Enzyme-Directed Biomineralization Coating on TiO2 Nanotubes and

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Article Cite This: ACS Biomater. Sci. Eng. 2019, 5, 2769−2777

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Enzyme-Directed Biomineralization Coating on TiO2 Nanotubes and its Positive Effect on Osteogenesis Jialing Wu,† Jingyan Huang,† Jiaojiao Yun,† Jiajun Yang,† Jinghong Yang,† Alex Fok,‡ and Yan Wang*,† †

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Guanghua School of Stomatology, Hospital of Stomatology, Guangdong Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510055, China ‡ Minnesota Dental Research Center for Biomaterials and Biomechanics, School of Dentistry, University of Minnesota, Minneapolis, Minnesota 55455, United States S Supporting Information *

ABSTRACT: Hydroxyapatite (HA)-coated TiO2 nanotubes (TNTs) have been reported to enhance osteogenesis. However, the nanoscale topography of TNTs usually vanishes due to the uncontrollable mineralization on the surface. In this study, TNTs with different diameters(small, 25 nm; medium, 55 nm; and large, 85 nm) were fabricated by anodization in 3 different voltages. Enzyme-directed biomineralization was adopted to deposit calcium phosphate on the above TNTs. The surface structures and properties of the coatings were characterized by scanning electron microscopy, dispersive X-ray spectrometry, X-ray diffraction, and Fourier-transform infrared spectroscopy. The osteogenesis effect of the hybrid TNT/HA and the original TNTs were evaluated. The results showed that hydroxyapatite deposited homogeneously along the TiO2 nanotubes while preserving the intrinsic nanotopography. Mechanically, alkaline phosphatase(ALP) played a critical role in the mineralization and large nanotube size is more favorable for the mineralizing process because of more ALP absorption. Besides, the hybrid nanosurface TNT/HA coating was found to improve the adhesion, proliferation, and osteogenic differentiation of MC3T3-E1 cells compared to pure TNTs. Our study suggests that the hybrid TNT/HA coating constructed by enzyme-directed biomineralization on TiO2 nanotubes is a promising modification strategy for titanium implants. KEYWORDS: TiO2 nanotubes, hydroxyapatite, alkaline phosphatase, biomineralization, dental implant

1. INTRODUCTION Titanium and its alloys have been widely used as implantation or orthopedic materials because of their superior mechanical properties, corrosion resistance, and biocompatibility.1 However, the bioactivity of titanium still needs to be improved to further shorten clinical healing time. To this end, surface modification of titanium has attracted enormous attention. Biochemically, biomimetic coatings containing calcium phosphate (CaP) or bioactive molecules were reported to facilitate osteointegration. Biophysically, nanotopographies such as TiO2 nanotubes (TNTs) could also improve the osteogenesis of titanium implants.2−4 Bone is primarily composed of hydroxyapatite (HA), collagen, and noncollagenous proteins, which are organized in hierarchical structure from the nano, micro, to macro scales.5 Implied by this physical cue in the bone microenviron© 2019 American Chemical Society

ment, TNTs have been explored for surface modifications of titanium for decades. In our previous work, titanium implants coated with TiO2 nanotubes showed superior hydrophilicity and sustainable osteogenic activity both in vitro and in vivo with adequate mechanical strength.6 Additionally, the chemical cue of bone has inspired research on coating CaP including HA onto the titanium surface either by plasma spraying, sol− gel dip coating or depositing stimulated body fluid (SBF).7 Such bioactive coatings also endowed titanium implants with improved biocompatibility.8 Moreover, combined strategies were developed by depositing CaP layers onto the surface of TNTs to induce a synthetic effect.9−11 Highly ordered TNTs Received: March 25, 2019 Accepted: May 17, 2019 Published: May 17, 2019 2769

DOI: 10.1021/acsbiomaterials.9b00418 ACS Biomater. Sci. Eng. 2019, 5, 2769−2777

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ACS Biomaterials Science & Engineering

dispersive spectrometer (EDS) and scanning electron microscope (SEM). To test the hypothesis that the mineralization on TNTs with different diameters relates to the different affinity of ALP to the TNT substrates, the protein adsorption of ALP on TNTs with different diameters was measured by enzyme-linked immunosorbent assay (Elisa). TNT specimens with the above 3 diameters were immersed in 30 μL of ALP (Sigma, Aldrich) solution(0.2 μg/mL) at 37 °C for 4 h. After washing three times with PBS, the elution containing the unbound protein was collected for measurement of the amount of ALP with an Elisa kit (Telenbiotech, China) according to the manufacturer’s instructions. The amount of adsorbed ALP was calculated according to the following formula: The adsorption amount = the initial amount of ALP used − the amount of ALP in the elution 2.3. Surface Characterization. The morphologies and chemical compositions of the samples were observed and detected by SEM (LEO 1530VP FESEM, Zeiss, Germany) equipped with an energydispersive spectrometer (EDS) before and after mineralization in the complete medium for the various periods. From the SEM images, the inner diameters of TNTs were calculated by ImageJ (Rawak Software, Inc. Germany). To analyze the crystalline phase of the coatings, X-ray diffraction (XRD, Bruker D8 ADVANCE, Germany) was used with Cu−Kα radiation in a 2θ range from 20° to 45° as well as Raman spectroscopy (Horiba Jobin Yvon XploRA) at the wavelength of 532 nm of an Ar ion laser (15 mW). The chemical structures of the coatings were detected with Fourier-transform infrared spectra (FTIR, Vector 33, Germany). Roughness and hydrophilicity of annealed TNTs were measured by a profilometer of a laser scanning confocal microscope (LSM700, Zeiss, Germany) and a contact angle analyzer (OCAl5, Data Physics, Germany), respectively. 2.4. Cell Culture. Mouse calvarial preosteoblastic (MC3T3-E1) cells (Chinese Academy of Science, Shanghai, China) were cultured in the complete medium mentioned above at 37 °C in a humidified atmosphere of 5% CO2. The medium was replaced every 3 days. The cells were passaged using 0.25% trypsin when they reached 85% confluence. 2.5. In Vitro Cell Viability and Morphology. Cells were seeded on the surfaces of TNT substrates of 3 different diameters with or without biomineralization via complete medium. Meanwhile, substrates of culture polystyrene (TCPS) and pure Ti were established as control. Cell viability was evaluated using a Cell Counting Kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan) according to the instructions. MC3T3-E1 cells were seeded on the substrates at a density of 2 × 104 cells/mL and cultured for 4 h, 7 days, and 14 days in 48-well plates. At each time point, cells on the substrates were rinsed with PBS and incubated in 165 μL of CCK-8 solution each well (48-well plate) for 2 h, the wavelength of the supernatants at 450 nm was determined with a spectrophotometric microplate reader (Infinite200, Tecan, Switzerland). To observe the morphology of MC3T3-E1 cells on various surfaces, the cytoskeleton of actin was fluorescently stained. After being seeded onto different substrates at the density of 5 × 103 cells/ ml and cultured for 24 h, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min and permeabilized by 0.1% Triton-100 for 10 min. Next, the samples were washed with PBS three times and incubated in 5% bovine serum albumin (BSA) for 1 h. The actin cytoskeleton was then stained with fluorescein isothiocyanate (FITC)-labeled phalloidin (Sigma) diluted in 5% BSA at 1:100 for 2h and DAPI for 5 min. A confocal laser scanning microscope (CLSM, LSM780, Zeiss, Germany) was used to observe cell morphologies. 2.6. In Vitro Osteogenesis Evaluation. 2.6.1. Alkaline Phosphatase Activity of MC3T3-E1 on Different Surfaces. To test the early osteoblast differentiation on various surfaces, we measured alkaline phosphatase (ALP) activity of MC3T3-E1 cells. The cells were seeded on the substrates at the density of 1 × 104 cells/mL and cultured in the osteoblast-inducing medium containing 0.1 μM dexamethasone, 50 μg/mL ascorbic acid and 10 mM β-GP for 7, 14,

were reported to stimulate the nucleation and growth of apatite from SBF solutions.12 Besides, Parcharoen et al.13 demonstrated that TNTs, as an interlayer, increased the bonding strength of the HA coating to the substrate. However, when these two coatings were combined, the advantage of nanotopography of TNTs was usually compromised because of uncontrollable deposition of CaP on the surface.14 Thus, alternative methods for controllable biomineralization on nanotopography are required. Enzyme-directed biomineralization has been widely used in preparing organic bone substitutes such as sol−gel scaffolds or hydrogels.15,16 Thereinto, enzymes play a key role in regulating the forming features and microstructures of apatite in a controllable pattern. Besides the organic material, this technique has also been applied in metal templates. Li et al.17 constructed an amorphous calcium phosphate (ACP) layer on nanotextured titanium surface while retaining the original nanotopography via biomineralization assisted by enzymes in fetal calf serum (FBS) including alkaline phosphate (ALP) and a nucleating agent. Nevertheless, the addition of the nucleating agent makes the process of biomineralization complex and renders the biosafety a concern. Hence, in this study, we aimed to construct a hybrid TNT/ HA coating on titanium surface via controllable biomineralization directed by enzymes on TiO2 nanotubes without using additional nucleating agents. Meanwhile, the regulatory effect of nanotube diameter on the biomineralization process as well as the osteogenic bioactivity of the hybrid coating was investigated.

2. MATERIALS AND METHODS 2.1. Fabrication of TNTs. Commercial pure Ti foils (Baoji Titanium Industry, China) with a diameter of 1 cm were used as experimental substrates. After being chemically cleaned with 10% HF and 15% HNO3 and polished with SiC sandpapers (#400, #600, #1000, #1500), the foils were ultrasonically cleaned in acetone, alcohol, and distilled water, respectively, and dried at 80 °C for further use. Anodic oxidation was performed on a two-electrode conjugation in electrolyte containing 0.5 wt % HF. Voltages of 10, 14.5, and 20 V were selected to form TiO2 nanotubes with three different diameters. After anodization, all samples were rinsed with distilled water and heated at 450 °C for 6 h. Annealed TNTs were used in the following experiments including the surface characterization unless specifically stated. 2.2. Enzyme-Directed Biomineralization of TNTs. 2.2.1. Enzyme-Directed Biomineralization of TNTs via Complete Medium. To controllably deposit CaP on TNTs, we adopted the method of enzyme-directed biomineralization. In brief, annealed TNT substrates were immersed in the mineralizing solution, consisting of complete medium (α-Minimal Essential Media in the presence of 1% penicillinstreptomycin and 5% FBS containing 255 mU/ml ALP) supplemented with 10 mM β-glycerophosphate (β-GP) and 20 mM CaCl2 at 37 °C for 3, 6, 11, and 18 days, respectively.15 The medium was changed every 3 days. The mineralization was performed under aseptic conditions for further bioactivity tests. Meanwhile, pure Ti was also mineralized with the same method as control. 2.2.2. Enzyme-Directed Biomineralization of TNTs via ALP Solution. To determinate the specific role of ALP, one crucial enzyme in the complete medium, during the mineralizing process above, ALP powder (Sigma, Aldrich) dissolved in tris-buffer (pH 7.4) was used as mineralizing solution instead. TNTs were immersed in the tris-buffer solution containing 10 mM β-GP and 20 mM CaCl2 with or without 0.2 μg/mL ALP at 37 °C for 6 days. The mineralization on TNT samples was then analyzed by detecting the chemical compositions and observing the deposition by energy2770

DOI: 10.1021/acsbiomaterials.9b00418 ACS Biomater. Sci. Eng. 2019, 5, 2769−2777

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Figure 1. Surface characterizations of TNTs with different diameters. (A) SEM images of TNTs (scale bar = 200 μm). (B) Mean roughness values. (C) Quadratic average roughness values. (D) Contact angles of TNTs having different diameters. (E) XRD patterns of TNTs with different diameters (inset: detailed view of XRD pattern). (F) Roman spectra of TNTs with different diameters. * indicates P < 0.05. and 21 days. Subsequently, the cells were lysed with 1% Triton X-100 at 4 °C overnight and the ALP activity was measured with An ALP kit (Jiancheng, Nanjing, China) according to the manufacturer’s instruction. The total protein of lysates was also measured using MicroBCA Protein Assay with the wavelength at 450 nm. The ALP activity was then normalized to the total intracellular protein amount. 2.6.2. Immunofluorescence Staining of Osteopontin (OPN). OPN production on various substrates was also visualized by immunofluorescence microscopy. After 14 days of culture, the cells seeded on the substrates were fixed with 4% paraformaldehyde at room temperature for 30 min, permeabilized with 0.1% Triton X-100 for 10 min and incubated with 5% BSA at room temperature for 1 h. Subsequently, the OPN on the samples were stained by incubation with rabbit monoclonal antibody against osteopontin (1:200 in 5% BSA, Abcam, American) at 4 °C for 16 h and mouse-anti rabbit FITC-conjugated secondary antibody (1:200 in 5% BSA, Abcam, American) at room temperature for 1 h. Also, the nuclei were stained with DAPI for 5 min before the samples were observed with CLSM. 2.7. Statistical Analysis. The data were statistically analyzed with SPSS 22.0 (IBM, USA) software and were expressed as the mean ± standard deviation for continuous variables. An ANOVA one-way analysis or Kruskal−Wallis test was used to analyze the differences between groups, and P < 0.05 was considered to be significant.

3. RESULTS 3.1. Surface Characterizations of Nanotube Substrates. The SEM images of TNT samples prepared with anodization at 10, 14.5, and 20 V and annealing were shown in Figure 1A. All the TNTs appeared to be highly ordered. The inner diameters of TNTs increased accordingly with the anodization voltages from 25 to 85 nm (Table 1). Each group was named 25 nm TNT, 55 nm TNT, and 85 nm TNT, respectively. As for the roughness (Figure 1B, C), no significant difference was found between each group except Table 1. Inner Tube Diameters and Wall Thickness of TNTs with Different Diametersa potential (V)

inner tube diameters (nm)

wall thickness (nm)

10.0 14.5 20.0

25.42 ± 2.19 55.44 ± 4.75b 85.18 ± 5.90c

10.53 ± 1.46d 11.16 ± 1.85d 11.93 ± 1.85d

a

a

There was statistical difference between the groups marked with different letters. 2771

DOI: 10.1021/acsbiomaterials.9b00418 ACS Biomater. Sci. Eng. 2019, 5, 2769−2777

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ACS Biomaterials Science & Engineering

Figure 2. SEM images of TNTs after mineralization for 3, 6, 11, and 18 days. All groups of nanotubes maintained their nanotopographies after mineralization for 6 days.

significantly higher Sa value of 55 nm TNT than 25 nm TNT (P < 0.05). Water contact angles (Figure 1D) of all the surfaces with TNTs were lower than 20°, indicating their superior hydrophilicity. Additionally, the contact angle of 85 nm TNT is statistically lower and thus more hydrophilic than the other groups (P < 0.05). The XRD patterns (Figure 1E) showed the phase of TNTs after annealing at 450 °C. The peak of anatase phase at 25.28° was observed in all groups of nanotubes while rutile phase at 27.52° could only be found in 25 nm TNT. Raman spectroscopy was also used to further determine the crystalline phase (Figure 1F). Anatase phase characterized by wavenumbers at 144(3Eg), 397(2B1g), 513(1A1g) and 639(3Eg) cm−118 were found in both 55 nm TNT and 85 nm TNT. As for the surface of 25 nm TNT, brookite phase indicated by Raman active modes at 320(B1g) cm−1, and rutile phase at 143(B1g), 447(Eg), 612(A1g) cm−1, as well as anatase phase at 144(3Eg), 196(3Eg) cm−1 were observed. To sum up, annealed 25 nm TNT contained mixed-crystal phases, especially anatase and rutile, whereas 55 nm TNT and 85 nm TNT formed primarily anatase phase. 3.2. Enzyme-Directed Biomineralization of TNTs. Controllable biomineralization on TNTs was achieved by enzyme-directed biomineralization. The SEM images (Figure 2) showed that after enzyme-directed biomineralization for 3, 6, 11, and 18 days, deposits were observed on the surfaces of the nanotubes, spreading homogeneously by the edge. Furthermore, the deposits became denser and the inner tube diameters decreased as the mineralizing process went on. To the extreme, all the tubes of TNT substrates were completely covered by deposits after 18 days of mineralization. In comparison, the hybrid nanosurface coating of TNT/CaP was fabricated after mineralization for 6 days in view of the well-maintained nanotopographies. In spite of the decrease in inner tube diameters in each group because of the thickened nanotube wall after mineralization, the diameters still increased significantly from group 25 nm TNT to 85 nm TNT (Table 2). Consequently, 6 days was chosen in the following cellular experiment as the optimal mineralization time. The chemical compositions of all coatings were analyzed by EDS in particular of calcium and phosphorus. The results showed that the Ca/P ratio of the deposits on all the samples approximated 1.67 (Figure 3A), which is close to that of HA.

Table 2. Inner Tube Diameters and Wall Thickness of TNTs with Different Diameters after Mineralization for 6 Daysa 25 nm TNT 55 nm TNT 85 nm TNT

inner tube diameters (nm)

wall thickness (nm)

9.71 ± 1.28 34.08 ± 2.36b 63.86 ± 5.72c

17.02 ± 1.58d 22.65 ± 4.05e 22.04 ± 3.45e

a

a

There was statistical difference between the groups marked with different letters.

Therefore, the deposits were supposed to be HA. Furthermore, deposits with notably higher calcium content were observed on 85 nm TNT at all time points than the other groups (Figure 3B). Thus, 85 nm TNT presented the highest level of mineralization. To further determine the crystalline phase of the CaP layer, we then subjected TNT samples after mineralization for 6 and 18 days to XRD and FTIR analysis. The XRD spectra of the mineralized TNTs with different diameters showed peaks assigned to crystalline HA ((002) plane) at 25.82° (Figure 3C, D). The FTIR analysis (Figure 3E, F) showed phosphate stretching frequencies characterized by an absorption band in the region of 980−1090 cm−119 (v1 and v3 vibrational mode of P−O) appeared in all mineralized groups. Weak bands located at 1450 and 1646 cm−1 representing amide III and I could also be observed. Thus, CaP coatings of all groups were deduced to be HA. Synthetically, hybrid coatings combining TNT topography and HA component (TNT/HA) were constructed on the surfaces of titanium by enzyme-directed biomineralization of 6d (denoted as 25 nm TNT-6dm, 55 nm TNT-6dm, and 85 nm TNT-6dm). 3.3. Role of ALP in the Mineralization of TNTs. The role of ALP enzyme contained in the complete medium during the mineralizing process was explored. The results showed that deposits grew on the tube surfaces in the ALP-supplemented solution and reproduced the nanotopographies of TNTs. By contrast, no deposits were observed on the surfaces treated by the solution without ALP (Figure S2, Table S2). Furthermore, the precipitation rate indicated by the ratio of Ca/Ti and ALP absorption on TNTs with different diameters were inves2772

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Figure 3. Chemical analysis of mineralized TNTs with different diameters. (A) Ca/P ratio of the deposits on all the samples approximated 1.67. (B) Ca/Ti ratio of the deposits on TNTs after mineralizing for 3, 6, 11, and 18 days. (C, D) XRD patterns of TNTs after mineralization for 6 and 18 days (insets denote the detailed view of the XRD pattern). (E, F) FTIR spectra of TNTs after mineralization for 6 and 18 days. ***indicates P < 0.001.

tigated. The results showed that the highest ratio of Ca/Ti ratio were found in 85 nm TNT after mineralizing for 6d and the highest amount of ALP absorption (Figure S3) was shown in 85 nm TNT. This is consistent with the results of mineralization directed by the complete medium. 3.4. In Vitro Cell Response to the TNT/HA Coatings. 3.4.1. Cell Viability and Morphology on Different Substrates. MC3T3-E1 cells were used to explore the effect of the hybrid nanosurface coatings on cellular behaviors. Cell viability determined by a CCK-8 kit (Figure 4A) showed that after mineralization, there was a significant or mild increase in the number of initially adhered cells at 4 h on 25 nm TNT, 55 nm TNT, and 85 nm TNT, respectively. Besides, the adhesion levels of all TNT/HA groups were significantly higher than those of pure titanium (P < 0.01). As for cellular proliferation (Figure 4B), all the mineralized TNT groups showed

significantly higher proliferation level than those of pristine TNTs after culturing for 7 days, whereas the lowest level was observed on pure titanium surfaces (P < 0.01). The cell proliferation level at 14 days shared a similar trend with that at 7 d ays, except for no significant difference between 25 nm TNT-6dm and 25 nm TNT. Visualized actin by Fluorescence staining (Figure 4C) showed that the cells on the TNTs and TNT/HA surfaces were polygonal as well as more branched than those on pure titanium. Between the groups of TNT substrates, the cells on the nanotubes with small diameters were well-spread, whereas they were more elongated and spindle-like on larger nanotubes. 3.4.2. Cell Differentiation. To study the osteoblastic differentiation ability of MC3T3-E1 on original and mineralized TNTs with different diameters, we examined the activity 2773

DOI: 10.1021/acsbiomaterials.9b00418 ACS Biomater. Sci. Eng. 2019, 5, 2769−2777

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corresponding TNT substrates, indicating the enhanced osteogenic differentiation ability of mineralized TNTs.

Figure 4. Cell adhesion, proliferation, and morphology on different substrates: (A) cell adhesion on different substrates after culture of 4 h; (B) cell proliferation on different substrates after culture of 7 and 14 days; (C) immunofluorescent images of MC3T3-E1 on different substrates at day 1. The nucleus was stained blue, the cytoskeleton (Factin) was green (scale bar = 50 μm). * indicates P < 0.05 and *** indicates P < 0.001.

of ALP and the expression of OPN. TCPS and pure Ti were used as control groups. The ALP activity of cells cultured on different substrates for 7, 14, and 21 days was measured (Figure 5). The ALP activity

Figure 5. ALP activity on different substrates after culture for 7, 14, and 21 days. The cells cultured on the TNT/HA substrates showed significantly greater levels of ALP than those on the corresponding original TNT substrates on 21 days. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. Figure 6. OPN expression of MC3T3-E1 cells seeded onto different substrate surfaces after culture of 14 days (scale bar = 50 μm). Cell nuclei stain (blue) and OPN (green). The mineralized TNT groups expressed more OPN than the original TNT groups.

of cells on almost all surfaces peaked on day 14. Notably, the ALP activity of cells on 25 nm TNT-6dm was significantly higher than that of 25 nm TNT (P < 0.05), whereas no significant difference between the premineralized and postmineralized surfaces of other groups. However, after 21 days of osteoinduction, ALP activity of the cells cultured on all the TNT/HA substrates was almost doubled compared with those on the corresponding TNT substrates (P < 0.01). No significant difference was found among the TNT/HA groups. The osteogenic differentiation was also evaluated by the immunohistochemical staining of OPN expressed by cells on various surfaces after culturing for 14 d (Figure 6). All the TNT/HA substrates induced more OPN production than the

4. DISCUSSION In this study, anodization and enzyme-directed biomineralization were applied to fabricate a hybrid TNT/HA coating on the titanium surface without sacrificing nanoscale features of TNTs. The impact of this hybrid coating on the adhesion, proliferation and differentiation of osteoblast cells was evaluated. Such enzyme-directed biomineralization have been applied in gel matrix to emulate the hydroxyapatite mineralization 2774

DOI: 10.1021/acsbiomaterials.9b00418 ACS Biomater. Sci. Eng. 2019, 5, 2769−2777

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chemistry, etc.).4 The synergic effect of nanotubes and HA coatings on cell adhesion was obvious in groups of 25 nm TNT-6dm and 55 nm TNT-6dm. We assume that mineralized TNTs possessing smaller dimension tubes than corresponding original TNTs might facilitate the formation focal adhesion. It was reported32 that cell adhesion was severely impaired on nanotube layers with a diameter larger than 50 nm, which may induce cell death. Also, Ca2+ has been verified to enhance cell adhesion and spreading through the effect of Ca2+ bridging in the binding of proteins, which affected cellular interactions with the microenvironment.7 Cell shapes can be altered by the topography of the culture substrates. In this study, cells were more spread-out on the TNTs and mineralized TNTs than smooth Ti. It might relate to the higher surface energy of nanotopography.33,34 In addition, the filopodia of cells went into the structure of the nanotubes, resulting in the interlocked cell configuration. This study further revealed that cells were more elongated on large diameter nanotubes, indicating better osteogenesis inducing ability.35 The nanotopographic cues36 and chemical cues from hybrid coating are perceived by cells through focal adhesion and then transmitted, thus initiating the intracellular signal transduction associated with the subsequent celluar response. From the results of our study, all mineralized TNTs enhanced the proliferation of cells compared with original TNTs, similar with Chernozem and co-workers’ report.10 Our results indicated that cells’ response to biomimetic surface affected by both chemistry and geometry of biomimetic surface of titanium. It has been reported that HA and nanotopography simultaneously triggered bone-related signaling cascades such as Wnt signaling, resulting in the improved proliferation, differentiation and maturation of cells.37 As for original TNTs with different diameters, cell proliferated the fastest on 25 nm TNT, closing to the optimal nanotube diameter of 15 nm for cell behaviors reported in Park et. al’s work.38 However, the effect of tube dimension on cell proliferation was diluted among the mineralized TNTs. Thus, HA seems to play a dominant role on the hybrid coating rather than nanotopography. Besides an increase in cell adhesion and proliferation, a significant increase in the cell differentiation markers, namely ALP activity and OPN expression, was also observed on the surface of mineralized TNTs. The ALP activity is associated with differentiation stage of bone cells.39 Our result showed that the ALP activity of all groups reached a climax at 14 days and then decreased at 21 days, which is in accordance with the study reported by Dolder et. al.40 The ALP activity of mineralized TNTs was significantly enhanced at 21d as compare to the original TNTs, implying that the hybrid TNT/HA nanosurface may affected the late stage of osteogenic differentiation of MC3T3-E1. Furthermore, an up-regulated expression of OPN was also detected in the mineralized TNT groups compared with original TNTs or pristine Ti groups. The possible explanation is that the dissolved Ca2+ from HA increased the extracellular Ca2+ concentration, which raised intracellular Ca2+ through the Ltype calcium channel, activated the CaM-CaMK2 pathway and increased the osteoblast differentiation level.41 All the above increased expression of the cell markers in the TNT/HA groups implied a more favorable bioactivity of this hybrid coating.

process in natural bones. The biomineralization involved the nucleation induced by fiber gel and the temporal control of phosphate ions by enzyme entrapped within the gel.20 Inspired by this, we innovatively applied this system on the metal substrate with TNTs rather than the organic template. The system of enzyme-directed biomineralization consists of enzymes, organic phosphate and calcium ions. In this system, inorganic phosphate ions are gradually released by the hydrolysis of organic phosphate via enzymes in a timedependent manner, avoiding uncontrollable precipitation in solution. The released inorganic phosphate is then combined with calcium ions, initiating the nucleation and growth of the crystal.21 Hence, the process of biomineralization directed by enzymes is more controllable, compared with the simple deposition of HA on TNT surfaces where small particles form and grow into crystals.22 ALP is speculated to play a crucial role in such a process, based on the results that mineralization only occurred in ALP-supplemented solution (Figure S2). The ALP enzyme existing in FBS can cleave off the organic phosphate, increasing the local concentration of inorganic phosphate. The released inorganic phosphate then reacted with the calcium ions, inducing the precipitation of CaP on the nanotube surfaces. Furthermore, previous studies showed that the geometric constraints associated with the morphology of nanostructures might effectively control the nucleation and growth of HA.23 Thus, we further explored the effect of nanotube diameters on the mineralization. In our study, compared with mineralized 25 nm TNT and mineralized 55 nm TNT, the mineralized 85 nm TNT exhibited the most intense peak in XRD and FTIR spectra. This result indicated that the biomimetic mineralization became faster and higher-quality as the tube diameter increased. Consistently, Kodama and co-workers’ work showed that 100 nm TNT favored the apatite formation over that of 15 nm TNT.24 Additionally, titanium with TNTs showed higher rate of mineralization than machined polished pure Ti (Figure S1), demonstrating the importance of nanotubes’ structure in the mineralization. One explanation for this phenomenon is that TNTs with large diameters possess more available areas and empty tube volumes, suggesting more nucleating sites. Another hypothesis is that the better wettability of 85 nm TNT allowed more attraction and adhesion of effective and critical proteins such as ALP, which participated in a reaction onto the surface.25 Our study demonstrated that more ALP was absorbed on 85 nm TNT than 25 nm TNT or 55 nm TNT (Figure S3), which is consistent with previous reports.26,27 Meanwhile, the crystal phase of TNTs can also affect the mineralization. The primary component of 55 nm TNT and 85 nm TNT was anatase titania, which has been reported to be more effective for apatite formation than rutile,28−30 whereas 25 nm TNT mainly consists of rutile. Roughness can be another factor affecting mineralization. The roughness of 55 nm TNT was statistically higher than that of 25 nm TNT, which might result from the normal distribution of surface heights of 25 nm TNT and more distinct leptokurtic distribution with many high peaks of 55 nm TNT.31 However, the roughness of 85 nm TNT showed no difference from the other groups (Figures 1B, C), which may negligibly affect the mineralization. This biologically directed precipitation of HA coating on nanotubes enhanced the adhesion of osteoblasts. Cells adhesion to the titanium surface induced by the focal adhesion is dependent upon surface characteristics (topography, 2775

DOI: 10.1021/acsbiomaterials.9b00418 ACS Biomater. Sci. Eng. 2019, 5, 2769−2777

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ACS Biomaterials Science & Engineering

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5. CONCLUSION In summary, hybrid TNT/HA coatings maintaining the nanotopography of nanotubes were fabricated on the surfaces of titanium via enzyme-directed biomineralization. Within the scope of this study, TNTs with larger diameters were more favorable for mineralization below 100 nm. The substrates with hybrid coatings of TNT/HA promoted the adhesion, proliferation and osteogenic ability of osteoblasts compared with solely nanotube substrates. Thus, this coating can be considered a promising modification strategy for developing highly efficient and bioactive implant surfaces.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.9b00418.

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SEM images of different titanium surfaces before and after mineralization via complete medium; chemical elements of mineralized pure Ti and 85 nm TNT-6dm; surface characterization and chemical elements of TNTs in mineralizing solutions with/without ALP; amount of adsorbed protein on TNTs with different diameters (PDF)

AUTHOR INFORMATION

Corresponding Author

*Email: [email protected] (Y.W.). Fax: 02083822807. ORCID

Yan Wang: 0000-0002-7278-740X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant NSFC 81550013).

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REFERENCES

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DOI: 10.1021/acsbiomaterials.9b00418 ACS Biomater. Sci. Eng. 2019, 5, 2769−2777