Hz. The laser beam was focused on the surface of the sample placed on an xyz-stage using a beam shaper (Focal  shaper 9TiS, AdlOptica GmbH, Germany) and a lens with a focal length of 300 mm. The xyz-stage controlled the irradiation position in the x- (horizontal) and y- (vertical) directions. 5

ACCEPTED MANUSCRIPT The focused laser profile was measured by a beam profiler consisting of a CCD camera and an analyzing software. The focused beam was elliptical because of the astigmatism of the laser beam. The intensity profile on the sample was adjusted with the beam shaper to be

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near-Gaussian with a homogeneous part around the beam center. The peak fluence Fpeak of the near-Gaussian beam was estimated using Fpeak =2E/( reff 2)

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(1)

reff = (rx・ry)0.5,

(2)

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where E is the pulse energy, reff is the effective beam radius, and rx and ry are the horizontal and vertical beam radii at 86.5% energy transmission, respectively. By controlling the pulse

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energy E using a variable attenuator and the effective beam radius reff using the z position of the sample, the peak laser fluence Fpeak was set to ~ 4 J/cm2 per pulse; based on the previous

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work, this is higher than the ablation threshold (1.5 J/cm2 per pulse) and falls within the

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effective range (2.7‒ 7.7 J/cm2 per pulse) for the production of the periodic grating structure

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on the surfaces of Y-TZP ceramics [26]. The effective range of the peak laser fluence was experimentally determined by changing the laser energy and the focused beam size on the Y-TZP sample followed by surface morphological observation [26].

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Laser irradiation was conducted on the sample in ambient air with two different scanning modes: the stamp-scan mode and the line-scan mode. In the stamp-scan mode [26], 40 pulses were irradiated onto the same focused region of the sample surface without moving the sample. Irradiation using this stamp-mode generated an ablated elliptical crater on the sample surface with lateral dimensions of ~75 m (x-direction) × ~105 m (y-direction) and a depth of ~8 m. After each stamp-mode irradiation, the sample was repeatedly moved by 60 m steps in the x-direction. When the horizontal stamp scanning was finished at the edge of the sample, the sample was moved by 90 and 30 m in the y- and x-directions, respectively, to irradiate the next row in the same manner. In the line-scan mode, the focusing position was continuously moved in the x-direction at 6

ACCEPTED MANUSCRIPT a constant speed of 0.7 mm/s. The primal line-scan irradiation generated an ablated line with a width of ~105 m and a depth of ~8 m on the sample surface. When the horizontal line scan was finished at the edge of the sample, the sample was moved by 70 m in the y-direction to

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start the scanning of the next line.

For both scanning modes, laser irradiation was repeated to treat the entire surface of the

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sample. The stamp-scan mode generated elliptical craters with a checkered micro-pattern on the sample, as shown in the central image in Figure S1 (this sample is denoted as TZP-Fs).

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The line-scan mode generated an ~70-m-pitch parallel grooving micro-pattern on the sample as shown in the right image in Figure S1 (this sample is denoted as TZP-Fl). The laser-treated

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samples were ultrasonically washed three times with acetone and then dried in air.

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2.3. Alternate dipping in Ca and P solutions (CaP dipping process treatment) for precursor

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precoating

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One of the laser-treated samples (TZP-Fs) and the untreated Y-TZP sample used as a control were subjected to the CaP dipping process [19-22] prior to immersion in the CP solution. The CaP dipping process was conducted for precoating the sample with apatite

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precursors using a simplified version of Taguchi’s method [28]. First, each sample was dipped in 20 mL of an aqueous 200 mM CaCl2 (Nacalai Tesque, Inc., Japan) solution for 10 seconds, dipped in ultrapure water for 1 second, and then dried. The sample was then dipped in 20 mL of an aqueous 200 mM K2HPO4·3H2O (Nacalai Tesque, Inc., Japan) solution for 10 seconds, dipped again in ultrapure water for 1 s, and then dried. The above alternate dipping process in Ca and P solutions was repeated three times. The Y-TZP sample subjected to both the laser treatment (stamp-scan mode) and the CaP dipping process is denoted as TZP-Fs-CaP, and that subjected to the CaP dipping process only is denoted as TZP-CaP. We performed conventional oxygen gas plasma treatment to modify the surface of the Y-TZP sample to compare the results with those obtained using the laser treatment. The 7

ACCEPTED MANUSCRIPT plasma-treated sample was then subjected to the CaP dipping process described above (the resulting sample is denoted as TZP-P-CaP). The plasma treatment was conducted on the untreated Y-TZP sample for 60 seconds in oxygen gas (99.999%) at a pressure of 30 Pa under

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an electric field operating at 13.56 MHz using a compact ion etcher (Model FA-1, Samco

2.4. Immersion in CP solution for apatite coating

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International Inc., Japan). The energy density was adjusted to 1.5 W/cm2.

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The untreated Y-TZP sample and the samples subjected to the different treatments (Figure 1) were immersed in 3 mL of the CP solution at 25 C. After immersion for 1 or 7 days, the

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samples were removed from the CP solution and gently washed with ultrapure water. During the immersion process of up to 7 days, the CP solution remained transparent, indicating the

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absence of homogeneous CaP precipitation.

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To prepare the CP solution, we dissolved reagent grade chemicals (obtained from Nacalai

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Tesque Inc., Japan): NaCl (final concentration = 142 mM), K2HPO4·3H2O (1.50 mM), HCl (40 mM), and CaCl2 (3.75 mM) in ultrapure water under stirring [27]. The solution was finally buffered to pH 7.40 at 25.0C with tris(hydroxymethyl)aminomethane (50 mM) and a

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necessary amount of extra HCl. Prior to use, the prepared CP solution was stored at 4C and was used within 4 weeks after preparation.

2.5. Surface analyses The surfaces of the samples prepared as described in the previous sections were examined using scanning electron microscopes (SEM; Model XL30, FEI Company, USA and S-4800, Hitachi High-Technologies Corporation, Japan), a contact angle meter (Drop Master DM500, Kyowa Interface Science Co. Ltd., Japan), an X-ray photoelectron spectrometer (XPS; VersaProbe, ULVAC-PHI, Inc, Japan), and a thin-film X-ray diffractometer (TF-XRD; Rint-Ultima X, Rigaku Co., Japan). The TF-XRD measurements were carried out at 40 kV 8

ACCEPTED MANUSCRIPT and 40 mA with CuK X-rays with the incident beam angle of 0.7°. The scan speed, step size, and 2 range were 1 or 2°/min, 0.04°, and 3‒ 50°, respectively. In the contact angle measurement, an image of a droplet of ultrapure water was captured 2 seconds after the 1 μL

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droplet established contact with the sample surface. To examine the variation of the surface wettability with time, the contact angle of water droplet on the laser-treated sample (TZP-Fl)

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was measured after storing the sample in air for various periods up to 27 weeks. The contact angle measurement was conducted at 3‒ 4 different points on the sample surface to obtain the

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average and standard deviations of the measured data.

Thickness of the apatite layer was measured using a 3D color laser scanning microscope

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(VK-9700, KEYENCE Corporation, Japan). Before observations, one or more slits were made in the apatite layer using a metallic knife. Three dimensional images were captured at 9

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or more different points for each sample to calculate an average value and standard deviation

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of the layer thickness. For selected fully coated samples, adhesion of the apatite layer to the

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sample surface was examined by a preliminary tape-detaching test. Scotch® mending tape (3M, USA) was attached onto the half region of the sample surface and then detached from the surface. The tape-detached surfaces were examined using an SEM and an energy-dispersive

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X-ray analyzer (EDX; Genesis 2000, AMETEK, Inc., USA).

3. Results and Discussion 3.1. Surface structural changes due to laser treatment The TZP-Fs and TZP-Fl samples, with different submicro- and micro-structured surfaces, were fabricated by the femtosecond laser processing using the stamp- and line-scan modes, respectively. As shown in the center image of Figure 2, a periodic grating submicro-structure was formed by stamp-scan mode processing in the inner region of each elliptical crater in the checkered micro-pattern. Approximately half of the surface area exhibited such a periodic grating structure while the other half showed a non-periodic rough surface. The period 9

ACCEPTED MANUSCRIPT between the adjacent grates was approximately 0.8

1 μm, and the direction of the grates

was parallel to that of the linear polarization, as previously reported [26]. Such a structure is known as a laser-induced periodic surface structure (LIPSS), and has been reported for

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various materials including metals, semiconductors, and dielectrics [29-31]. The most widely accepted mechanism of LIPSS formation is that the surface-scattered laser field including the

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surface plasmon or surface plasmon polariton or the electromagnetic wave generated by carriers interferes with the incident laser field causing periodically strengthened and

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weakened surface ablation with a period determined by the wavelengths of the incident and scattered fields [32]. The geometrical profile of LIPSS reaches a certain steady state during

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repeated laser pulse irradiations. In addition to the grating structure, other structures including ripples and dots can be produced depending on the materials and irradiation conditions such

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as polarization and incidence angle [33]. In this study, we fabricated a periodic grating

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submicro-structure on the Y-TZP sample (TZP-Fs) by employing the irradiation peak fluence

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of ~ 4 J/cm2 per pulse, which is in the reported appropriate range for generating the LIPSS [26]. Different from previous reports on other materials [26-28], the period of the LIPSS observed on the Y-TZP sample was larger than the laser wavelength. Such a large period is

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difficult to be explained only by the commonly accepted surface plasmon polariton model. Surface melting and re-solidification processes might also be involved in the formation of LIPSS on the Y-TZP sample [33]. Detailed experimental and theoretical research is required to fully understand the mechanism of LIPSS formation. By changing the laser scanning mode to the line-scan mode, we fabricated a random porous submicro-structure in the parallel grooving micro-pattern on the Y-TZP sample (TZP-Fl). As shown in the right image in Figure 2, a submicro-scale porous structure without an apparent periodicity was formed on the inner region of each line of the grooving micro-pattern on the TZP-Fl surface. With the exception of the use of the scanning mode, the laser irradiation conditions were the same as those used to prepare the TZP-Fs sample. Thus, 10

ACCEPTED MANUSCRIPT LIPSS observed on the TZP-Fs surface could be formed on the TZP-Fl surface, considering that the irradiation peak fluence (~ 4 J/cm2 per pulse) was within the range (2.7‒ 7.7 J/cm2 per pulse [26]) appropriate for the LIPSS formation. However, this was not the case, most likely

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because of the post surface ablation by the outer part of the focused beam, where the fluence

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was higher than the ablation threshold (1.5 J/cm2 per pulse [26]) of the Y-TZP surface but

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insufficient (less than 2.7 J/cm2 per pulse [26]) for the LIPSS formation. Note that the laser beam with a near-Gaussian intensity profile exhibits a lower fluence in the outer (near-edge)

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part than that in the central part. With the line-scan mode, the central part of the beam that is effective in LIPSS formation is always followed by the lower fluence outer part of the same

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beam during the continuous movement of the irradiated beam position. Therefore, even if irradiation with the central part of the beam (~ 4 J/cm2 per pulse) temporarily produces LIPSS

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on the sample surface, the following irradiation with the outer part of the same beam (1.5–2.7

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J/cm2 per pulse) ablates the surface and destroys the produced LIPSS, leaving a random

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porous submicro-structure without LIPSS on the surface after the line-scan mode irradiation. This conclusion was supported by the appearance of LIPSS only in the central region of the finally stopped beam position on the Y-TZP surface when the scanning was stopped during

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the line-scan mode irradiation.

3.2. Surface water wettability of the laser-treated samples Both laser-treated samples (TZP-Fs and TZP-Fl) showed higher water wettability than the untreated Y-TZP sample. The contact angle of water droplet on the untreated Y-TZP sample was as high as approximately 80⁰; remarkably, this value decreased to approximately 20⁰ after the femtosecond laser processing irrespective of the scanning mode (Figure 3a). The water contact angle values of water droplet observed for the TZP-Fs and TZP-Fl surfaces were comparable. Thus, femtosecond laser processing for the Y-TZP ceramics was found to be effective in increasing surface water wettability and surface roughness. Controlled surface 11

ACCEPTED MANUSCRIPT wettability by LIPSS formation has been reported also for metallic materials [34, 35]. It is not clear yet why the contact angle of water droplet on the Y-TZP surface decreased by the laser treatment. There are various factors affecting contact angles of water droplet on a

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material, including surface morphology, surface chemistry (surface free energy, surface contamination, etc.), reactions with water (swelling, degradation, etc.), and measurement

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conditions (amount of water droplet, ambient temperature, etc.). We consider that the latter two factors are ignorable, because Y-TZP is chemically stable and measurements were carried

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out under constant conditions. The former two factors might affect contact angles of water droplet on the Y-TZP surface. As described in the preceding section, surface roughness of the

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Y-TZP sample was drastically increased after the laser treatment due to surface ablation (Figure 2). The laser-induced surface ablation involving plasma may also affect surface

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chemistry of the Y-TZP surface by eliminating hydrophobic contaminants from the surface

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and/or producing polar Zr-OH groups on the surface. It should be emphasized here that the

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crystalline phase of the Y-TZP surface remained almost unchanged (the monoclinic phase content at the surface was less than 1 vol% even after the laser treatment [26]). We examined the variation of the surface wetting property of the laser-treated TZP-Fl sample with time.

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When this sample was stored in air, the contact angle of water droplet on the sample surface increased to the level comparable to that found for the untreated Y-TZP sample with increasing storing period of up to 27 weeks (Figure 3b4). A similar phenomenon, i.e., transition from wetting to non-wetting of the TZP-Fl surface with time, was reproduced by three independent experiments using different plates. The decrease in surface water wettability with time is probably due to the decrease in surface free energy caused by adsorption of hydrophobic contaminants and/or elimination of polar Zr-OH groups on the surface. SEM observations confirmed that the submicro- and micro-structures on the TZP-Fl surface remained unchanged even after the above storing process. Thus, the observed time-dependent decrease in surface wettability is independent of the surface morphology. 12

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3.3. Apatite-forming ability of the laser-treated samples Partial coating of apatite in the CP solution was attained on the laser-treated TZP-Fs and

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TZP-Fl samples. Apatite-forming ability of the laser-treated samples and the untreated sample used as a control was assessed 7 days after the immersion of the samples in the CP solution.

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Neither SEM (Figure 4, left image) nor TF-XRD (Figure 5) results showed any sign of apatite formation on the surface of the untreated sample. This result is a natural consequence of the

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bioinert nature of Y-TZP ceramics and is in agreement with the previous report [13]. By contrast, a low crystalline apatite layer with a microscale plate-like structure partially

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covering the surfaces of the laser-treated TZP-Fs and TZP-Fl samples was observed (Figure 4, center and right images). For both samples, the coating coverage was less than two thirds of

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the entire surface, with the rest of the surface area remaining uncoated. The surface layers

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were composed of calcium phosphate as proven by their XPS spectra (Figure S2). As shown

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in Figure 5, XRD peaks ascribed to hydroxyapatite (marked with open circles) were observed for the laser-treated TZP-Fs and TZP-Fl samples. Other sub-peaks ascribed to hydroxyapatite (at 11, 17, 40, and 47⁰) were very weak in intensity, because of the low crystallinity of the

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apatite phase that was formed in the CP solution at normal temperature and pressure. According to the results of laser microscopy, the apatite layers on the TZP-Fs and TZP-Fl samples had a thickness of 4.8 ± 1.2 μm and 4.8 ± 0.6 μm, respectively. These results show that femtosecond laser processing with both scanning modes is an effective tool for pre-modification of Y-TZP ceramics’ surfaces in the biomimetic apatite coating process using the CP solution. We propose a following mechanism for apatite formation on the laser-treated samples. In metastable supersaturated CaP solutions such as the CP solution, nano-sized prenucleation clusters can stably exist without inducing any homogeneous CaP nucleation [36-38]. Owing to the better wettability of the laser-treated Y-TZP surfaces, these nanoclusters and their 13

ACCEPTED MANUSCRIPT aggregates accumulate more easily on the laser-treated Y-TZP surface than on the untreated Y-TZP surface in the CP solution. The Zr-OH groups exposed to the laser-treated Y-TZP surface via ablation plasma might be involved in the surface accumulation of the CaP

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nanoclusters [11, 39]. The accumulated CaP nanoclusters are then densified and fused to form a continuous layer of amorphous CaP (ACP) on the surface [38]. Because the CP solution is

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supersaturated with respect to not only apatite but ACP, tThe ACP layer spontaneously grows upward and thickens by further incorporating calcium and phosphate ions and/or CaP

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nanoclusters in the CP solution. Simultaneously, the ACP transforms into crystalline apatite, because apatite is the most thermodynamically stable phase among all the CaP compounds

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under neutral pH conditions [40]. As a result of the 7-days immersion in the CP solution, a micro-thick apatite layer composed of well-developed plate-like crystals is thus formed on the

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laser-treated Y-TZP surface. The uncoated area of the surface may not have a sufficient

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amount of CaP nanoclusters that act as seeds on the growth of apatite layer in the CP solution.

3.4. Apatite-forming ability of the laser-treated and CaP-dippedsamples A full coating of apatite in the CP solution was successfully accomplished by applying the

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CaP dipping process to the laser-treated sample. The CaP dipping processinvolves the precoating of nanoparticulate ACP that is a precursor of the crystalline apatite on a material surface [41]. The CaP dipping process was applied to the TZP-Fs sample (the resulting sample is denoted as TZP-Fs-CaP) and the untreated Y-TZP (the resulting sample is denoted as TZP-CaP) as a control. The apatite-forming ability of the CaP-dipped samples was assessed 24 hours after immersion in the CP solution. The TZP-Fs-CaP sample formed an apatite layer on almost the entire surface within 24 hours in the CP solution, as shown by the SEM (Figure 6, right image) and TF-XRD (Figure 7) results. The XPS analysis confirmed that the surface layer was composed of calcium phosphate (data not shown). Thickness of the apatite layer was 3.0 ± 0.7 μm, which was thinner than the apatite layer formed on the TZP-Fs sample 14

ACCEPTED MANUSCRIPT because of the shorter immersion period in the CP solution. Note that the TZP-Fs sample formed an apatite layer only on a part of the surface in the absence of the CaP dipping process even after a longer immersion period (7 days) in the CP solution (Figure 5, center image).

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These results verify the effectiveness of the additional CaP dipping process in enhancing the apatite-forming ability of the laser-treated sample in the CP solution.

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In addition to the CaP dipping process, the use of the laser treatment contributed to the successful formation of the apatite coating on the surface of the TZP-CaP-Fs sample in the CP

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solution. In fact, in the absence of the primal laser treatment, the CaP-dipped sample (TZP-CaP) did not form apatite at all on its surface as shown in Figure 6 (left image) and

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Figure 7. This might be attributed to the insufficient water wettability of the untreated Y-TZP sample (see Figure 3a) for CaP precoating in the CaP dipping process. It has been reported

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that the CaP dipping process can yield a precoating layer of CaP on surfaces with sufficient

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water wettability [19]. Thus, laser-induced surface wetting (see Figure 3a) plays an essential

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role in the precursor-assisted biomimetic process by making the surface suitable for the CaP precoating. The precoated CaP layer can act then as seeds for apatite growth in the CP solution that is supersaturated with respect to CaPs [41]. Aas described in Section 3.3., the

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precoated CaP layer grows upward and matured crystallochemically with time in the CP solution. As a result of this, a micro-thick layer of low crystalline apatite was formed on the entire surface of the TZP-CaP-Fs sample within 24 hours in the CP solution (Figures 6 and 7). Precursor-assisted biomimetic processes similar to the process described above have been used to create apatite coating on various polymeric materials in combination with a plasma pretreatment [19-22]. Here, we demonstrated that femtosecond laser processing for Y-TZP ceramics is an effective surface pre-modification tool for ensuring sufficient wettability for overall apatite coating in the precursor-assisted biomimetic process.

3.5. Apatite coating adhesion 15

ACCEPTED MANUSCRIPT The roughening of Y-TZP ceramics’ surfaces generated by femtosecond laser processing reinforces the adhesion of apatite coating obtained by the precursor-assisted biomimetic process. The coating adhesion was examined by a preliminary tape-detaching test for the

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TZP-Fs-CaP sample and for the TZP-P-CaP sample used as a control. The control TZP-P-CaP sample was prepared by applying the conventional oxygen gas plasma treatment instead of the

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laser treatment prior to the CaP dipping process (see Figure 1). The plasma treatment increased water wettability of the sample surface (contact angle of water droplet decreased to

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approximately 5⁰); however, it did not cause any surface morphological changes in submicroand micro-scales (Figure S3). Therefore, their surface morphologies show a critical difference

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between the laser-treated and plasma-treated samples: the former exhibits a checkered micro-structure containing periodic grating submicro-structures (center images in Figures S1

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and 2), whereas the latter exhibits a flat surface on submicro- and micro-scales (Figure S3).

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After the CaP dipping process treatment followed by 24-h immersion in the CP solution, both

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samples (TZP-Fs-CaP and TZP-P-CaP) were fully coated with an apatite layer. The surface apatite layer formed on the TZP-P-CaP sample was peeled off from the surface as depicted in the upper right image in Figure 8 (see the lower half region of the image where the tape was

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attached and then detached). No Ca or P was detected by EDX measurements on the tape-detached region of this sample. In contrast, such a complete detachment of the coating was not observed on the TZP-Fs-CaP sample by the same tape-detaching test (upper left image in Figure 8), although the surface microstructure changed as shown in the magnified images (compare lower right two images in Figure 8). The presence of Ca and P was detected by the EDX measurements on this layer remaining on the tape-detached region of the sample. It is considered from these results that the apatite layer adhered to the TZP-Fs-CaP sample so strongly that the tape-detaching test resulted in the occurrence of fracture not at the layer-sample interface but within the layer. Mechanical interlocking effects due to the laser-induced surface roughening may be responsible for the better coating adhesion for the 16

ACCEPTED MANUSCRIPT TZP-Fs-CaP sample than for the TZP-P-CaP sample. While the interfacial structure remains to be clarified, we suggest that the apatite nano-crystals in the coating layer filled the space throughout the periodic grating submicro-structure of the TZP-Fs-CaP surface, with the

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resulting interlocking interface reinforcing the coating adhesion.

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3.6. Possible future applications

Apatite-coated Y-TZP ceramics can be created by the present combination of femtosecond

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laser processing and precursor-assisted biomimetic process. Y-TZP ceramics are intrinsically bioinert and exhibit no bone-bonding ability in vivo; hence, their use in orthopedic and dental

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implants often requires cement fixation that is associated with a risk of allergic side effects. A previous report demonstrated the bone-bonding ability of apatite-coated zirconia/alumina

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nanocomposite which was prepared by a similar precursor-assisted biomimetic process in

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combination with acid and heat treatments [12]. It is expected that the apatite-coated Y-TZP

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ceramics, prepared by our laser- and precursor-assisted biomimetic process, also exhibit bone-bonding ability through the interfacial apatite layer, eliminating the need for the cement fixation.

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The proposed apatite coating technique using the laser- and precursor-assisted biomimetic process has advantages of low thermal effects on Y-TZP ceramics and highly safe coating media without any toxic reagents. The primal femtosecond laser treatment can modify the Y-TZP surface without inducing significant tetragonal-to-monoclinic phase transformation. The monoclinic content at the Y-TZP surface after the equivalent laser treatment was only approximately 1 vol% in the top 10-μm-thick surface layer examined by XRD [26]. The subsequent CaP dipping process for CaP precoating and the CP solution-immersion process for apatite coating are conducted under normal temperature and pressure conditions in coating media composed of water and serum ions. An apatite coating medium (CP solution in this study) with added biomolecules (proteins, antibacterial agents, nucleic acids, etc.) can 17

ACCEPTED MANUSCRIPT produce biomolecule-immobilized apatite layers [42, 43]. Depending on the type of the immobilized biomolecule, the apatite-coated Y-TZP ceramics could possibly be tailored to exhibit higher bone-bonding ability and/or additional biological functions.

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In the proposed apatite coating technique, the femtosecond laser processing played an essential role in the formation of the apatite coating because of the laser-induced surface

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wetting (Figures 6 and 7). In addition, femtosecond laser processing had a reinforcing effect on apatite coating adhesion because of the laser-induced surface roughening (Figure 8). More

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detailed surface reactions in the present laser processing and effects on mechanical properties of the Y-TZP ceramics as well as in vivo biological responses to the apatite-coated Y-TZP

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ceramics are subjects for future studies.

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4. Conclusions

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Apatite coating on the Y-TZP ceramics was successfully accomplished by the

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precursor-assisted biomimetic process in combination with the preliminary femtosecond laser The

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processing

induced

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wetting

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submicro-/micro-roughening via ablation plasma, enabling the formation of a complete and

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strongly bonded coating of apatite on the Y-TZP ceramics in the CP solution. The coating technique with low thermal effects demonstrated in this work would be useful for fabrication of apatite-coated Y-TZP ceramics with bone-bonding ability.

Acknowledgements This study was supported by KAKENHI (25108517, 15H00906) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. A part of this study was conducted at the AIST Nano-Processing Facility, supported by the "Nanotechnology Platform Program" of MEXT, Japan.

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ACCEPTED MANUSCRIPT Appendix A. Supplementary data

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Supplementary data to this article can be found online at

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ACCEPTED MANUSCRIPT Figure captions Figure 1 Diagram of the experimental procedure.

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Figure 2 SEM images of the surfaces of the untreated Y-TZP sample (left image) and of the TZP-Fs (center image) and TZP-Fl (right image) samples. The inset shows the magnified image. Figure 3 (a) Water cContact angle of water droplet on the surfaces of the untreated Y-TZP sample and of the TZP-Fs and TZP-Fl samples (average ± standard deviation). (b)

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Variation in water contact angle of water droplet on the untreated Y-TZP sample and the TZP-Fl sample with storing period of up to 27 weeks (average ± standard deviation).

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Figure 4 SEM images of the surfaces of the untreated Y-TZP sample (left image) and of the TZP-Fs (center image) and TZP-Fl (right image) samples that were immersed in the CP solution for 7 days. The insets show the magnified images.

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Figure 5 TF-XRD patterns of the surfaces of the untreated Y-TZP sample and of the TZP-Fs and TZP-Fl samples that were immersed in the CP solution for 7 days.

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Figure 6 SEM images of the surfaces of the TZP-CaP (left image) and TZP-Fs-CaP (right image) samples that were immersed in the CP solution for 24 hours. The insets show the magnified images. Figure 7 TF-XRD patterns of the surfaces of the TZP-CaP (left image) and TZP-Fs-CaP (right image) samples that were immersed in the CP solution for 24 hours. The insets show the magnified images.

Figure 8 SEM images of the surfaces of the apatite-coated TZP-Fs-CaP (upper left image) and TZP-P-CaP (upper right image) samples after the tape-detaching test. The lower half surfaces below the dotted line denote the tape-detached regions. The lower three images are the magnified images of the surface of the apatite-coated TZP-Fs-CaP sample.

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Highlights

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We developed an apatite coating technique with low thermal effects on Y-TZP ceramics. Femtosecond laser processing effectively modified the Y-TZP surface for coating. A continuous apatite coating adhered firmly to the Y-TZP surface. Laser-induced surface wetting & roughening contributed to full & firm apatite coating. Y-TZP ceramic implants with bone-bonding ability might be created.

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