TiO 2 Nanonetwork on Rough Ti Enhanced Osteogenesis In Vitro and In Vivo

Abstract

The objective in this study was to enhance osteogenic responses (in vitro and in vivo) to roughened titanium (Ti) dental implants through the formation of superhydrophilic TiO₂ nanonetwork surface structure. Sandblasting and acid etching (SLA) was used to roughen the Ti surface. An electrochemical anodization process was then used to form a superhydrophilic TiO₂ nanonetwork on the SLA Ti surfaces. The pore size of the nanonetwork structure ranged from a few nanometers to more than 100 nm, which is on the same scale as many biological species. Human bone marrow mesenchymal stem cells were used as an in vitro test model. The TiO₂ nanonetwork structure was shown to have a significantly positive effect on hydrophilicity, protein adsorption, cell adhesion, cell migration, cell mineralization, and the gene and protein expression of osteogenic markers. The osseointegration of an anodized SLA screw-type Ti dental implant was investigated in vivo via implantation in the femur of New Zealand white rabbits for durations of 4 or 12 wk. The presence of a superhydrophilic surface TiO₂ nanonetwork was shown to significantly enhance the bone-to-implant contact of the roughened SLA screw-type Ti dental implants. Overall, the proposed superhydrophilic TiO₂ nanonetwork structure on the roughened SLA Ti surface proved highly effective in enhancing osteogenic responses in vitro and in vivo.

Keywords

dental implants surface properties nanostructures hydrophilicity bone-implant interface osseointegration

Introduction

Surface modification is crucial to the long-term osseointegration of titanium (Ti) dental implants, particularly for patients with poor bone quality. Various surface modifications have been developed for Ti dental implants (Najeeb et al. 2019). However, the resulting implants differ considerably in terms of surface topography, chemistry, wettability, and porosity, all of which are crucial to osseointegation (Karjuna and Ganapathy 2020). A macro-rough topography provides good mechanical interlocking between implant and surrounding bone (Langowski et al. 2019). Cells on hydrophilic porous Ti surfaces show greater attachment, extracellular synthesis, and osteogenic marker production (Zhao et al. 2005). Different cellular responses are affected by specific surface nanopore diameters of the substrate (Oh et al. 2009; Lv et al. 2015; Bello et al. 2017; Nasrollahi et al. 2017). Overall, it appears that a surface combining a micro-/macro-rough topography with a nanoporous structure, biocompatible chemistry (e.g., TiO₂), and good wettability would provide an ideal surface for cell response.

Sandblasting and acid etching (SLA) is commonly used to produce a combination of macro- and micro-rough surface features on Ti dental implants. As for increasing the hydrophilicity of Ti dental implants, a global well-known superhydrophilic Straumann SLActive implant surface, with a water contact angle of approximately 0°, is processed by rinsing the SLA surface in a nitrogen chamber and then storing in isotonic sodium chloride solution with no atmospheric contact (Alayan et al. 2017; Masrouri et al. 2020). In our previous research (Yang and Huang 2019), we applied an electrochemical anodization process to produce a superhydrophilic TiO₂ nanonetwork on a smooth Ti surface to enhance in vitro biocompatibility and surface bioactivity. The current study is based on the hypothesis that a superhydrophilic TiO₂ nanonetwork with a mixed pore size range on a roughened SLA Ti surface would enhance osteogenesis in vitro and in vivo. We investigated the osteogenic responses of human bone marrow mesenchymal stem cells (hBMSCs), including adhesion, migration, proliferation, mineralization, and differentiation. We also studied the osseointegration in vivo of the same material in the form of a roughened SLA screw-type Ti dental implant, in terms of bone-to-implant contact (BIC).

Materials and Methods

Material Preparations

Commercial-grade II Ti (Ø 15 × 1 mm) disks (Ultimate Materials Technology) were used as specimens for in vitro analysis. Specimen surfaces underwent sequential grinding using SiC papers until #1200. SLA Ti surfaces were prepared via sandblasting with large-grit alumina particles (size 250 µm; pressure 4 bar), followed by acid etching in HF/HNO₃ solution (1% HF/30% HNO₃/H₂O = 50%:30%:20%, v/v/v) (Sigma). The specimens then underwent sequential electrochemical anodization treatments in 5 M NaOH solution under anodic currents of I₁ (<0.1 A) or I₂ (<0.2 A) by a potentiostat (ECW-5600; Jiehan) in less than 30 min. The resulting SLA Ti disk-shape specimens were designated SAI₁ and SAI₂. The roughened SLA Ti disk-shape specimen without anodization treatment was designated SA. All specimens were sterilized using UV light (Germicidal lamp 15 W; Sankyo Denki) for 30 min just before in vitro testing.

In vivo testing was performed using screw-type Ti dental implants (Ø 3.5 × 8 mm), which had undergone the same surface modifications described above. The SLA screw-type Ti dental implants were designated SA-I (without anodization treatment), SAI₁-I (with anodization current I₁), and SAI₂-I (with anodization current I₂). All implants were sterilized with a dose of 25 kGy of gamma irradiation (Appendix Text 1) (China Biotech Corporation) just before in vivo testing. Detailed procedures for cleaning and storage of test specimens, including disks and implants, are shown in Appendix Text 2.

Surface Characterizations

Surface topography of specimens was characterized using field emission scanning electron microscopy (FE-SEM) (JSM-6700F; JEOL). Surface crystal structure was analyzed using a thin-film X-ray diffractometer (TF-XRD) (D-max/IIB; Rigaku). Surface hydrophilicity was based on the water contact angle on the specimen, which was measured using a contact-angle goniometer (100SB; Sindatek) based on the image of a sessile drop (2 µL) at the points of intersection between the drop profile and the projection of the specimen surface. Roughness values, including arithmetical mean height (Sa), root mean square height (Sq), and maximum height (Sz), were obtained using a 3-dimensional (3D) profilometer (Profilm 3D; KLA-Filmetrics) with a scanning area of 400 × 150 µm and Gaussian filter size (cutoff) of 25 µm (1/5 evaluation length). All measurements were repeated 2 times, and the number of specimens for each test group per measurement was 3.

For the surface protein adsorption assay, specimens were immersed for 5 min in 1 mL phosphate-buffered saline (PBS) with 5 mg fibronectin or bovine serum albumin (BSA) and then rinsed in deionized water. Protein adsorption was characterized using X-ray photoelectron spectroscopy (XPS) spectra (N1s) and quantified using a bicinchoninic acid (BCA) (Sigma) assay. Following incubation of protein solution (200 µL) on specimens at 37°C for 1, 6, or 24 h, nonadherent protein was mixed with BCA, maintained at 37°C for 30 min, and then analyzed using an ELISA reader (Thermo Scientific Multiskan FC) (570 nm).

In Vitro hBMSC Responses

hBMSCs were used for in vitro cell responses, including adhesion, migration, proliferation, mineralization, and osteogenic gene and protein expressions, as briefly described below. Detailed experimental procedures for evaluation of hBMSCs responses are shown in Appendix Text 3. Cell migration was assessed using hBMSCs transduced with green fluorescent protein (GFP) gene (Perng et al. 2008). Osteogenic differentiation was assessed by switching to differentiation induction medium of low-glucose Dulbecco’s modified Eagle’s medium (DMEM) with 50 µg/mL ascorbic-2 phosphate, 10⁻⁸ M dexamethasone, and 10 mM β-glycerophosphate (all from Sigma).

Cell adhesion morphology was observed using FE-SEM after a 24-h cell incubation. Wound-healing assay was used to observe the migration of GFP-labeled cells on specimens after cell incubation for 12, 24, 36, and 48 h. Cell proliferation was monitored during 7 d using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma) assay. Cell mineralization was assessed via Alizarin red S staining for 7, 14, or 21 d. Osteogenesis-related gene expression was examined via reverse transcription polymerase chain reaction (RT-PCR) using Omniscript RT Kit (Qiagen) for 3, 5, 7, 14, or 21 d. Primers and the corresponding annealing temperature and PCR cycle number used for RT-PCR are listed in Appendix Table 1. Osteogenesis-related protein expression was detected using antibodies (anti-bone sialoprotein, anti–type I collagen, anti-osteopontin, anti-osteocalcin, and anti-GAPDH) via chemiluminescence using the ECL detection kit (Applied Biological Materials) for 7, 14, or 21 d.

In Vivo Osseointegration

In vivo osseointegration was assessed by implanting screw-type Ti dental implants in the femurs of adult male New Zealand white rabbits for 4 or 12 wk. Specimen preparation for histological analysis is listed in Appendix Text 4. Histological analysis was performed using FE-SEM/backscattered electron imaging (BEI) with toluidine blue staining focusing on the formation of bone at the bone/implant interface. Energy dispersive spectrometry (EDS) (Xplore; Oxford Instruments) was used to differentiate between Ti implant and surrounding calcium/phosphorus (Ca/P)–containing bone. Optical microscopy was used to observe the BIC region and quantify the BIC (%) value (i.e., ratio of bone-implant contact length to total thread profile length). The animal test was approved by the Institutional Animal Care and Use Committee (IACUC) of National Yang-Ming University (No. 1021111).

Statistical Analysis

All measurements were performed in triplicate. The number of specimens for each test group at each time point per measurement was 3. Test values were expressed as mean ± standard deviation (SD). A 2-way analysis of variance (ANOVA) was used to analyze the effects of “surface treatment” and “time point” on biological responses with significance at α = 0.05, except that cell migration was analyzed using repeated-measure ANOVA (JMP v.14.2 software; SAS Institute). Tukey’s test was used for pairwise comparisons.

Results

Surface Characterizations

Figure 1A presents FE-SEM images of test specimens. The anodized Ti specimens presented a mixed nanonetwork structure, identified as anatase phase TiO₂ by TF-XRD (Fig. 1B), with pore sizes ranging from the nanoscale to the submicron scale with the following mean ± SD: SAI₁ (66 ± 24 nm) and SAI₂ (81 ± 37 nm). Contact angle measurements were as follows: SA (24 ± 2°; indicative of hydrophilicity) and SAI₁ and SAI₂ (<5°; indicative of superhydrophilicity) (Fig. 1C). After 4 wk of storage in a dry box at room temperature and atmospheric pressure, SAI₁ and SAI₂ surfaces still maintained their superhydrophilicity (Appendix Fig. 1). As shown in Appendix Table 2, no significant differences in surface roughness (in micrometers) were observed among the disk and implant specimens, showing Sa ~0.9−1.0, Sq ~1.1−1.2, and Sz ~7.7−9.5.

Figure 1D presents the XPS N1s spectra indicating albumin and fibronectin adsorption following 5-min immersion. The N1s intensity of SAI₁ and SAI₂ was 1.3 to 1.5 times higher than that of SA, indicating higher protein adsorption. The N_1S intensity ratio for SA:SAI₁:SAI₂ was approximately 1.0:1.5:1.4 (albumin) and 1.0:1.3:1.3 (fibronectin). The BCA assay results in Figure 1E indicate that treatment and time significantly increased albumin and fibronectin adsorption (P < 0.05), while no significant differences in protein adsorption were observed between SAI₁ and SAI₂ following immersion for 24 h.

In Vitro Cell Responses

Figure 2A presents FE-SEM micrographs of hBMSCs incubated on specimens for 24 h: micrographs in the right column were higher magnifications of white dotted rectangles in the left column. The spreading of attached cells was more pronounced on SAI₁ and SAI₂ than on SA, indicating superior biocompatibility. The migration of the green fluorescent protein (GFP)–labeled hBMSCs on specimens was evaluated during 48 h via wound-healing assay (Appendix Fig. 2, Fig. 2B). The number of cells that directionally migrated toward the central wound on SAI₁ and SAI₂ significantly exceeded the number on SA (P < 0.05) at 36 and 48 h, while no significant differences were observed between SAI₁ and SAI₂.

Figure 3A presents hBMSC proliferation following cell incubation for 7 d. The proliferation of hBMSCs on SA significantly exceeded that on SAI₁ and/or SAI₂ at 4 and 7 d (P < 0.05). Figure 3B presents hBMSC differentiation assay based on Alizarin red S staining following incubation for 7, 14, and 21 d. Extracellular calcium deposits were more extensive on SAI₁ and SAI₂ than on SA at 21 d (P < 0.05), while no significant differences were observed between SAI₁ and SAI₂.

Figure 4A presents the osteogenesis-related gene expression of hBMSCs after culturing for 3, 5, 7, 14, and 21 d. Runx2 and Osterix messenger RNA (mRNA) levels were higher on SAI₁ and SAI₂ than on SA after 3 d (P < .05), particularly on SAI₂ after 5 d (P < 0.05). The expression levels of bone sialoprotein, type I collagen, and/or osteocalcin mRNA were higher on SAI₁ and/or SAI₂ than on SA after 7, 14, and/or 21 d (P < 0.05). Surface treatment and cell incubation time had no significant effect on osteopontin mRNA expression (P > 0.05). Figure 4B presents the results of Western blot analysis, indicating the protein levels of osteogenesis-related markers after culturing for 7, 14, and 21 d. The protein expression levels of bone sialoprotein, type I collagen, and/or osteocalcin were higher on SAI₁ and/or SAI₂ than on SA after 7, 14, and 21 d. The expression of osteopontin (an early osteogenic marker) was significantly higher on SAI₁ and SAI₂ than on SA after 7 d, particularly on SAI₂ (P < 0.05).

In Vivo Osseointegration

The FE-SEM micrographs in Appendix Figure 3 show the presence of nanonetwork structure on screw-type SAI₁-I and SAI₂-I Ti implants, indicating good topographic similarity with disk-shape specimens. The FE-SEM/BEI images in Figure 5A show the interface between screw-type Ti implants (SA-I, SAI₁-I, and SAI₂-I) and surrounding tissue after 4 and 12 wk postimplantation. The contact area of Ca/P-containing bone on implant surfaces was in the following order: SAI₂-I > SAI₁-I > SA-I. Figure 5B presents histological sections obtained at 4 and 12 wk, confirming the formation of new bone (indicated by blue stain) around the Ti implants. Figure 5C presents the corresponding BIC (%) values. The treatment and time point had significant effects on BIC value (P < 0.05). The BIC (%) values, mean (SD), were as follows: (4-wk implantation) SAI₂-I 74% (8) > SAI₁-I 71% (12) > SA-I 46% (15); (12-wk implantation) SAI₂-I 94% (5) > SAI₁-I 86% (13) > SA-I 76% (10).

Discussion

Results demonstrate that the electrochemical anodization did not change the surface roughness at the micrometer scale; nevertheless, it created a superhydrophilic anatase TiO₂ nanonetwork with a mixed pore size ranging from nanoscale to submicron scale (Fig. 1A–C). The TiO₂ nanonetwork was shown to reduce the water contact angle: SA (24°; hydrophilic) and SAI₁/SAI₂ (<5°; superhydrophilic). A mixed HCl/H₂SO₄ solution is widely used for processing SLA on Ti dental implants. This SLA surface has a water contact angle of 70° to 120° (Tugulu et al. 2010; Hou et al. 2020) and surface roughness Sa of 1.18 µm (Tugulu et al. 2010). This study used a mixed HF/HNO₃ solution for the SLA process, which produced a similar surface roughness (Sa 0.9 µm) but a more hydrophilic surface (water contact angle ~24°).

Rough SLA Ti surfaces with good hydrophilicity are ideally suited to early cellular adhesion (Blatt et al. 2018). Thus, we expected to see good cell proliferation on SA with good hydrophilicity and micron-scale roughness, while we did not expect to see a decrease in cell proliferation on SAI₁ and SAI₂ (Fig. 3A) with superhydrophilic surfaces. The retarded proliferation (Fig. 3A) and advanced mineralization (Fig. 3B) of cells on SAI₁ and SAI₂ suggest that the anodized surfaces accelerated the cell growth cycle.

The electrochemical anodization in this study produced specimens that differed only in terms of pore size: 66 ± 24 nm (SAI₁) and 81 ± 37 nm (SAI₂). No significant differences were observed between SAI₁ and SAI₂ in terms of hydrophilicity, protein adsorption, and cell responses (Figs. 1 –3). Nonetheless, the expression of some osteogenic markers was higher on SAI₂ than on SAI₁ (Fig. 4).

Transcription factors Runx2 and Osterix are crucial to bone maturation (Harada et al. 1999; Nakashima et al. 2002). Osteoblast differentiation is mediated by Runx2, which regulates the expression of bone extracellular matrix protein genes encoding for bone sialoprotein, type I collagen, osteopontin, and osteocalcin (Harada et al. 1999). Osteopontin is expressed in the early stage of osteoblast differentiation; however, osteopontin expression decreases and osteocalcin expression increases during osteoblast maturation (Ducy and Karsenty 1998; Zhu et al. 2008). In this study, the high Runx2 and/or Osterix gene expression of hBMSCs on SAI₁ and SAI₂ after 3 and 5 d (Fig. 4A) triggered cell differentiation, resulting in the expression of some osteogenic makers after 7, 14, and/or 21 d (Fig. 4A, B). Overall, these results indicate that the anodized SAI₁ and SAI₂ were better able to induce osteogenic differentiation in hBMSCs than were the untreated SA, except that SAI₁ and SAI₂ had lower osteopontin protein expression than SA after 14 and 21 d (Fig. 4B).

SLA surface modification is regarded as the gold standard for screw-type Ti dental implants (Herrero-Climent et al. 2013). We were therefore not surprised by the good osseointegration of the SLA Ti dental implants (SA-I in Fig. 5A), which presented BIC values of 46% after 4-wk implantation and 76% after 12-wk implantation. Thus, our BIC values for SA-I are in good agreement with or even better than the results in the previous studies (Alayan et al. 2017; Nevins et al. 2018; Wu et al. 2020). The presence of a superhydrophilic TiO₂ nanonetwork on SAI₁-I and SAI₂-I surfaces produced BIC values beyond 70% after only 4-wk implantation, which increased to beyond 85% or even reached 94% after 12-wk implantation.

Different cellular responses are affected by specific surface nanopore diameters of the substrate (Oh et al. 2009; Lv et al. 2015; Bello et al. 2017; Nasrollahi et al. 2017). In this study, the pore size distribution on the anodized Ti surface ranged from a few to more than 100 nm, which is sufficient to cover the range of scales associated with various proteins essential for different cellular responses, such as albumin and fibronectin (Fig. 1D, E). Nanoscale proteins on the cell membrane can sense the nanoscale topography, thus inducing cell adhesion sites for the formation of focal adhesion complexes. This stimulates the activation of FAK and ERK1/2, resulting in enhancing cell migration and following responses (Yang and Huang 2019).

Furthermore, the hydrophilic surface can also promote the cell responses (Zhao et al. 2005; Jiang et al. 2019). The proposed superhydrophilic nanonetwork on the anodized Ti surface still maintained its superhydrophilicity regardless of the storage duration (<4 wk) and sterilization treatment. This was believed to be related to the presence of OH groups on the Ti surface after anodization in alkali solution. Similar results have been reported, demonstrating that the SLA Ti surfaces affectively maintain the superhydrophilicity through NaOH immersion (Tugulu et al., 2010; Jiang et al. 2019).

Combining the superhydrophilicity and the mixed nanoscaled network structure on the anodized SLA Ti surfaces affectively promoted the osteogenesis in vitro and in vivo. Both anodized SLA Ti surfaces with different applied currents I₁ and I₂ showed similar superhydrophilicity, protein adsorption ability, cell adhesion, cell migration, and cell mineralization, while the surfaces with higher applied current I₂ showed higher expressions of some major osteogenic genes and proteins, as well as revealed higher average bone-implant contact than that with lower applied current I₁. Therefore, using a higher applied current I₂ for anodization treatment is recommended in potential dental implant applications.

A well-known superhydrophilic SLActive surface (water contact angle of approximately 0°) on the global market is processed in a nitrogen chamber and uses storage in an isotonic sodium chloride solution with no atmospheric contact (Alayan et al. 2017; Masrouri et al. 2020). The hydrophilicity of the SLA Ti surface can also be significantly improved by coating with biodegradable pluronic F127 to obtain a water contact angle of approximately 0° (Hou et al. 2020); however, this uncontrollable degradation behavior of polymer F127 after implantation may limit its clinical application. As for BIC value, the SLActive Ti implants have a BIC percentage of 57% after 30 d and 67% after 60 d in the articular femoral knee joint of rabbits (Scarano et al. 2017). After 4 wk of implantation in sheep oral cavity, BIC of the SLActive Ti implant is around 38% and SLA is around 26% (Alayan et al. 2017), while BIC of the SLActive Ti implant is up to 82% after 4 wk of implantation in sheep iliac crest (Sartoretto et al. 2020).

In this study, even the untreated SA surface was still very hydrophilic (water contact angle ~24°). A simple and rapid alkali anodization treatment (<30 min) was applied to make the SLA Ti surface superhydrophilic (water contact angle <5°). The alkali-treated SLA Ti surfaces efficiently maintained their superhydrophilicity in a dry box at room temperature and atmospheric pressure for at least 4 wk (Appendix Fig. 1). Furthermore, the pore size distribution on the anodized Ti surface ranged from a few to more than 100 nm, which is sufficient to cover the range of scales associated with various proteins essential for different cellular responses. The SLA Ti implant (SA-I) had a BIC of 46% after 4 wk of implantation in femurs of rabbits, while the superhydrophilic anodized SLA Ti implants (SAI₁-I and SAI₂-I) had BIC values beyond 70% after only 4 wk of implantation, which was 1.5-fold higher osseointegration, in terms of BIC, than the untreated SLA Ti implants. Overall, the proposed TiO₂ nanonetwork structure, with long-lasting superhydrophilicity and mixed pore size range, on the roughened SLA Ti surface proved highly effective in enhancing osteogenic responses in vitro and in vivo. However, compared with the well-known superhydrophilic SLActive Ti surface, there is still room for improvement in the BIC value of the Ti surfaces proposed in this study.

Author Contributions

W.E. Yang, contributed to data acquisition, analysis, and interpretation, drafted the manuscript; H.H. Huang, contributed to conception and design, critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.

Supplemental Material

sj-pdf-1-jdr-10.1177_00220345211001017 – Supplemental material for TiO2 Nanonetwork on Rough Ti Enhanced Osteogenesis In Vitro and In Vivo

Supplemental material, sj-pdf-1-jdr-10.1177_00220345211001017 for TiO2 Nanonetwork on Rough Ti Enhanced Osteogenesis In Vitro and In Vivo by W.E. Yang and H.H. Huang in Journal of Dental Research

Footnotes

Acknowledgements

We thank Prof. Suh-Woan Hu, Graduate Institute of Oral Sciences, Chung Shan Medical University, Taichung, Taiwan, for assistance in statistical analysis; and Prof. Jyh-Wei Lee, Department of Materials Engineering, Ming Chi University of Technology, New Taipei City, Taiwan and Prof. Chau-Chang Chou, Department of Mechanical and Mechatronic Engineering, National Taiwan Ocean University, Keelung, Taiwan, for assistance in surface roughness analysis.

A supplemental appendix to this article is available online.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was financially supported by the Ministry of Science and Technology (MOST), Taiwan (MOST 105-2314-B-030-MY3; MOST 109-2731-M-/EM0000007900).

ORCID iD

H.H. Huang

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