Surface-treated titanium base material and method for manufacturing same, composite, culture bed, implant, and acicular nano-crystal of hydroxyapatite and method for manufacturing same

Abstract

The present invention pertains to: a surface-treated titanium base material which has a stratified nano-rough surface, and in which the stratified nano-rough surface is formed by including amorphous titanium oxide, the average distribution density of nano-projections forming the stratified nano-rough surface is 20-50 projections / μm2, and the distribution anisotropy of the nano-projections is 0.70-1.20; a method for manufacturing the surface-treated titanium base material; a composite; a culture bed; an implant; and acicular nano-crystals of hydroxyapatite, and a method for manufacturing the same.

Description

Surface-treated titanium substrate and method for producing the same, composite, culture bed, implant, and nano-needle crystals of hydroxyapatite and method for producing the same

[0001] The present invention relates to a surface-treated titanium substrate and a method for producing the same, a composite, a culture bed, an implant, and nano-needle crystals of hydroxyapatite and a method for producing the same.

[0002] Hydroxyapatite (hereinafter also abbreviated as "HAp") is a major component of bones and teeth, and because it can directly bond with bone tissue, it is used as a bone filling material, artificial dental root, and biological hard tissue replacement material (artificial bone, etc.). HAp is not only an excellent biomaterial, but is also known to exhibit a variety of functions, such as ion exchange properties, ion adsorption properties, catalytic properties, and ion conduction properties, and its applications as a variety of functional materials outside the body are expanding.

[0003] For example, titanium and titanium alloys are lightweight and exhibit high fracture toughness, and are widely used in artificial dental implants, artificial bones, and the like, but do not bond to bone. Therefore, the application of a HAp coating layer on titanium and titanium alloys has been considered, and various coating methods, such as plasma coating and chemical coating, have been investigated. As a representative example of a chemical coating method, Non-Patent Document 1 describes a technique in which an amorphous titanium oxide layer having countless porous nanoprotrusions is formed on the surface of titanium or a titanium alloy by alkaline etching, and the resulting nano-roughened titanium or titanium alloy is immersed in a simulated body fluid to precipitate HAp on the surface of the nano-roughened titanium or titanium alloy by electrochemical reaction. Patent Document 1 also describes a method (dip coating method) in which nanoparticles containing HAp are deposited on an implant that has been subjected to an acid etching process to form a micro-roughened surface. Furthermore, Patent Document 2 describes an apatite-coated bioactive implant in which the surface of titanium or a titanium alloy is subjected to acid treatment to form irregularities with pore sizes of 0.1 to 10 μm, and then further subjected to alkali treatment to process the titanium or titanium alloy so that hydroxyapatite can be easily precipitated, and the titanium or titanium alloy is then alternately immersed in an aqueous solution containing calcium ions and an aqueous solution containing phosphate ions to form a hydroxyapatite coating layer.

[0004] Kim HM, et al. J Biomed Mater Res 32:409-17(1966)

[0005] JP-T-2009-515600A JP-A-2006-255319A

[0006] The inventors have found that when titanium or titanium alloys with a microscale roughened surface formed by the acid etching treatment described in Patent Document 1 are immersed in a simulated body fluid, HAp is hardly precipitated. Furthermore, even when titanium or titanium alloys with a nanoscale amorphous titanium oxide layer roughened by the alkaline etching treatment described in Non-Patent Document 1 are immersed in a simulated body fluid, HAp precipitation is insufficient, and room for improvement is needed. Patent Document 2 describes the formation of a HAp layer, but does not describe the precipitation of nanoneedle crystals. Furthermore, Patent Document 2 also describes that a HAp crystal diffraction pattern was not observed after immersion in a simulated body fluid for three weeks. Furthermore, Patent Document 2 describes that the sintering treatment was performed in a vacuum and that titanium hydride disappeared after the sintering treatment, suggesting that an amorphous titanium oxide layer is not formed on the surface of the pure titanium article.

[0007] An object of the present invention is to provide a surface-treated titanium substrate and a method for producing the same, which enable more efficient deposition of hydroxyapatite nanoneedle crystals on the titanium substrate. Another object of the present invention is to provide a composite having highly crystalline hydroxyapatite nanoneedle crystals on the surface of a titanium substrate. Another object of the present invention is to provide a culture bed capable of promoting the differentiation of bone marrow stromal cells into osteoblasts. Another object of the present invention is to provide an implant capable of exhibiting high bioactivity and high tissue binding strength. A further object of the present invention is to provide a method for producing highly crystalline hydroxyapatite nanoneedle crystals, which enables more efficient production of highly crystalline hydroxyapatite nanoneedle crystals. A further object of the present invention is to provide highly crystalline hydroxyapatite nanoneedle crystals.

[0008] The present inventors have conducted extensive research in light of the above-mentioned problems and have found that, in a surface-treated titanium substrate having a hierarchical nano-roughened surface, the hierarchical nano-roughened surface is composed of amorphous titanium oxide, and by increasing the anisotropic distribution of the nanoprotrusions that make up the hierarchical nano-roughened surface, electrical reactivity is enhanced, and highly crystalline needle-like hydroxyapatite (HAp) crystals can be more efficiently obtained by immersion in a simulated body fluid.The present invention was completed based on these findings and through further research.

[0009] The object of the present invention has been achieved by the following means: <1> A surface-treated titanium substrate having a hierarchical nano-rough surface, wherein the hierarchical nano-rough surface is comprised of amorphous titanium oxide, and the average distribution density of nanoprotrusions constituting the hierarchical nano-rough surface is 20 to 50 / μm ２ <2> The hierarchical nano-rough surface has an average zeta potential of -5.0 to -2.0 mV, and the average contact potential difference between the hierarchical nano-rough surface and a silicone probe measured by Kelvin probe force microscopy is 1.60 to 2.00 V / 10 nm. ２<3> A composite comprising the surface-treated titanium substrate according to <1> or <2> and nanoneedle-shaped crystals of hydroxyapatite formed on the hierarchical nano-roughened surface of the titanium substrate. <4> A culture bed comprising the surface-treated titanium substrate according to <1> or <2> or the composite according to <3>. <5> A method for producing the surface-treated titanium substrate according to <1> or <2>, comprising: subjecting a titanium substrate having a mechanically polished surface to a heat treatment with an acid to impart micro-roughness to the mechanically polished surface of the titanium substrate; further subjecting the titanium substrate to a heat treatment with a 7.5 to 12.5 M alkali; and then sintering the titanium substrate under atmospheric pressure to form a hierarchical nano-roughened surface on the surface of the titanium substrate. <6> A method for producing a surface-treated titanium substrate according to <5>, wherein the heat treatment with an acid is treatment with an aqueous sulfuric acid solution at 100 to 150°C, the heat treatment with an alkali is treatment with a 7.5 to 12.5 M aqueous sodium hydroxide solution at 60 to 100°C, and the sintering treatment is heat treatment at 500 to 700°C under atmospheric pressure. <7> A method for producing nanoneedle-shaped hydroxyapatite crystals, comprising immersing the surface-treated titanium substrate according to <1> or <2> in a simulated body fluid containing calcium and phosphorus to precipitate nanoneedle-shaped hydroxyapatite crystals. <8> An implant comprising the surface-treated titanium substrate according to <1> or <2> or the composite according to <3>. <9> Nanoneedle-shaped hydroxyapatite crystals, wherein the ratio of calcium atoms to phosphorus atoms calculated by HAADF-STEM and EDX analysis is 1.00 or more, and the interplanar spacing of the (100) plane determined by electron diffraction structure analysis is 8.25 Å or less. <10> The average area of ​​the nano-needle crystals of hydroxyapatite obtained by analyzing the TEM image using image analysis software is 2000 nm ２ The above nano needle crystals of hydroxyapatite have an average long diameter of 200 nm or more and an average aspect ratio of 6 to 20.

[0010] In the present invention, a numerical range expressed using "to" means a range that includes the numerical values ​​written before and after "to" as the lower and upper limits. In the present invention, when multiple numerical ranges are set in stages for the content of a component, physical properties, etc., the upper and lower limits forming the numerical range can be combined as appropriate. The size and shape of the surface-treated titanium substrate, composite, culture bed, and implant of the present invention can be adjusted as appropriate to suit the desired application.

[0011] The surface-treated titanium substrate of the present invention allows for more efficient precipitation of acicular hydroxyapatite crystals on the surface of the surface-treated titanium substrate. According to the method for producing a surface-treated titanium substrate of the present invention, a surface-treated titanium substrate having a hierarchical nano-roughened surface can be obtained, in which the nanoprotrusions constituting the hierarchical nano-roughened surface have enhanced anisotropic distribution and electrical reactivity. Furthermore, the composite of the present invention has highly crystalline acicular hydroxyapatite crystals on the surface of the surface-treated titanium substrate. Furthermore, the culture bed of the present invention can promote differentiation of bone marrow stromal cells into osteoblasts. Furthermore, the implant of the present invention can exhibit high bioactivity and high tissue-bonding strength. Furthermore, according to the method for producing acicular hydroxyapatite crystals of the present invention, acicular hydroxyapatite crystals can be precipitated more efficiently and with high crystallinity. Furthermore, the acicular hydroxyapatite crystals of the present invention have high crystallinity.

[0012] 2(a) and 2(b) show scanning electron microscope (SEM) images of the surface of each surface-treated titanium disk in Example 1A. The figures show the average distribution density and distribution anisotropy of the nanoprotrusions determined from the brightness of secondary electron images taken with an SEM in Example 1A. FIG. 2(a) shows the average distribution density of the nanoprotrusions, and FIG. 2(b) shows the distribution anisotropy of the nanoprotrusions. The figures show bar graphs showing the average zeta potential of the surface of each surface-treated titanium disk in Example 1A. The figures show the results of surface potential microscope (KFM) analysis of each surface-treated titanium disk in Example 1A. The upper row of FIG. 4 shows an image of the surface shape of each surface-treated titanium disk, and the lower row of FIG. 4 shows an image of the surface potential of each surface-treated titanium disk. The figures show the distribution, average value, and standard deviation of the contact potential difference obtained by surface potential microscope (KFM) analysis of each surface-treated titanium disk in Example 1A. 7(a) and 7(b) show overall photographs of the surfaces of a polished titanium disk and a surface-treated titanium disk having a nano-rough surface in Reference Example 1, as well as scanning electron microscope (SEM) images of the surfaces and a random model pattern. In Reference Example 1, the distribution anisotropy and average distribution density of nanoprotrusions calculated from the brightness of a secondary electron beam image taken with an SEM, and the distribution anisotropy and average distribution density of nanoprotrusions calculated from the random model pattern are shown. FIG. 7(a) shows the distribution anisotropy of the nanoprotrusions constituting the rough surface, and FIG. 7(b) shows the average distribution density of the nanoprotrusions. In Reference Example 1, the spectra obtained by Fourier transform infrared spectroscopy (FTIR analysis) for the polished titanium disk and the surface-treated titanium disk having a nano-rough surface are shown. The wavenumber at which OH groups are observed is approximately 3200 to 3300 cm. －１In the graph, the data showing the highest absorbance is Nano (15M), followed by Nano (5M), then Nano (10M), and data showing 0 absorbance is Machined data. Transmission electron microscope (TEM) images and energy dispersive X-ray spectroscopy (EDX) analysis are shown for a surface-treated titanium disk having a nano-roughened surface treated with a 5.0 M aqueous sodium hydroxide solution in Reference Example 1. FIG. 9( a) shows a transmission electron microscope (TEM) image of a longitudinal section, and FIGS. 9( b) to 9( d) show enlarged images and / or energy dispersive X-ray spectroscopy (EDX) analysis results for each of the microregions b to d indicated by circles in FIG. 9( a). Transmission electron microscope (TEM) images and energy dispersive X-ray spectroscopy (EDX) analysis are shown for a surface-treated titanium disk having a nano-roughened surface treated with a 10.0 M aqueous sodium hydroxide solution in Reference Example 1. Figure 10(a) shows a transmission electron microscope (TEM) image of a longitudinal section, and Figures 10(b) to 10(e) show enlarged images and / or energy dispersive X-ray spectroscopy (EDX) analysis results for each of the microregions b to e indicated by circles in Figure 10(a). Scanning electron microscope (SEM) images of the surface of each surface-treated titanium disk in Example 1B-1 are shown. The anisotropy of the distribution of nanoprotrusions determined from the brightness of secondary electron images taken with an SEM of each surface-treated titanium disk in Example 1B-1 is shown. The zeta potential of the surface of each surface-treated titanium disk in Example 1B-1 is shown. The results of surface potential microscope (KFM) analysis of each surface-treated titanium disk in Example 1B-1 are shown. The upper row of Figure 14 shows an image of the surface shape, the middle row of Figure 14 shows an image of the surface potential, and the lower row of Figure 14 shows the distribution of contact potential difference. 16A and 16B show scanning electron microscope (SEM) images of the surface of each surface-treated titanium disk having a hierarchical nano-rough surface obtained by changing the base concentration in Example 1B-2, and the surface of a surface-treated titanium disk obtained by sintering under reduced pressure. The figures also show the average distribution density and distribution anisotropy of the nanoprotrusions determined from the brightness of secondary electron beam images taken with the SEM of each surface-treated titanium disk in Example 1B-2. Figure 16A shows the average distribution density of the nanoprotrusions, and Figure 16B shows the distribution anisotropy of the nanoprotrusions.1C shows scanning electron microscope (SEM) images of the surfaces of the polished titanium disk and the surface-treated titanium disk with a hierarchical nano-roughened surface for the pure titanium disk and the Ti-6Al-4V titanium alloy disk in Example 1C. 1D shows the average distribution density and distribution anisotropy of the nanoprotrusions determined from the brightness of secondary electron images taken with an SEM, as well as the results of energy dispersive X-ray spectroscopy (EDX) for the pure titanium disk and the Ti-6Al-4V titanium alloy disk with a hierarchical nano-roughened surface for Example 1C. 1C shows the average distribution density of the nanoprotrusions, (b) the anisotropy of the nanoprotrusion distribution, (c) the percentage of Ti among the constituent elements of the hierarchical nano-roughened surface, and (d) the percentages of Al and V among the constituent elements of the hierarchical nano-roughened surface. 1D shows scanning electron microscope (SEM) images of the surfaces of the surface-treated titanium disks in Example 2A before and after 28 days of immersion in simulated body fluid. 20 shows SEM images of a surface-treated titanium disk having a nano-roughened surface and a surface-treated titanium disk having a hierarchical nano-roughened surface after 28 days of immersion in a simulated body fluid in Example 2A. The upper row of Fig. 20 shows SEM images of the surface-treated titanium disks viewed perpendicular to the surface, and the lower row of Fig. 20 shows SEM images of the surface-treated titanium disks viewed obliquely to the surface. The results of energy dispersive X-ray spectroscopy (EDX) analysis of the needle-shaped structured material deposited on the surface of the surface-treated titanium disks having a hierarchical nano-roughened surface after 28 days of immersion in a simulated body fluid in Example 2A are shown. The calcium and phosphorus ion concentrations obtained by performing colorimetric assays of calcium ions and phosphate ions on solutions containing dissolved needle-shaped structured material deposited on the surface of each surface-treated titanium disk and a standard culture dish (made of polystyrene) at 5, 14, and 28 days after immersion in the simulated body fluid in Example 2A are shown. The atomic ratios of calcium, phosphorus, and titanium (the proportion of each element among all detected atoms) were obtained by energy dispersive X-ray spectroscopy (EDX) analysis of the needle-shaped structure substances precipitated on the surface of each surface-treated titanium disk and a regular culture dish (made of polystyrene) 5, 14, and 28 days after immersion in the simulated body fluid in Example 2A.

[0033] Figure 1 shows the results of depth-direction elemental analysis by glow discharge optical emission spectroscopy (GD-OES analysis) of the needle-shaped structure material deposited on the surface of each surface-treated titanium disk after 28 days of immersion in simulated body fluid in Example 2A.

[0034] Figure 2 shows X-ray diffraction (XRD) data for the needle-shaped structure material, hydroxyapatite crystal structure, and titanium oxide crystal structure deposited on the surface of each surface-treated titanium disk after 14 days of immersion in simulated body fluid in Example 2A.

[0035] Figure 3 shows Fourier transform infrared spectroscopy (FTIR) data for each surface-treated titanium disk after 28 days of immersion in simulated body fluid in Example 2A.

[0036] Figure 4 shows FTIR spectra of various calcium phosphates.

[0037] Figure 5 shows the results of scanning electron microscope (SEM) analysis of the surface of each surface-treated titanium disk before immersion in simulated body fluid and after 14 and 28 days of immersion in simulated body fluid in Example 2B. The upper row of Figure 28 shows an SEM image before immersion in simulated body fluid, the middle row shows an SEM image after 14 days of immersion in simulated body fluid, and the lower row of Figure 28 shows an SEM image after 28 days of immersion in simulated body fluid. SEM images of each surface-treated titanium disk in Example 2B viewed from an oblique angle are shown. The upper row of Figure 29 shows an SEM image after 14 days of immersion in simulated body fluid, and the lower row of Figure 29 shows an SEM image after 28 days of immersion in simulated body fluid. The atomic ratio of calcium on the surface of each surface-treated titanium disk in Example 2B obtained by energy dispersive X-ray spectroscopy (EDX) analysis is shown. For each surface-treated titanium disk in Figure 30, the left side shows the atomic ratio of calcium after 14 days of immersion in simulated body fluid, and the right side shows the atomic ratio of calcium after 28 days of immersion in simulated body fluid. 31(a) and 31(b) show scanning electron microscope (SEM) images of the surface of a surface-treated Ti-6Al-4V titanium alloy disk having a hierarchical nano-roughened surface before and after 28 days of immersion in simulated body fluid in Example 2C. The upper row of Fig. 31 shows the SEM image before immersion in simulated body fluid, and the lower row of Fig. 31 shows the SEM image (at different magnifications) after 28 days of immersion in simulated body fluid. Fig. 32(a) shows the result of energy dispersive X-ray spectroscopy (EDX) of the surface of the surface-treated Ti-6Al-4V titanium alloy disk having a hierarchical nano-roughened surface after 28 days of immersion in simulated body fluid in Example 2C.Figure 32(b) shows the atomic ratios of calcium, phosphorus, and titanium (the proportion of each element among all detected atoms) obtained by energy dispersive X-ray spectroscopy (EDX) analysis of the needle-shaped structure material precipitated on the surface of a surface-treated Ti-6Al-4V titanium alloy disk with a hierarchical nano-roughened surface after 28 days of immersion in simulated body fluid in Example 2C. Scanning electron microscope (SEM) images of the surface of each surface-treated titanium disk before and after BMSC culture in Example 3 are shown. For each surface-treated titanium disk in Figure 33, the left side shows an SEM image of the surface of the surface-treated titanium disk before culture, and the right side shows an SEM image of the surface of the surface-treated titanium disk after 30 days of culture. Scanning electron microscope (SEM) images of the surface of each surface-treated titanium disk after 30 days of BMSC culture in Example 3 are shown. The right side of Fig. 34 shows SEM images of the surfaces of a surface-treated titanium disk having a HAp-coated nano-roughened surface and a surface-treated titanium disk having a HAp-coated hierarchical nano-roughened surface after 30 days of culture, and the left side of Fig. 34 shows SEM images of the surfaces of a surface-treated titanium disk having a nano-roughened surface and a hierarchical nano-roughened surface after 30 days of culture, respectively. This figure shows the atomic ratios (%) of calcium, phosphorus, and titanium, and the atomic ratio (%) of calcium to phosphorus, obtained by energy dispersive X-ray spectroscopy (EDX) analysis of the mineralized matrix formed by BMSC culture for 30 days in Example 3. This figure shows the results of calcium quantification obtained by energy dispersive X-ray spectroscopy (EDX) analysis of the mineralized matrix formed by BMSC culture for 30 days in Example 3. This figure shows the results of XRD analysis of the mineralized matrix obtained after BMSC culture for 30 days in Example 3. Figure 37(a) shows data in the range of 2θ = 0 to 70°, and Figure 37(b) is an enlarged view of the range of θ = 25 to 40° in Figure 37(a), showing an overlay of data for BMSCs cultured on surface-treated titanium disks with HAp-coated hierarchical nano-roughened surfaces and data for BMSCs cultured on surface-treated titanium disks with HAp-coated nano-roughened surfaces. The left side of Figure 38 shows absorbance at 370 nm after 2 and 4 days of culture, as determined by BrdU (Bromodeoxyuridine) analysis.The right side of Figure 38 shows the cell counts after 1 day, 5 days, and 7 days of culture by WST-1 cell proliferation assay. In Example 3, (a) to (c) of Figure 39 show the results of real-time RT-PCR (reverse transcription PCR) analysis after 5 days of culture, and (d) of Figure 39 shows the results of quantitative analysis of ALP (Alkaline Phosphatase) after 5 days of culture. Figure 40(a) shows a schematic diagram of the implant base material used in Example 4, and Figures 40(b) to (d) show schematic diagrams showing the implantation site of the implant shown in Figure 40(a) into a rat. Figure 40(b) shows a schematic diagram of the implant implantation site in an overall image of a rat, Figure 40(c) shows a photographic image of one implant implanted in each maxillary bone site immediately after extraction of the left and right maxillary first molars of a rat, and Figure 40(d) shows a schematic diagram of the site where one implant is implanted in the central part of the femoral shaft of a rat. 44(a) and (b) are a TEM image and an enlarged image of the hierarchical nano-rough surface thin slice sample, and FIG. 44(c) is an electron diffraction pattern of the hierarchical nano-rough surface thin slice sample. Also, FIG. 44(d) and (e) are a TEM image and an enlarged image of the hierarchical nano-rough surface thin slice sample, and FIG. 44(f) is an electron diffraction pattern of the hierarchical nano-rough surface thin slice sample. Figure 45(a) is an HAADF-STEM image of a thin slice sample with a layered nano-rough surface in Example 2A, Figure 45(b) is the EDX spectrum and the results of simple quantitative composition analysis of a thin slice sample with a layered nano-rough surface, Figure 45(c) is an HAADF-STEM image of a thin slice sample with a nano-rough surface in Example 2A, and Figure 45(d) is the EDX spectrum and the results of simple quantitative composition analysis of a thin slice sample with a nano-rough surface, and Figure 46 is a TEM image used for image analysis of the shape of the needle-like crystals of HAp.The upper row of Fig. 46 is a TEM image of a thin sample with a nano-rough surface in Example 2A, and the lower row of Fig. 46 is a TEM image of a thin sample with a layered nano-rough surface in Example 2A. Fig. 47 is a diagram showing, surrounded by a line, needle-like structures selected using image analysis software for the TEM image of the thin sample with a layered nano-rough surface in Example 2A shown at the left end of the lower row of Fig. 46. Fig. 48 is a dot plot and box plot of various parameters (area, major axis, and aspect ratio) measured by image analysis using image analysis software for the shape of the needle-like crystals of HAp in the TEM images of the thin sample with a layered nano-rough surface and the thin sample with a nano-rough surface shown in Fig. 46. Fig. 48(a) is a diagram showing area, Fig. 48(b) is a diagram showing major axis, and Fig. 48(c) is a diagram showing aspect ratio, respectively.

[0013] [Surface-treated titanium substrate] The surface-treated titanium substrate of the present invention is a surface-treated titanium substrate having a hierarchical nano-rough surface, wherein the hierarchical nano-rough surface is composed of amorphous titanium oxide, and the average distribution density of nanoprotrusions constituting the hierarchical nano-rough surface is 20 to 50 / μm ２and the nanoprotrusions have a distribution anisotropy of 0.70 to 1.20. In the present invention, a "hierarchical nano-roughened surface" refers to a roughened surface such as that shown in the scanning electron microscope (SEM) images of the surface of a titanium disk that has been subjected to a hierarchical nano-roughening treatment, as shown in Figures 1, 11, 15, and 17. In the present invention, a "nano-roughened surface" refers to a surface having a microstructure of approximately 1 to several hundred nanometers (nano-order), such as undulations, sharp protrusions, and pores, and a "hierarchical nano-roughened surface" refers to a structure in which the nano-roughened surface is formed on a surface of a micro-order surface structure (micro-roughened surface) of 1 to 100 μm. In the present invention, a "hierarchical nano-roughened surface" is determined by whether the nanoprotrusions constituting the hierarchical nano-roughened surface satisfy the above-mentioned average distribution density and distribution anisotropy. As long as these properties are satisfied, there are no particular limitations on the physical structure in more detail. In other words, in the present invention, the term "hierarchical nano-roughened surface" is used to describe a roughened surface in which the nanoprotrusions constituting the roughened surface satisfy the above-mentioned average distribution density and distribution anisotropy. The hierarchical nano-roughened surface of the surface-treated titanium substrate of the present invention can be formed by subjecting a desired surface of the titanium substrate on which the hierarchical nano-roughened surface is to be formed, for example, by mechanical polishing, heat treatment with an acid, heat treatment with a 7.5 to 12.5 M alkali, and sintering treatment under atmospheric pressure, as described in the method for producing a surface-treated titanium substrate of the present invention. Details are as described below in the method for producing a surface-treated titanium substrate of the present invention.

[0014] (Average Distribution Density and Distribution Anisotropy of Nanoprotrusions Constituting the Hierarchical Nano-rough Surface) The average distribution density of the nanoprotrusions constituting the hierarchical nano-rough surface of the surface-treated titanium substrate of the present invention is 20 to 50 / μm ２ and preferably 25 to 50 particles / μm ２ and more preferably 28 to 50 particles / μm ２ and more preferably 30 to 50 particles / μm ２ The "average distribution density of nanoprotrusions constituting the hierarchical nano-roughened surface" is determined by obtaining a scanning electron microscope (SEM) image of the hierarchical nano-roughened surface of the surface-treated titanium substrate, and calculating the distribution density of the nanoprotrusions (number / μm) from the number of nanoprotrusions per square 1 μm. ２) and then averaging the distribution density of the nanoprotrusions in three different 1 μm squares. Details of the measurement conditions are as described in the Examples below. Furthermore, the distribution anisotropy of the nanoprotrusions constituting the hierarchical nano-roughened surface of the surface-treated titanium substrate of the present invention is 0.70 to 1.20, preferably 0.80 to 1.20, more preferably 0.90 to 1.20, and even more preferably 0.90 to 1.10. The "distribution anisotropy of the nanoprotrusions constituting the hierarchical nano-roughened surface" is calculated by Voronoi tessellating an image obtained by extracting vertices from the brightness of a secondary electron beam image of the hierarchical nano-roughened surface of the surface-treated titanium substrate taken at a magnification of 5,000 to 10,000 times using a scanning electron microscope (SEM), and dividing the standard deviation of the area of ​​each divided region by the average value (to determine the coefficient of variation). The secondary electron beam image to be Voronoi tessellated is an image with a magnification of 5,000 to 1,000 times. Details of the measurement conditions are as described in the Examples below. That is, the hierarchical nano-rough surface of the surface-treated titanium substrate of the present invention has nanoprotrusions present at a high density and with a highly anisotropic distribution.

[0015] The hierarchical nano-roughened surface of the surface-treated titanium substrate of the present invention, which is composed of an amorphous titanium oxide layer, acquires a negative surface potential and determines its electrochemical reactivity with simulated body fluid. Furthermore, as described above, the hierarchical nano-roughened surface of the surface-treated titanium substrate of the present invention is composed of nanoprotrusions present at a high density and with a high anisotropic distribution. Therefore, it is believed that the work function is lower and the electrical reactivity is higher than that of conventional surface-treated titanium substrates with roughened surfaces. Therefore, when the surface-treated titanium substrate of the present invention is immersed in a simulated body fluid, the electrochemical reactivity in the simulated body fluid is enhanced, and the crystal growth rate and crystallinity of HAp are increased, resulting in improved precipitation efficiency of HAp nanoneedle crystals. Specifically, a low work function and high electrical reactivity are believed to mean that the hierarchical nano-roughened surface of the surface-treated titanium substrate of the present invention satisfies the average zeta potential described below and the average contact potential difference with a silicone probe measured by Kelvin probe force microscopy analysis described below. This is thought to be due to the fact that, as described in Bartocci L, et al. Journal of Chemical Physics 142:184304-13(2015), the surface charge of a solid is concentrated at the apex of protrusions on the surface, and the anisotropic electron distribution on the solid surface promotes electron transfer reactions.

[0016] (Average Zeta Potential of the Hierarchical Nano-Rough Surface) The average zeta potential of the hierarchical nano-rough surface of the surface-treated titanium substrate of the present invention is preferably −5.0 to −2.0 mV, more preferably −5.0 to −3.0 mV, and even more preferably −5.0 to −4.0 mV. The “average zeta potential of the hierarchical nano-rough surface” is the average value of the zeta potentials of three measurement samples obtained using a flat sample cell at a temperature of 25°C, and the measurement conditions are as described in detail in the Examples below. When the surface-treated titanium substrate of the present invention has an average zeta potential of −5.0 to −2.0 mV, it is believed that when the surface-treated titanium substrate of the present invention is immersed in a simulated body fluid, calcium ions in the simulated body fluid are more likely to be adsorbed to the hierarchical nano-rough surface of the surface-treated titanium substrate through electrostatic interaction, thereby promoting the precipitation of HAp crystals.

[0017] (Average Contact Potential Difference of Layered Nano-Rough Surface with Silicone Probe Measured by Kelvin Probe Force Microscope Analysis and Its Unbiased Variance Value) The average contact potential difference of the layered nano-rough surface of the surface-treated titanium substrate of the present invention with a silicone probe measured by Kelvin Probe Force Microscope analysis (also simply referred to as "average contact potential difference") is 1.60 to 2.00 V / 10 nm. ２ is preferably 1.68 to 2.00 V / 10 nm, and more preferably 1.68 to 2.00 V / 10 nm. ２ and more preferably 1.71 to 2.00 V / 10 nm. ２ The contact potential difference is calculated by subtracting the work function of the hierarchical nano-rough surface on the surface-treated titanium substrate from the work function of the silicon probe. Therefore, it is believed that the larger the contact potential difference, the smaller the work function of the hierarchical nano-rough surface on the surface-treated titanium substrate, resulting in increased electrical reactivity. Furthermore, as shown in Figures 4 and 14, the apexes of the nanoprotrusions that make up the hierarchical nano-rough surface are the locations that exhibit a high contact potential difference. The "average contact potential difference between the hierarchical nano-rough surface and the silicone probe as determined by Kelvin probe force microscope analysis" is a value measured using a silicone probe with a Kelvin probe force microscope, and the measurement conditions are as described in detail in the Examples below. The unbiased variance of the average contact potential difference is preferably 0.0050 to 0.0300, more preferably 0.0100 to 0.0300, and even more preferably 0.0175 to 0.0300. The average contact potential difference between the hierarchical nano-rough surface on the surface-treated titanium substrate of the present invention and the silicone probe as determined by Kelvin probe force microscope analysis is 1.60 to 2.00 V / 10 nm. ２ Therefore, when the surface-treated titanium substrate of the present invention is immersed in a simulated body fluid, it is thought that the electrochemical reactivity in the simulated body fluid is enhanced, thereby improving the precipitation efficiency of HAp nanoneedle crystals.

[0018] (Titanium substrate) In the surface-treated titanium substrate of the present invention, the titanium constituting the titanium substrate to be surface-treated may be pure titanium or a titanium alloy, and examples thereof include pure titanium of Grade I or II and titanium alloy (Ti-6Al-4V) of Grade V according to the ASTM (American Society for Testing and Materials) standard. Among these, pure titanium of Grade I or II according to the ASTM standard is preferred. Note that the surface of the titanium substrate usually contains TiO ２ A passive film is formed on the surface of the titanium substrate before the surface treatment of the present invention.

[0019] (Amorphous Titanium Oxide) The hierarchical nano-rough surface of the surface-treated titanium substrate of the present invention is comprised of amorphous titanium oxide. The hierarchical nano-rough surface of the surface-treated titanium substrate of the present invention contains amorphous titanium oxide, thereby acquiring a negative surface potential and determining its electrochemical reactivity with simulated body fluid. Examples of the amorphous titanium oxide include amorphous alkali titanates, with amorphous sodium titanate and amorphous potassium titanate being preferred, and amorphous sodium titanate being more preferred. Note that amorphous alkali titanates can be considered to be titanium oxide crystals containing alkali ions. Therefore, in the present invention, the term "amorphous titanium oxide" is used to include amorphous alkali titanates in addition to ordinary amorphous titanium oxide (titanium oxide with low crystallinity). The surface-treated titanium substrate of the present invention can be suitably obtained, for example, by the method for producing a surface-treated titanium substrate of the present invention described below. In this case, a titanium substrate having a mechanically polished surface is subjected to heat treatment with an acid to remove TiO present on the surface of the titanium substrate. ２ The passive film of TiH is reduced. ２ A layer is formed, and then, by contact with moisture in the air or water in the medium, TiH ２A thin new oxide layer is formed on the layer. Further heat treatment with 7.5 to 12.5 M alkali produces a hydrated gel of alkali titanate, which is then converted into amorphous titanium oxide by sintering under atmospheric pressure. Thus, by producing a surface-treated titanium substrate by the method for producing a surface-treated titanium substrate of the present invention, at least the outermost surface (outermost surface) of the hierarchical nano-rough surface of the surface-treated titanium substrate of the present invention is composed of amorphous titanium oxide, and the hierarchical nano-rough surface contains amorphous titanium oxide. Note that the hierarchical nano-rough surface may exhibit variations, such as increased crystallinity or the inclusion of components other than amorphous titanium oxide, as it moves away from the outermost surface side of the hierarchical nano-rough surface and closer to the titanium substrate. For example, according to the method for producing a surface-treated titanium substrate of the present invention, after the heat treatment with acid, the TiO formed before the heat treatment with alkali may be removed. ２ Examples of such a form include a form in which a passive film (not corresponding to amorphous titanium oxide) remains partially.

[0020] (Shape, etc.) The shape of the surface-treated titanium substrate of the present invention is not particularly limited and can be designed according to the application. Examples include mesh, porous, particulate, and shapes suited to applications such as artificial dental roots or artificial synthetic bones.

[0021] (Uses) As described below, the uses of the surface-treated titanium substrate of the present invention include use as a material for more efficiently precipitating HAp nanoneedle crystals on the surface and as a source of HAp nanoneedle crystals. Furthermore, by directly implanting the surface-treated titanium substrate of the present invention obtained using a titanium substrate of a desired shape into a living body, HAp nanoneedle crystals are precipitated on the hierarchical nano-roughened surface of the surface-treated titanium substrate in body fluids, thereby enhancing the affinity and adhesion of the surface-treated titanium substrate to the living body, and the surface-treated titanium substrate can also be used as an implant, such as an osseointegrated implant. Furthermore, by using the surface-treated titanium substrate of the present invention as a culture bed and culturing bone marrow stromal cells, differentiation into osteoblasts can be promoted.

[0022] [Method for producing surface-treated titanium substrate] The surface-treated titanium substrate of the present invention is produced by a method comprising: subjecting a titanium substrate having a mechanically polished surface to a heat treatment with an acid to impart micro-roughness to the mechanically polished surface of the titanium substrate; further subjecting the titanium substrate to a heat treatment with a 7.5 to 12.5 M alkali; and then subjecting the titanium substrate to a sintering treatment under atmospheric pressure to form a hierarchical nano-roughened surface on the surface of the titanium substrate.

[0023] The chemical and structural changes that occur until the surface-treated titanium substrate of the present invention is obtained will be described in detail below in relation to each step included in the method for producing a surface-treated titanium substrate of the present invention. First, a titanium substrate having a mechanically polished surface is heat-treated with an acid to remove TiO 2 present on the surface of the titanium substrate. ２ The passive film of Ti dissolves and the exposed Ti reacts with the acid, resulting in TiH ２ A layer is generated, forming a micro-roughened surface. Then, when the layer comes into contact with moisture in the air or water in the medium, the TiH ２ A thin new oxide layer is formed on the layer. Next, by heat treatment with 7.5 to 12.5 M alkali, a layer of hydrated gel of alkali titanate is produced, and the morphology of a hierarchical nano-roughened surface is formed. The structural features of the average distribution density and anisotropic distribution of nanoprotrusions that constitute the hierarchical nano-roughened surface of the surface-treated titanium substrate of the present invention are formed at approximately this stage. Further sintering under atmospheric pressure converts the hydrated gel of alkali titanate into amorphous titanium oxide, thereby obtaining the surface-treated titanium substrate of the present invention.

[0024] (Titanium substrate having a mechanically polished surface) The description of the titanium substrate in the surface-treated titanium substrate of the present invention described above can be applied to the titanium substrate as is. There are no particular limitations on the mechanical polishing performed on the titanium substrate, and lathe processing can be given as an example. Mechanical polishing performed on the titanium substrate leaves cutting marks on the surface of the titanium substrate. After mechanical polishing, the titanium substrate is preferably washed because machine oil adheres to it. There are no particular limitations on the washing method, and it is preferable to wash the titanium substrate successively with acetone, ethanol, and distilled water, for example. After washing, it is preferable to perform a heat treatment with an acid.

[0025] (Heat Treatment with Acid) By subjecting a titanium substrate having the above-described mechanically polished surface to heat treatment with acid, micro-roughness can be imparted to the mechanically polished surface of the titanium substrate. In the present invention, "micro-roughness" refers to the undulations (roughness) of the titanium substrate surface on the order of approximately 1 to 10 micrometers. For example, the micro-roughness shown in the scanning electron microscope (SEM) image of the rough surface of a titanium disk that has been subjected to micro-roughening treatment shown in Figure 1 can be obtained. Note that this acid heat treatment also removes TiO present on the surface of the titanium substrate. ２ The passive film of TiH is reduced ２ A layer is formed, imparting micro-roughness. Acids capable of reducing the passive film present on the surface of a mechanically polished titanium substrate and imparting micro-roughness include acids that do not supply oxygen (non-oxidizing acids), and preferred examples include sulfuric acid, hydrofluoric acid, hydrochloric acid, and phosphoric acid, with sulfuric acid being more preferred. The above acids may be used alone or in combination of two or more.

[0026] The acid heat treatment can be carried out, for example, by immersing a titanium substrate having a mechanically polished surface in an aqueous solution of the acid and heating it. The concentration of the acid aqueous solution can be adjusted appropriately depending on the type of acid used. For example, the concentration of the sulfuric acid aqueous solution can be 60 to 90 mass%, preferably 65 to 75 mass%, and more preferably 67 to 70 mass%. The temperature of the acid heat treatment can be adjusted appropriately depending on the type of acid used. For example, the temperature of the heat treatment with the sulfuric acid aqueous solution is preferably 100 to 150°C, more preferably 110 to 140°C, and even more preferably 120 to 140°C, from the viewpoint of increasing the anisotropy of the micro-roughened shape and further anisotropizing the structure of the nano-roughened surface by heat treatment with a 7.5 to 12.5 M alkali. The time of the acid heat treatment can be adjusted appropriately depending on the type of acid used and the heat treatment temperature. For example, the time of the heat treatment with the sulfuric acid aqueous solution can be 60 to 150 seconds, preferably 70 to 140 seconds, and more preferably 75 to 100 seconds. After the acid heat treatment, the titanium substrate is preferably washed to remove the acid treatment solution. There are no particular restrictions on the washing method, and it is preferable to use, for example, distilled water.

[0027] (7.5-12.5M Alkali Heat Treatment) A titanium substrate having a micro-roughened surface that has been subjected to the above-described mechanical polishing and acid heat treatment in sequence can be further subjected to a 7.5-12.5M alkali heat treatment (hereinafter simply referred to as "alkali heat treatment") to form the above-described hierarchical nano-roughened surface structure on the surface of the titanium substrate. This alkali heat treatment produces a layer of alkali titanate hydrate gel on the titanium oxide layer having micro-roughness. Preferred alkalis that can form a layer of alkali titanate hydrate gel on the surface of a titanium substrate that has been subjected to the above-described mechanical polishing and acid heat treatment in sequence include sodium hydroxide and potassium hydroxide, with sodium hydroxide being more preferred. The above alkalis may be used alone or in combination.

[0028] The alkali heat treatment can be carried out, for example, by immersing a titanium substrate having a micro-roughened surface that has been mechanically polished and heat-treated with an acid in the alkali aqueous solution and then heating it. The alkali heat treatment is preferably a water bath treatment in the alkali aqueous solution. By adjusting the concentration of the alkali aqueous solution to 7.5 to 12.5 M, a hierarchical nano-roughened surface composed of nanoprotrusions that satisfy the aforementioned average distribution density and distribution anisotropy can be formed. A concentration of 8.0 to 12.0 M is preferred, 8.5 to 11.5 M is more preferred, and 9.0 to 11.0 M is even more preferred. In particular, as is clear from a comparison of the TEM images shown in Figures 9 and 10 of the Examples described below, when the concentration of the sodium hydroxide aqueous solution is 10 M (Figure 10), the layer containing amorphous titanium oxide obtained as a result of the acid heat treatment, alkali heat treatment, and sintering under atmospheric pressure is approximately 1.7 times thicker and denser than when the concentration of the sodium hydroxide aqueous solution is 5 M (Figure 9). The temperature of the alkali heat treatment can be adjusted appropriately depending on the type of alkali used. For example, 60 to 100°C is preferred. From the viewpoint of suppressing alkali crystallization that may occur during the alkali heat treatment, a temperature above 80°C but not exceeding 100°C is more preferred, and 85 to 100°C is even more preferred. The alkali heat treatment time can be adjusted appropriately depending on the type of alkali used and the heat treatment temperature. For example, it can be 20 to 30 hours, preferably 20 to 28 hours, and more preferably 22 to 28 hours. After the alkali heat treatment, the titanium substrate is preferably washed to remove any alkali remaining on the surface of the titanium substrate. There are no particular restrictions on the washing method, and distilled water is preferred, for example. After the alkali heat treatment, the titanium substrate is preferably dried before the sintering treatment (preferably after the washing and before the sintering treatment). There are no particular restrictions on the drying method, and examples include natural drying under dry air.

[0029] (Sintering Treatment Under Atmospheric Pressure) The titanium substrate, which has been subjected to the above-mentioned mechanical polishing, heat treatment with an acid, and heat treatment with an alkali in sequence, and on which a hierarchical nano-rough surface structure has been formed, is then subjected to a sintering treatment under atmospheric pressure (hereinafter also simply referred to as "sintering treatment"), whereby the hydrated gel of alkali titanate that constitutes the hierarchical nano-rough surface is converted into amorphous titanium oxide.

[0030] The means for carrying out the sintering treatment are not particularly limited, and for example, a heating furnace such as an electric furnace, a gas heating furnace, or an induction heating furnace can be used. The temperature for the sintering treatment is, for example, preferably 500 to 700°C, more preferably 550 to 650°C, and even more preferably 580 to 620°C. The sintering treatment can be carried out under atmospheric pressure. In the present invention, "under atmospheric pressure" refers to atmospheric pressure (-10 to 0 PaG) or a sealed, pressurized atmosphere (greater than 0 kPaG but not exceeding 19.6 kPaG), with atmospheric pressure being preferred. If the sintering treatment is carried out under vacuum or reduced pressure (less than -10 PaG), particularly under vacuum, the hydrated gel of the alkali titanate will not be converted to amorphous titanium oxide, and the surface-treated titanium substrate of the present invention will not be obtained. The sintering time is not particularly limited as long as the hydrated gel of the alkali titanate is converted to amorphous titanium oxide, and can be, for example, 30 minutes to 2 hours, preferably 45 minutes to 1.5 hours, and more preferably 50 minutes to 1.2 hours. The method for lowering the temperature of the titanium substrate after the sintering treatment to room temperature is not particularly limited as long as the surface-treated titanium substrate of the present invention can be obtained, and examples thereof include air cooling.

[0031] [Composite] The composite of the present invention comprises the surface-treated titanium substrate of the present invention and nano-needle crystals of hydroxyapatite (HAp) formed on the hierarchical nano-roughened surface of the surface-treated titanium substrate. In the present invention, "nano-needle crystals" refers to needle crystals with a nano-scale short diameter of about 1 to 1000 nm. The short diameter is defined as described below. In the present invention, "hydroxyapatite (HAp)" refers to Ca １０ (P.O. ４ ) ６ (OH) ２ In addition to hydroxyapatite itself, Ca１０ (P.O. ４ ) ６ (OH) ２ The term "calcium phosphate" is used to include calcium phosphate that exhibits an XRD pattern similar to the crystal structure of the above, and is insoluble in water (neutral) but soluble in acidic water.

[0032] (Applications) The composite of the present invention can be used as a bioactive implant material for artificial bones, artificial dental roots, synthetic bone replacement materials, bone defect fillers, artificial joints, blood filter materials, catheters, stents, and other applications. For example, the composite of the present invention obtained by using titanium having a desired shape, such as particulate, as the titanium substrate can be used as the above-mentioned material. The composite of the present invention can exhibit enhanced bioactivity due to the synergistic effect of the hierarchical nano-roughened surface of the surface-treated titanium substrate of the present invention and the nano-needle crystals of HAp formed on the hierarchical nano-roughened surface. In the present invention, "bioactivity" refers to high affinity or adhesion with the living body. Furthermore, the composite of the present invention can be used as a source of HAp. For example, HAp extracted from the composite of the present invention can be used for various applications as a functional material. Furthermore, differentiation into osteoblasts can be promoted by culturing bone marrow stromal cells using the composite of the present invention as a culture bed.

[0033] [Method for producing a composite and method for producing needle-like hydroxyapatite crystals] The composite of the present invention can be produced by a method comprising immersing a surface-treated titanium substrate of the present invention in a simulated body fluid containing calcium and phosphorus (hereinafter also referred to simply as "simulated body fluid") to precipitate needle-like hydroxyapatite (HAp) crystals on the hierarchical nano-roughened surface of the surface-treated titanium substrate of the present invention. Furthermore, the needle-like HAp crystals of the present invention can be produced by a method comprising immersing a surface-treated titanium substrate of the present invention in a simulated body fluid containing calcium and phosphorus to precipitate needle-like HAp crystals. That is, the needle-like HAp crystals of the present invention can be produced by precipitating needle-like HAp crystals on the hierarchical nano-roughened surface of a surface-treated titanium substrate of the present invention, similar to the method for producing the composite of the present invention, and then recovering the resulting needle-like HAp crystals.

[0034] In the surface-treated titanium substrate of the present invention, the hierarchical nano-rough surface is composed of amorphous titanium oxide, and the apexes of the nanoprojections that make up the hierarchical nano-rough surface are arranged with high density and high anisotropy in their distribution, which enhances the intermolecular forces and electrostatic forces between the nanoprojections. As a result, it is believed that immersing the surface-treated titanium substrate of the present invention in a simulated body fluid efficiently attracts calcium ions to the hierarchical nano-rough surface of the surface-treated titanium substrate of the present invention, and subsequently attracts phosphate ions. As a result, simply by immersing the surface-treated titanium substrate of the present invention in a simulated body fluid, it is possible to more efficiently precipitate HAp nano-needle crystals, i.e., to more efficiently promote the self-organization of HAp nano-needle crystals. In particular, as can be seen from the SEM images shown in Figures 19, 20, 28, 29, and 31 of the Examples described below, the GD-OES analysis (depth-direction component analysis) shown in Figure 24, the XRD analysis shown in Figure 25, and the electron diffraction structure analysis shown in Figure 44, the HAp nanoneedle crystals obtained by the composite manufacturing method of the present invention are thick and highly crystalline precipitated on the surface of the surface-treated titanium substrate of the present invention. Furthermore, the STEM HAADF analysis shown in Figure 45 also reveals that the HAp nanoneedle crystals obtained by the composite manufacturing method of the present invention are biomimetic HAp needle crystals with a high Ca / P ratio and a stable crystal structure. Furthermore, the image analysis using ImageJ shown in Figure 48 also reveals that the HAp nanoneedle crystals obtained by the composite manufacturing method of the present invention are longer and wider in shape.

[0035] (Immersion of Surface-Treated Titanium Substrate in Simulated Body Fluid) The "simulated body fluid containing calcium and phosphorus" used in the method for producing a composite of the present invention specifically refers to a simulated body fluid having the ion composition shown in Table 3 in the Examples below. The simulated body fluid can be prepared according to S.B. Cho, K. Nakanishi, T. Kokubo, N. Soga, C. Ohtsuki, T. Nakamura, T. Kitsugi, and T. Yamamuro, J. Am. Ceram. Soc., 78, 1769-1774 (1995). The pH of the simulated body fluid may be 7.4 to 7.5, with 7.4 being preferred. The pH can be adjusted by conventional methods using hydrochloric acid, sodium hydroxide, or the like. The conditions (temperature, time) for immersing the surface-treated titanium substrate of the present invention in the simulated body fluid are not particularly limited, as long as the hierarchical nano-roughened surface of the surface-treated titanium substrate of the present invention is immersed in the simulated body fluid and nano-needle crystals of HAp are precipitated. The temperature conditions for immersion in the simulated body fluid are, for example, preferably 35 to 38° C., more preferably 36 to 37° C. The time for immersion in the simulated body fluid is, for example, preferably 1 to 4 weeks, more preferably 2 to 4 weeks.

[0036] (Recovery and purification of HAp nanoneedle crystals) The HAp nanoneedle crystals precipitated on the surface-treated titanium substrate of the present invention can be peeled off and recovered from the surface-treated titanium substrate of the present invention by physical peeling or the like. There are no particular limitations on the physical peeling, and it can be carried out by a conventional method, for example, peeling by scraping. The recovered HAp nanoneedle crystals can usually be used as is without treatment such as purification. In addition, if the purity is to be further improved, they can be purified by a conventional method such as natural precipitation and used.

[0037] For example, when the composite of the present invention obtained by immersing the surface-treated titanium substrate of the present invention in a simulated body fluid at 37°C for 4 weeks is subjected to glow discharge optical emission spectroscopy (GD-OES analysis), elemental analysis in the depth direction from the outermost surface (depth 0 nm) of the HAp nano-needle crystals toward the surface-treated titanium substrate reveals that the emission intensity of calcium is observed up to a depth of about 300 to 400 nm, and the emission intensity of phosphorus is observed up to a depth of about 250 to 350 nm.

[0038] [HAp nanoneedle crystals] The HAp nanoneedle crystals of the present invention exhibit high crystallinity, and examples thereof include HAp nanoneedle crystals that satisfy at least one of the average interplanar spacing and various parameters analyzed by image analysis software (average area, average major axis, and average aspect ratio) listed below. Furthermore, since the HAp nanoneedle crystals of the present invention have a stable crystal structure in addition to high crystallinity, examples thereof include HAp nanoneedle crystals that satisfy the following Ca / P ratio. The HAp nanoneedle crystals of the present invention refer to an aggregate of HAp nanoneedle crystals. Therefore, the Ca / P ratio, interplanar spacing, and various parameters analyzed by image analysis software (average area, average major axis, and average aspect ratio) also refer to the values ​​as an aggregate of HAp nanoneedle crystals. The HAp nanoneedle crystals of the present invention can be produced, for example, by the method for producing the HAp nanoneedle crystals of the present invention.

[0039] (Face spacing) The HAp nanoneedle crystal of the present invention may be a HAp nanoneedle crystal having a face spacing (distance between crystal faces) of 8.25 Å or less on the (100) plane obtained by electron diffraction structure analysis. The smaller the face spacing of the (100) plane, the more stable the crystal structure becomes, and 8.20 Å or less is preferable. The lower limit of the face spacing is determined by the Ca ５ [P.O. ４ ] ３ The a-axis lattice constant of [OH] is 8.15 to 8.25 Å, for example.

[0040] (Analysis items by image analysis software: area, major axis, and aspect ratio) The HAp nanoneedle crystals of the present invention exhibit high crystallinity, and therefore can become larger crystals (crystals with longer and wider shapes) than HAp needle crystals in conventional nano-roughened surface thin flake samples. Specifically, examples of the HAp nanoneedle crystals of the present invention include HAp nanoneedle crystals obtained by analyzing TEM images with image analysis software, whose average area, average major axis, and average aspect ratio satisfy the following: Image analysis software used was ImageJ 1.53t (open source Java software, Wayne Rasband and contributors National Institutes of Health, USA, available for download at https: / / imagej.net / ij / , Java 1.8.0_345 (64-bit)). Needle structures with the same contrast as the needle-like crystals confirmed to be HAp by electron diffraction structure analysis (i.e., needle-like crystals of HAp, not needle-like Ti substrates) were visually identified, and one of these HAp nanoneedle crystals (primary needle-like crystals of HAp) was selected using the Polygon selection tool in ImageJ 1.53t, and the area, major axis, and minor axis were measured. The average area, average major axis, and average aspect ratio are all average values ​​for 17 or more primary needle-like crystals of HAp. The average area of ​​the HAp nanoneedle crystals was 2000 nm ２ or more, 2000 to 14000 nm ２ is preferred, and 3000 to 14000 nm ２ More preferably, 4000 to 14000 nm ２ More preferably, 5000 to 14000 nm ２ is particularly preferred, and 6000 to 14000 nm ２ The upper limit of the average area of ​​the HAp nanoneedle crystals is 12,000 nm ２ It can also be 11000 nm or less, ２ It is also preferable to set it to 10,000 nm or less. ２ It is more preferable to set it to 9000 nm or less. ２It is even more preferable that it is less than 200 nm. The average major axis of the HAp nanoneedle crystals is 200 nm or more, preferably 200 to 700 nm, more preferably 250 to 700 nm, even more preferably 300 to 700 nm, and particularly preferably 350 to 700 nm. The upper limit of the average major axis of the HAp nanoneedle crystals can be 650 nm or less, preferably 600 nm or less, more preferably 550 nm or less, and even more preferably 500 nm or less. The average aspect ratio of the HAp nanoneedle crystals is 6 to 20, for example, preferably 7 to 16, and more preferably 8 to 14. The "major axis" of the nanoneedle crystals refers to the maximum Feret diameter (the distance between two parallel lines sandwiching the nanoneedle crystal), which is the Euclidean distance between two points combining all points detected on the contour of the object, and the minor axis of the nanoneedle crystal refers to the length of the minor axis of an ellipse fitted to the object by the least squares method. The aspect ratio of the nanoneedle crystals refers to the ratio of the long diameter of the nanoneedle crystals to the short diameter of the nanoneedle crystals (long diameter of the nanoneedle crystals / short diameter of the nanoneedle crystals). The area of ​​the nanoneedle crystals refers to the area within the outline selected as the region of interest. The coefficients of variation (values ​​obtained by dividing the standard deviation by the average value and multiplying by 100 to express as a percentage) of the data used to calculate the average area, average long diameter, and average aspect ratio as described above are 35% or less for the average area, 30% or less for the average long diameter, and 30% or less for the average aspect ratio, respectively.

[0041] (Ca / P ratio) The HAp nanoneedle crystals of the present invention include HAp nanoneedle crystals having a ratio of calcium atoms to phosphorus atoms (also simply referred to as "Ca / P ratio" in the present invention) of 1.00 or more, calculated by HAADF-STEM (high-angle annular dark-field scanning transmission microscope) and EDX (energy dispersive X-ray spectroscopy) analysis. The higher the Ca / P ratio, the more stable the crystalline structure of HAp becomes, and the ratio is preferably 1.20 or more, more preferably 1.35 or more, and even more preferably 1.40 or more. The upper limit of the Ca / P ratio is the ratio of calcium atoms to phosphorus atoms (also simply referred to as "Ca / P ratio" in the present invention). ５ [P.O. ４ ] ３The stoichiometric ratio of [OH] is Ca / P = 5 / 3 = approximately 1.67. Therefore, a preferred range of the Ca / P ratio is, for example, 1.00 to 5 / 3. It is known that the Ca / P ratio of HAp in vivo is approximately 1.47 due to the outflow of Ca. From the viewpoint of achieving a more biomimetic crystal structure, the upper limit of the Ca / P ratio is preferably 1.62 or less, more preferably 1.57 or less, and even more preferably 1.52 or less.

[0042] (Uses) The HAp nanoneedle crystals obtained by the manufacturing method of the present invention and the HAp nanoneedle crystals of the present invention can be used in a variety of applications. For example, they can be used as a material for remineralizing dental enamel and a dental pulp capping material. In this case, there is no particular limitation, but an example is a form in which the HAp nanoneedle crystals obtained by the manufacturing method of the present invention or the HAp nanoneedle crystals of the present invention are mixed as nanoparticles with a drug and used. They can also be used as an artificial synthetic bone filling material. In this case, there is no particular limitation, but an example is a form in which the HAp nanoneedle crystals obtained by the manufacturing method of the present invention or the HAp nanoneedle crystals of the present invention are used in an aggregated form. They can also be used as cosmetics, catalyst materials, research reagents, etc. They can also be used as adsorbents for columns separating biological macromolecules such as amino acids, proteins, lipids, and sugars.

[0043] [Implant] The implant of the present invention comprises the surface-treated titanium substrate of the present invention or the composite of the present invention. Implants such as artificial bones, artificial dental roots, artificial synthetic bone filling materials, bone defect fillers, artificial joints, blood filtering materials, catheters, and stents, which comprise the surface-treated titanium substrate of the present invention or the composite of the present invention, can exhibit high bioactivity (high affinity or adhesion to the living body) and high tissue bonding strength (high bonding strength with bone tissue and periodontal tissue).

[0044] [Culture Bed] The culture bed of the present invention comprises the surface-treated titanium substrate of the present invention or the composite of the present invention. By using the surface-treated titanium substrate of the present invention or the composite of the present invention as a culture bed for bone marrow stromal cells and culturing the bone marrow stromal cells in an osteoblast differentiation-inducing medium, it is possible to promote the differentiation of bone marrow stromal cells into osteoblasts. As a result, a mineralized matrix with a high calcium content and high crystallinity can be obtained. Therefore, by implanting these culture beds in a living body, it is expected that bone formation will occur in the living body.

[0045] The present invention will be described in more detail based on examples. The present invention should not be construed as being limited to the following examples, except as otherwise specified in the present invention. In the figures, "P < 0.05 between different letters, Tukey HSD test" indicates that there is a significant difference in the Tukey HSD test between data marked with different letters (a, b, c, etc.) in each figure. In the figures, "P < 0.05 between different circles, Tukey HSD test" indicates that there is a significant difference in the Tukey HSD test between data surrounded by different circles in each figure. In the graphs in the figures, unless otherwise specified, the bars indicate the mean values, and the error bars indicate the standard deviation.

[0046] The following instruments were used in each analysis. Scanning electron microscope (SEM) analysis: Model XL30 microscope (instrument name), manufactured by Philips Zeta potential analysis: ELS-Z2 (instrument name), manufactured by Otsuka Electronics Co., Ltd. Surface potential microscope (KFM) analysis: Dimension icon XR (instrument name), manufactured by Bruker Fourier transform infrared spectroscopy analysis (FTIR analysis): IRT7000 linear array imaging microscope (instrument name), manufactured by JASCO Corporation Transmission electron microscope (TEM) analysis: HF-2000 (instrument name), manufactured by HITACHI Corporation Energy dispersive X-ray spectroscopy analysis (EDX analysis): EX-94300S4L1Q (instrument name), manufactured by JEOL Ltd. HAADF-STEM (High Angle Scattering Annular Dark-Field Scanning Transmission Microscope) and EDX (Energy Dispersive X-ray Spectroscopy) analysis: JEM-ARM200F (Cold-FEG) (instrument name) and SDD detector CENTURIO (instrument name, large area 100 mm ２ Silicon drift detector), both manufactured by JEOL Ltd. Colorimetric assay analysis using colorimetric reactions of calcium ions and phosphate ions: Calcium assay kit (BioChain), Malachite Green Phosphate assay (R&D System), SpectraMax M5 ｅ (Device name), manufactured by Molecular Devices Glow discharge optical emission spectroscopy (GD-OES analysis): GD-Profiler 2 (Device name), manufactured by HORIBA X-ray diffraction (XRD) analysis: SmartLab (Device name), manufactured by Rigaku Electron diffraction structure analysis: JEM-ARM200F (Device name), manufactured by JEOL Ltd.

[0047] Example 1A: Preparation of surface-treated titanium disc (1) Preparation of titanium disc with mechanically polished surface A titanium disc (ASTM (American Society for Testing and Materials) standard Grade II Ti (pure titanium), diameter 20 mm x thickness 1 mm) was lathe-machined to prepare a titanium disc with a mechanically polished surface. Hereinafter, the titanium disc with a mechanically polished surface prepared as described above will be referred to as a "polished titanium disc", and the following surface roughening treatment was carried out so that the mechanically polished surface of the polished titanium disc was roughened. (2) Surface roughening treatment of polished titanium disc The polished titanium disc obtained in (1) above was subjected to one of the following surface roughening treatments (i) to (iii). The roughened surface of the surface-treated titanium disks subjected to (i) hierarchical nano-roughening treatment or (ii) nano-roughening treatment has amorphous titanium oxide formed on the outermost layer (the layer constituting the outermost surface). (i) Hierarchical nano-roughening treatment: A polished titanium disk was immersed in 60% concentrated sulfuric acid heated to 120°C for 90 seconds and then washed with distilled water to produce a surface-treated titanium disk with a micro-roughened surface. The surface-treated titanium disk with a micro-roughened surface was then immersed in a 10 M aqueous sodium hydroxide solution at 90°C for 24 hours. The disk was then sintered at 600°C under atmospheric pressure to produce a surface-treated titanium disk with a hierarchical nano-roughened surface. (ii) Nano-roughening treatment: A polished titanium disk was immersed in a 10 M aqueous sodium hydroxide solution at 90°C for 24 hours. The disk was then sintered at 600°C under atmospheric pressure to produce a surface-treated titanium disk with a nano-roughened surface. (iii) Micro-roughening Treatment The polished titanium disk was immersed for 90 seconds in hot 60% concentrated sulfuric acid heated to 120° C. to prepare a surface-treated titanium disk having a micro-roughened surface.

[0048] The surfaces of the surface-treated titanium disks prepared as described above were analyzed by scanning electron microscope, zeta potential analysis, and Kelvin probe force microscope. The "roughened surface of the surface-treated titanium disk" is also referred to simply as the "surface of the surface-treated titanium disk." In the evaluations of Examples 1A to 1C, Reference Example 1, and Examples 2A and 2B below, unless otherwise specified, in each figure, "Machined" refers to a polished titanium disk, "Nano" refers to a surface-treated titanium disk with a nano-roughened surface, "Micro" refers to a surface-treated titanium disk with a micro-roughened surface, "Micro / Nano" refers to a surface-treated titanium disk with a hierarchical nano-roughened surface, and "Polystyrene" refers to a polystyrene disk.

[0049] [Evaluation 1: Scanning Electron Microscope Analysis] Figure 1 shows scanning electron microscope (SEM) images of the surfaces of each surface-treated titanium disk. From the SEM images shown in Figure 1, hierarchical nanoprotrusions (hierarchical nanorough surfaces) were observed in the surface-treated titanium disks with a hierarchical nanorough surface, which were not observed in the surface-treated titanium disks with a microrough surface or a nanorough surface. Furthermore, vertices (vertices of nanoprotrusions) were extracted from the brightness of secondary electron images taken with an SEM at magnifications of 5,000 to 10,000 times. From these secondary electron images, the number of nanoprotrusions per 1 μm square (distribution density) was determined, and the average distribution density was calculated by averaging the distribution densities at three different locations. Furthermore, the secondary electron images were subjected to Voronoi division, and the anisotropy of the nanoprotrusion distribution was determined by dividing the standard deviation of the area of ​​each divided region by the average value (to obtain the coefficient of variation). Figure 2(a) shows the "average distribution density of nanoprotrusions," and Figure 2(b) shows the "distribution anisotropy of nanoprotrusions." As shown in Figure 2, the average distribution density of nanoprotrusions on the surface-treated titanium disk with a hierarchical nano-rough surface is 31 / μm. ２ The distribution anisotropy is 0.90, and the average distribution density of nanoprotrusions on the surface-treated titanium disk with nano-roughened surface is 16 / μm ２ , the average distribution density of the nanoprotrusions was increased by about 2 times and the distribution anisotropy of the nanoprotrusions was increased by about 1.5 times compared to the distribution anisotropy of 0.64.

[0050] [Evaluation 2: Zeta Potential Analysis] Figure 3 shows the average zeta potential of the surface of each surface-treated titanium disk. The zeta potential of the surface of the surface-treated titanium disk was measured by cutting a measurement sample measuring 5 mm long x 23 mm wide from each surface-treated titanium disk, washing the rough surface of each surface-treated titanium disk with ultrapure water, and placing the measurement sample in a flat sample cell so that the rough surface of the surface-treated titanium disk was in contact with the monitor particle solution. The measurement was performed by electrophoretic scattering. The monitor particle solution used was a measurement solution prepared by dispersing polystyrene latex monitor particles in a 10 mM sodium chloride aqueous solution at pH 6.0, and the measurement was performed at 25°C. The average zeta potential was calculated by averaging the zeta potentials of the rough surfaces of three measurement samples. As shown in Figure 3, the zeta potentials of the roughened surfaces of the polished titanium disks and the surface-treated titanium disks with micro-roughened surfaces were in the range of approximately -1 to 1 mV, whereas the zeta potentials of the roughened surfaces of the surface-treated titanium disks with hierarchical nano-roughened surfaces were in the range of approximately -1.8 to -5.6 mV, with an average zeta potential of -4.2 mV, indicating that the hierarchical nano-roughened surfaces (surfaces) had an increased negative charge.

[0051] [Evaluation 3: Surface Potential Microscope (KFM) Analysis] Using a scanning probe microscope (device name: Dimension icon XR, manufactured by Bruker), measurement mode: Peak Force Tapping Kelvin Probe Microscope (PF-KPFM) mode, measurement probe: silicon probe, the surface shape and surface potential in a measurement area of ​​1 μm square, as well as the contact potential difference (the difference between the work function value of the silicon probe and the work function value of the target point on the sample) in a measurement area of ​​10 nm square were measured. The upper part of Figure 4 shows an image of the surface shape of each surface-treated titanium disk, and the lower part of Figure 4 shows an image of the surface potential of each surface-treated titanium disk. In addition, Figure 5 shows the distribution, average value, and standard deviation of each contact potential difference (the average value and standard deviation are displayed as bars), and Table 1 shows the average value, unbiased variance, and sample number (the number of target points on the sample) of each contact potential difference. The arrows in FIG. 4 are used to make it easier to see the positions of the same protrusion apexes in the upper and lower rows.

[0052]

[0053] The white areas in the image of the surface shape in the upper part of Figure 4 indicate protrusions, and the white areas in the image of the surface potential in the lower part of Figure 4 indicate areas from which electrons are emitted. It was found that in the surface-treated titanium disk with a hierarchical nano-roughened surface, electron emission was enhanced (high contact potential difference) in areas corresponding to the apexes of the nano-protrusions of the hierarchical nano-roughened surface. Furthermore, as shown in Figure 5 and Table 1, the average contact potential difference of the roughened surface of the surface-treated titanium disk with a hierarchical nano-roughened surface was 1.8138 V / 10 nm. ２ and the average contact potential difference of the roughened surface of the surface-treated titanium disk having a nano-roughened surface is 1.6760 V / 10 nm. ２ 0.14V / nm ２ The unbiased dispersion value of the average contact potential difference of the rough surface of the surface-treated titanium disk with a hierarchical nano-roughened surface was 0.0177 V / 10 nm. ２ The unbiased dispersion value of the average contact potential difference of the roughened surface of the surface-treated titanium disk having a nano-rough surface is 0.0046 V / 10 nm. ２ 0.0131V / 10nm ２ It was found that the SiO2 content also increased, indicating higher anisotropy.

[0054] Thus, the surface-treated titanium disk with a hierarchical nano-roughened surface has a large negative charge with an average zeta potential of -4.2 mV (rating 2). Moreover, compared to the surface-treated titanium disk with a nano-roughened surface, the roughened surface has a smaller work function and exhibits high anisotropy (rating 3), which is thought to result in a higher electrical reactivity.

[0055] The above evaluations 1 to 3 show that the surface-treated titanium disks with a hierarchical nano-roughened surface have a higher average distribution density and distribution anisotropy of the nanoprotrusions compared to surface-treated titanium disks with a conventional micro-roughened or nano-roughened surface (Evaluation 1), and that the surface has a more negative surface potential compared to surface-treated titanium disks with a conventional micro-roughened surface (Evaluation 2). Furthermore, as a result of the higher average distribution density and distribution anisotropy of the nanoprotrusions, the work function of the roughened surface is smaller and its anisotropy is also higher compared to surface-treated titanium disks with a conventional micro-roughened or nano-roughened surface, which is thought to result in higher electrical reactivity.

[0056] Reference Example 1: Preparation of surface-treated titanium disks (change in nano-rough surface depending on base concentration) The polished titanium disks obtained in (1) of Example 1A above were immersed in aqueous sodium hydroxide solutions of specified concentrations (2.5 M, 5.0 M, 7.5 M, 10.0 M, 12.5 M, and 15.0 M) for 24 hours at 60° C. for 2.5 M and 5.0 M solutions, and at 90° C. for 7.5 M, 10.0 M, 12.5 M, and 15.0 M solutions. The disks were then sintered at 600° C. under atmospheric pressure to prepare surface-treated titanium disks with nano-rough surfaces. Figure 6 shows overall photographs of the surfaces of polished titanium disks and surface-treated titanium disks with nano-roughened surfaces, scanning electron microscope (SEM) images of the surfaces, and random model patterns created using Microsoft Excel programs with random points at a 30% occurrence rate within a 214 x 126 cell range (total of 26,964). Figure 7(a) shows the anisotropic distribution of nanoprotrusions constituting the roughened surface, and Figure 7(b) shows the average distribution density of the nanoprotrusions. As shown in Figure 7, the nano-roughened surfaces treated with 7.5M, 10.0M, or 12.5M aqueous sodium hydroxide solution exhibited anisotropic nanoprotrusion arrangements exceeding the anisotropy threshold (shown by the dashed line in Figure 7(a)) calculated from the random model pattern, with an average distribution density of 14 to 21 nanoprotrusions / μm. ２ On the other hand, as shown in Figure 7(a), the nanoprotrusion arrangement on the nano-roughened surface treated with 5.0 M or less aqueous sodium hydroxide solution did not exceed the anisotropy threshold calculated from the random model pattern and was isotropic. Furthermore, as shown in Figure 6, the formation of nanoprotrusions was not confirmed on the roughened surface treated with 15.0 M or more aqueous sodium hydroxide solution. Furthermore, as shown in the spectrum obtained by Fourier transform infrared spectroscopy (FTIR analysis) in Figure 8, there was almost no difference in the presence of OH groups, which are the source of the negative potential on the titanium surface, between concentrations of 5.0 M, 10.0 M, and 15.0 M.

[0057] For a surface-treated titanium disk having a nano-roughened surface treated with a 5.0 M aqueous solution of sodium hydroxide, Fig. 9(a) shows a transmission electron microscope (TEM) image of a longitudinal section of the surface of the surface-treated titanium disk, and Figs. 9(b) to 9(d) show enlarged images and / or energy dispersive X-ray spectroscopy (EDX) analysis results for each of the microregions b to d shown in Fig. 9(a). For a surface-treated titanium disk having a nano-roughened surface treated with a 10.0 M aqueous solution of sodium hydroxide, Fig. 10(a) shows a transmission electron microscope (TEM) image of a longitudinal section of the surface of the surface-treated titanium disk, and Figs. 10(b) to 10(e) show enlarged images and / or energy dispersive X-ray spectroscopy (EDX) analysis results for each of the microregions b to e shown in Fig. 10(a). The samples (longitudinal sections) for each TEM image were obtained using a GATAN 691 PIPS ion milling system (manufactured by GATAN Corporation) without disrupting the roughened surface structure of the surface-treated titanium disk. Figures 9(d) and 10(e) correspond to the untreated portion of the titanium disk (untreated titanium disk). As shown in Figures 9 and 10, regardless of whether the titanium disk was treated with 5.0M or 10.0M sodium hydroxide solution, the outermost layer of the roughened surface was composed of amorphous sodium titanate, and the crystallinity increased toward the titanium disk (inner side). On the other hand, the thickness of the porous structure on the surface of the surface-treated titanium disk was 600-700 nm when treated with 5.0 M aqueous sodium hydroxide solution (Fig. 9(a)), whereas it was 1000-1200 nm when treated with 5.0 M aqueous sodium hydroxide solution (Fig. 10(a)), showing an increase of about 1.7 times.

[0058] Example 1B-1: Preparation of Surface-Treated Titanium Disks (Changes in Hierarchical Nano-Rough Surface Depending on Base Concentration) The polished titanium disks obtained in Example 1A (1) above were immersed in a sodium hydroxide solution of a specified concentration (5.0 M or 10.0 M) at 90°C for 24 hours. Subsequently, they were sintered at 600°C under atmospheric pressure to prepare surface-treated titanium disks with nano-rough surfaces. Furthermore, the polished titanium disks obtained in Example 1A (1) above were immersed in 60% concentrated sulfuric acid heated to 120°C for 90 seconds and washed with distilled water to prepare surface-treated titanium disks with micro-rough surfaces. Furthermore, the surface-treated titanium disks with micro-rough surfaces were immersed in a sodium hydroxide solution of a specified concentration (5.0 M or 10.0 M) at 90°C for 24 hours. Subsequently, they were sintered at 600°C under atmospheric pressure to prepare surface-treated titanium disks with hierarchical nano-rough surfaces. Figure 11 shows scanning electron microscope (SEM) images of the surface of each surface-treated titanium disk, and Figure 12 shows the anisotropic distribution of nanoprotrusions constituting the rough surface. Figure 13 shows the average zeta potential of the surface of each surface-treated titanium disk, and Figure 14 shows images of the surface of each surface-treated titanium disk measured by scanning probe microscopy. The upper part of Figure 14 shows an image of the surface shape, the middle part shows an image of the surface potential, and the lower part shows the distribution of contact potential difference. Table 2 shows the average value, unbiased variance, and sample number (number of target points on the sample) of each contact potential difference. The evaluation methods were as described in Example 1A above.

[0059]

[0060] 11 and 12, when a 5 M aqueous solution of sodium hydroxide was used, no significant change was observed when the hierarchical nano-roughening treatment was performed compared to the nano-roughening treatment, and the anisotropy of the nanoprotrusion distribution merely changed from 0.44 to 0.52, which does not satisfy the anisotropy of the nanoprotrusion distribution specified in the present invention (see Micro / Nano(5M) versus Nano(5M) in FIGS. 11 and 12). In contrast, when a 10 M aqueous solution of sodium hydroxide was used, the anisotropy of the nanoprotrusion distribution increased from 0.6 to 0.9 by performing the hierarchical nano-roughening treatment compared to the nano-roughening treatment, which satisfies the anisotropy of the nanoprotrusion distribution specified in the present invention. Similarly, the average zeta potential increased in the negative direction from −3.6 mV to −4.2 mV, and the unbiased variance of the contact potential difference also increased from 0.005 to 0.018 (see Figures 11 to 14 and Table 2 for Micro / Nano (10 M) relative to Nano (10 M)).

[0061] Example 1B-2: Preparation of Surface-Treated Titanium Disks (Changes in Hierarchical Nano-Rough Surface Depending on Base Concentration, Comparison with Surface-Treated Titanium Disks Prepared by Sintering under Reduced Pressure Conditions) The polished titanium disk obtained in (1) of Example 1A above was immersed in 60% concentrated sulfuric acid heated to 120°C for 90 seconds and then washed with distilled water to prepare a surface-treated titanium disk with a micro-rough surface. The surface-treated titanium disk with a micro-rough surface was then immersed in a sodium hydroxide solution of a specified concentration (7.5 M, 10 M, or 12.5 M) at 90°C for 24 hours. The resulting solution was then sintered at 600°C under atmospheric pressure to prepare a surface-treated titanium disk with a hierarchical nano-rough surface. The polished titanium disk obtained in (1) of Example 1A above was immersed in a 48% aqueous sulfuric acid solution heated to 60°C for 1 hour, and the surface-treated titanium disk with a micro-rough surface was then immersed in a 5 M aqueous sodium hydroxide solution at 50°C for 24 hours. The disks were then sintered at 600°C for 10 minutes under reduced pressure (-100 kPaG) to produce surface-treated titanium disks sintered under reduced pressure. Figure 15 shows scanning electron microscope (SEM) images of the surfaces of the respective surface-treated titanium disks, while Figure 16(a) shows the average distribution density of the nanoprotrusions constituting the roughened surface, and Figure 16(b) shows the anisotropy of the distribution of the nanoprotrusions constituting the roughened surface. The evaluation methods were as described in Example 1A above. Surface-treated titanium disks sintered under reduced pressure are referred to as "sintered under reduced pressure" in the figures.

[0062] As shown in Figures 15 and 16, when any of 7.5 M, 10 M, and 12.5 M aqueous sodium hydroxide solutions was used, the average distribution density of the nanoprotrusions constituting the hierarchical nano-rough surface was 22 or 32 / µm ２The average nanoparticle density was high, and the distribution anisotropy was high at 0.85 to 0.95, satisfying the average nanoparticle distribution density and distribution anisotropy specified in the present invention (see Micro / Nano 7.5M, 10M, and 12.5M in Figures 15 and 16). On the other hand, in the surface-treated titanium disks sintered under reduced pressure, the conversion of the alkali titanate hydrate gel to amorphous sodium titanate hardly progressed at all. Furthermore, in the surface-treated titanium disks sintered under reduced pressure (which adopted the acid heat treatment and alkali heat treatment conditions described in Patent Document 2), the nanoparticle distribution anisotropy was low at 0.61, not satisfying the nanoparticle distribution anisotropy specified in the present invention (see Micro / Nano "Sintering under reduced pressure" in Figures 15 and 16).

[0063] Example 1C: Preparation of Surface-Treated Titanium Alloy Disks A titanium disk with a mechanically polished surface was prepared in the same manner as in Example 1A above, except that the titanium disk used in (1) preparation of the titanium disk with a mechanically polished surface was changed from ASTM Grade II Ti (pure titanium) to ASTM Grade V Ti (Ti-6Al-4V titanium alloy). Further, the titanium disk was subjected to the (i) hierarchical nano-roughening treatment to prepare a surface-treated titanium disk with a hierarchical nano-roughened surface. Scanning electron microscope (SEM) images of the surfaces of the polished titanium disk and the surface-treated titanium disk with a hierarchical nano-roughened surface are shown in Figure 17. The SEM image shown in Figure 17 confirms that hierarchical nanoprotrusions were present when a titanium disk made of ASTM Grade V Ti (Ti-6Al-4V titanium alloy) was used, just as they were when a titanium disk made of ASTM Grade II Ti (pure titanium) was used. Figure 18(a) shows the average distribution density of the nanoprotrusions that make up the hierarchical nano-roughened surface, and Figure 18(b) shows the distribution anisotropy of the nanoprotrusions that make up the hierarchical nano-roughened surface. In the surface-treated titanium disk with a hierarchical nano-roughened surface obtained using a titanium disk made of ASTM Grade V Ti (Ti-6Al-4V titanium alloy), the average distribution density of the nanoprotrusions that make up the hierarchical nano-roughened surface was 30 / µm. ２The nanoprotrusion distribution anisotropy was 0.73, indicating that a hierarchical nano-roughened surface was formed with an average density and distribution anisotropy of nanoprotrusions comparable to those of a titanium disk made of ASTM Grade II Ti (pure titanium). The evaluation methods were as described in Example 1A above. Figures 18(c) and 18(d) show the results of elemental analysis of the surface of the surface-treated titanium disk by energy dispersive X-ray spectroscopy (EDX). As shown in Figure 18(d), when a titanium disk made of ASTM Grade V Ti (Ti-6Al-4V titanium alloy) was used, vanadium (V) was detected, which was hardly detected in the Grade II titanium disk. Furthermore, as shown in Figures 18(c) and 18(d), the hierarchical nano-roughening treatment reduced the percentages (%) of Ti, Al, and V among the constituent elements of the hierarchical nano-roughened surface, replacing them with other elements.

[0064] Example 2A: Preparation of HAp nanoneedle crystals by immersion of surface-treated titanium disks in simulated body fluid Each of the surface-treated titanium disks prepared in Example 1A above was immersed for 28 days at 37°C in a simulated body fluid (pH 7.4) having the composition shown in Table 3 below. Hereinafter, "simulated body fluid" refers to the simulated body fluid (SBF) having the composition shown in Table 3 below.

[0065]

[0066] The time-dependent changes in the constituent atoms of the precipitates on the rough surface of the surface-treated titanium disks due to immersion in a simulated body fluid were analyzed using energy dispersive X-ray spectroscopy (EDX). After 28 days of immersion, the surface-treated titanium disks were analyzed using a scanning electron microscope (SEM), energy dispersive X-ray spectroscopy (EDX analysis, surface elemental analysis), glow discharge optical emission spectroscopy (GD-OES analysis, depth elemental analysis), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR).

[0067] [Evaluation 4: Scanning Electron Microscope Analysis] Figure 19 shows scanning electron microscope (SEM) images of the surfaces of each surface-treated titanium disk before and after 28 days of simulated body fluid immersion. The left side shows the SEM image before immersion, and the right side shows the SEM image after immersion. The SEM images shown in Figure 19 reveal that the polished titanium disk and the surface-treated titanium disk with a micro-roughened surface did not show any significant changes due to immersion in the simulated body fluid. Even the surface-treated titanium disk with a nano-roughened surface showed uneven precipitates due to immersion in the simulated body fluid, and the amount of precipitate was small. In contrast, the surface-treated titanium disk with a hierarchical nano-roughened surface showed uniform precipitates upon immersion in the simulated body fluid, and the amount of precipitate was large. 20 shows SEM images of the surface-treated titanium disks with a nano-roughened surface and the surface-treated titanium disks with a hierarchical nano-roughened surface after 28 days of immersion in a simulated body fluid, with the upper row showing SEM images taken perpendicular to the surface of the surface-treated titanium disk and the lower row showing SEM images taken obliquely to the surface of the surface-treated titanium disk. It can also be seen from the SEM images shown in Fig. 20 that the amount of precipitation of the needle-like structured material on the surface of the surface-treated titanium disk with the hierarchical nano-roughened surface is clearly greater than the amount of precipitation of the needle-like structured material on the surface of the surface-treated titanium disk with a nano-roughened surface.

[0068] [Evaluation 5: Energy Dispersive X-ray Spectroscopy (EDX) Analysis, Colorimetric Analysis of Calcium Ions and Phosphate Ions] Figure 21 shows the results of energy dispersive X-ray spectroscopy (EDX) analysis of the needle-shaped structure material deposited on the surface of the surface-treated titanium disk with a hierarchical nano-roughened surface after 28 days of immersion in simulated body fluid. The EDX analysis is a qualitative analysis of the surface of the measurement sample. As shown in Figure 21, the needle-shaped structure material deposited on the surface of the surface-treated titanium disk with a hierarchical nano-roughened surface after 28 days of immersion in simulated body fluid was found to contain calcium and phosphorus. Figure 22 also shows the calcium and phosphorus ion concentrations obtained by colorimetric analysis using a color reaction for solutions in which the needle-shaped structure material deposited on the surface of each surface-treated titanium disk and a conventional culture dish (made of polystyrene) was dissolved in 1 M hydrochloric acid at 5, 14, and 28 days after immersion in the simulated body fluid. Fig. 23 shows the atomic ratios of calcium, phosphorus, and titanium (the proportion of each element among all detected atoms) obtained by energy dispersive X-ray spectroscopy (EDX) analysis of the needle-shaped structure material deposited on the surface of each surface-treated titanium disk and a regular culture dish (made of polystyrene) 5, 14, and 28 days after immersion in the simulated body fluid. As shown in Fig. 22, the surface-treated titanium disk with a hierarchical nano-roughened surface (indicated by ● in Figs. 22 and 23) after 28 days of immersion in the simulated body fluid contained high concentrations of calcium ions (10 mg / dL) and phosphorus ions (33 mg / dL) in the needle-shaped structure material deposited on the surface. Furthermore, as shown in Fig. 23, the EDX analysis revealed that the atomic ratios of calcium and phosphorus on the surface of the measurement sample were higher than those of the other surface-treated titanium disks. The needle-like structured material (precipitate) deposited on the surface of the surface-treated titanium disk with a hierarchical nano-roughened surface after 14 and 28 days of immersion in simulated body fluid was then immersed in water or 1 M hydrochloric acid at 25°C for one week, and analyzed for calcium elution using a colorimetric assay using a color reaction to examine the solubility of the precipitate. As a result, both the precipitates after 14 and 28 days of immersion in simulated body fluid were soluble in acid but not in water (neutral), confirming that HAp, a calcium phosphate that is only soluble in acid, had precipitated.

[0069] [Evaluation 6: Glow Discharge Optical Emission Spectroscopy (GD-OES Analysis)] Figure 24 shows the results of glow discharge optical emission spectroscopy (GD-OES analysis) performed on the needle-shaped structure material deposited on the surface of each surface-treated titanium disk after 28 days of simulated body fluid immersion. The results show elemental analysis in the depth direction from the outermost surface (depth 0 nm) of the needle-shaped structure material toward the surface-treated titanium disk. As shown in Figure 24, in the surface-treated titanium disk with a hierarchical nano-roughened surface after 28 days of simulated body fluid immersion, the calcium emission intensity was observed up to a depth of about 360 nm, and the phosphorus emission intensity was observed up to a depth of about 290 nm. On the other hand, in the surface-treated titanium disk with a nano-roughened surface after 28 days of simulated body fluid immersion, the calcium emission intensity was observed up to a depth of about 280 nm, and the phosphorus emission intensity was observed up to a depth of about 230 nm. The presence or absence of each emission intensity was determined using the emission intensity of each element in the polished titanium disk (calcium: intensity of approximately 0.07, phosphorus: intensity of approximately 0.007) as a baseline. As described above, in the surface-treated titanium disk with a hierarchical nano-roughness, the depth at which the emission intensity of phosphorus and calcium was observed was approximately 50 nm deeper than in the surface-treated titanium disk with a nano-roughness, indicating that the needle-shaped structure of calcium phosphate compound had precipitated to a greater extent.

[0070] [Evaluation 7: X-ray diffraction (XRD) analysis, Fourier transform infrared spectroscopy (FTIR) analysis] Figure 25 shows X-ray diffraction (XRD) data for the needle-like structure material precipitated on the surface of each surface-treated titanium disk after 14 days of immersion in the simulated body fluid, and Figure 26 shows Fourier transform infrared spectroscopy (FTIR) data for each surface-treated titanium disk after 28 days of immersion in the simulated body fluid. At the bottom of Figure 25, as reference diffraction peak patterns, the diffraction peak pattern of the hydroxyapatite crystal structure is shown for HAp, and the diffraction peak pattern of the titanium oxide crystal structure is shown for TiO. ３25, the diffraction peaks characteristic of the crystalline structure of hydroxyapatite are marked with ●, and the diffraction peaks characteristic of the crystalline structure of titanium oxide are marked with ▼. As shown in FIG. 25, in the surface-treated titanium disk having a hierarchical nano-rough surface, more peaks matching the diffraction peak pattern of HAp were observed than in the surface-treated titanium disk having a nano-rough surface, indicating crystallinity closer to HAp. Furthermore, as shown in FIG. 26, in the FTIR spectrum of the surface-treated titanium disk having a hierarchical nano-rough surface, a peak derived from calcium phosphate (see the FTIR spectra of various calcium phosphates shown in FIG. 27) was observed at 1000 cm －１ This 1000 cm －１ The absorption near 1000 cm for the surface-treated titanium disk with nano-roughened surface －１ The intensity was approximately 1.3 times higher than that of the surrounding area. Thus, it was found that the surface-treated titanium disk with a hierarchical nano-roughened surface produced precipitates with crystallinity closer to that of HAp upon immersion in simulated body fluid than the surface-treated titanium disk with a nano-roughened surface.

[0071] From the above evaluations 4 to 7, it was confirmed that by immersing a surface-treated titanium disk having a hierarchical nano-roughened surface in a simulated body fluid, needle-like crystals of calcium phosphate containing HAp were precipitated on the roughened surface in a thicker and more highly crystalline state than when a surface-treated titanium disk having a nano-roughened surface was immersed in a simulated body fluid.

[0072] [Preparation of Thin Section Samples] Thin section samples were prepared using the various surface-treated titanium disks (surface-treated titanium disks with a nano-roughened surface and a surface-treated titanium disk with a hierarchical nano-roughened surface) prepared in Example 2A above after 28 days of immersion in simulated body fluid, based on the following method for preparing focused ion beam (FIB) samples. Note that the analytical data for the FIB samples obtained by the following method can be interpreted as analytical data for the HAp nano-needle crystals themselves, since the thinning process does not damage the HAp nano-needle crystals. Hereafter, up to the description of Evaluation 10, thin section samples in which the surface-treated titanium disks have a nano-roughened surface will be referred to as "nano-roughened surface thin section samples," and thin section samples in which the surface-treated titanium disks have a hierarchical nano-roughened surface will be referred to as "hierarchical nano-roughened surface thin section samples." In the following evaluations 8 to 10, "Nano" refers to a thin section sample with a nano-roughened surface, and "Micro / Nano" refers to a thin section sample with a layered nano-roughened surface. Figure 43 shows an SEM image of the obtained thin section sample with a layered nano-roughened surface. The thick sections on both sides of Figure 43 represent the unthinned portions of the composite, and the central section between these sections represents the thinned portion of the composite (thin section sample). The thin section sample is composed of, from top to bottom in Figure 34, an FIB-deposited film (the area indicated by the arrow in Figure 43), HAp, and a Ti substrate. The HAp region corresponds to the area of ​​several hundred nanometers below the FIB-deposited film. (FIB Sample Preparation Method) A scanning electron microscope was used to perform preliminary observations of the samples (various surface-treated titanium disks prepared in Example 2A above after 28 days of immersion in simulated body fluid) to determine the processing position. Next, a thick carbon deposition was performed on the sample surface to protect it from damage by the Ga ion beam. Then, a carbon deposition (carbon protective film) was deposited at the processing position determined above. The area around the processing position determined above was roughly cut using a focused ion beam (FIB) processing device with a Ga ion beam at an acceleration voltage of 30 kV, and the microprobe was placed in contact with the edge of the processing position, and the probe and sample were fixed using carbon deposition. The bottom and sides of the sample were cut off, and the sample cut out with the microprobe was lifted up.The cut sample was fixed onto a Cu mesh using carbon deposition, and then the probe and sample were separated using a Ga ion beam. The cut sample was then cooled and held until the temperature stabilized. The cut sample was thinned using Ga ion beams with acceleration voltages of 30 kV and 16 kV. Next, in order to remove the layer damaged by the Ga ion beam on the surface of the thinned sample, finishing was performed using a Ga ion beam with an acceleration voltage of 5 kV, and the sample was thinned to a thickness that could be transmitted by an electron beam. The obtained thin sample was returned to room temperature and stored in a vacuum desiccator.

[0073] [Evaluation 8: Electron Diffraction Structure Analysis] Figure 44(a) is a TEM image of a thin sample with a layered nano-rough surface, and Figure 44(d) is a TEM image of a thin sample with a nano-rough surface. In Figures 44(a) and 44(d), the lower right side of the image corresponds to the deposited film, and the upper left side of the image corresponds to the Ti substrate. Using the relationship between Figures 44(a) and 43, Figure 44(a) is an enlarged image of the thin sample shown in Figure 43, rotated 135° clockwise to show the HAp crystals deposited on the Ti substrate. Of the needle-like structures seen from the upper left to the lower right in Figures 44(a) and 44(d), the needle-like structures seen on the lower right side of the image (deposited film) are HAp nano-needle crystals, and the other needle-like structures seen on the upper left side of the image (Ti substrate) are the Ti substrate. Figure 44(b) is an enlarged TEM image of the area enclosed by a square in Figure 44(a). The black area in Figure 44(b) (the black area running from the upper left to the lower right in Figure 44(b)) is the crystalline portion of HAp, and Figure 44(c) shows the electron diffraction pattern obtained by electron diffraction structure analysis of the area enclosed by a square in this black area. As a result of the electron diffraction structure analysis, diffraction of (112) and (300) was observed in the HAp crystals of the hierarchical nano-rough surface, and Ca ５ [P.O. ４ ] ３It was found that the sample exhibited a hexagonal crystal structure of [OH]. Furthermore, in the electron diffraction pattern of FIG. 44(c), almost no streaks were observed, and the number of spots was limited, indicating high crystallinity. Furthermore, the interplanar spacing of the (100) plane was 8.18 Å. FIG. 44(e) is a TEM image enlarged from the area enclosed by a square in FIG. 44(d). The TEM image of FIG. 44(e) does not show as dark an area as in the TEM image of FIG. 44(b), but the relatively dark area (the black area running in a V-shape from the lower right to the upper left of FIG. 44(e)) is the crystalline portion of HAp. The electron diffraction pattern obtained by electron diffraction structure analysis of the area enclosed by a square in this black area is shown in FIG. 44(f). As a result of the electron diffraction structure analysis, diffractions of (112) and (300) were observed even in the nano-roughened HAp crystals, indicating the presence of Ca. ５ [P.O. ４ ] ３ It was found that the crystal exhibited a hexagonal crystal structure of [OH]. On the other hand, in the electron diffraction pattern of Figure 44(f), streaks were observed, and more spots were observed than in Figure 44(c), indicating low crystallinity. Furthermore, the interplanar spacing of the (100) plane was 8.26 Å, which was longer than in Figure 44(c). As a result of electron diffraction structure analysis of the thin section sample of the hierarchical nano-roughened surface and the thin section sample of the nano-roughened surface, it was confirmed that the needle-shaped crystals of calcium phosphate containing HAp obtained using the hierarchical nano-roughened surface have higher crystallinity and a shorter interplanar spacing, resulting in a more densely packed crystal structure, compared to the needle-shaped crystals of calcium phosphate containing HAp obtained using the nano-roughened surface.

[0074] [Evaluation 9: STEM HAADF Analysis] Figure 45(a) is an HAADF-STEM image of a thin specimen with a layered nano-rough surface. The white area in Figure 45(a) (the white area running from the upper left to the lower right of Figure 45(a)) is the crystalline portion of HAp. Figure 45(b) shows the EDX spectrum and the results of simple quantitative composition analysis for the circled area (point 1) within this white area. Figure 45(c) is an HAADF-STEM image of a thin specimen with a nano-rough surface. The white area in Figure 45(c) (the white area running from left to right of Figure 45(c)) is the crystalline portion of HAp. Figure 45(d) shows the EDX spectrum and the results of simple quantitative composition analysis for the circled area (point 2) within this white area. As mentioned above, it can be said that the higher the Ca / P ratio of HAp, the more stable the crystalline structure of HAp. Therefore, while the Ca / P ratio of the flake sample with the nano-rough surface was 19 / 23 (=0.83), the Ca / P ratio of the flake sample with the layered nano-rough surface was 27 / 19 (=1.42), which was higher and indicated a more stable crystal structure. It is known that the Ca / P ratio of HAp in vivo is approximately 1.47 due to Ca outflow, and the Ca / P ratio of the flake sample with the layered nano-rough surface (=1.42) is closer to 1.47, indicating a more biomimetic crystal structure.

[0075] [Evaluation 10: Image Analysis Using ImageJ] The upper row of Figure 46 shows a TEM image of a thin sample with a nano-rough surface, and the lower row of Figure 46 shows a TEM image of a thin sample with a layered nano-rough surface. Using these TEM images shown in Figure 46, image analysis of the shape of the HAp needle-like crystals was performed as follows. Specifically, using the above-mentioned ImageJ 1.53t as image analysis software, needle-like structures with the same contrast as the HAp structure whose crystallinity was analyzed (i.e., needle-like HAp crystals, rather than needle-like Ti substrates) were visually identified, and one of these HAp nano-needle crystals (primary needle-like HAp crystals) was selected using the Polygon selection tool in ImageJ 1.53t (an example of a primary needle-like HAp crystal selected in this manner is shown in Figure 47). The area, major axis, and minor axis of the selected HAp primary needle crystals were then measured, and various parameters (average area, average major axis, and average aspect ratio) were calculated. Note that the various parameters are the average values ​​of 21 HAp primary needle crystals in the TEM image of the hierarchical nano-roughened surface flake sample, and the average values ​​of 17 HAp primary needle crystals in the TEM image of the nano-roughened surface flake sample. Dot plots and box-and-whisker diagrams of the obtained various parameters are shown in Figure 48. In Figure 48, the bar at the bottom of the whiskers represents the minimum value, the bar at the top of the whiskers represents the maximum value, the dots outside the bars represent outliers, the bottom of the box represents the bottom 25% value, the horizontal line inside the box represents the median (50% value), the top of the box represents the bottom 75% value, and the x marks represent the average value. Table 4 also shows the minimum, maximum, and average values ​​for the area, major axis, and minor axis, as well as the coefficient of variation (the standard deviation divided by the average value multiplied by 100 to express it as a percentage).

[0076]

[0077] 48 and Table 4, when various parameters of the HAp needle crystals in the flake sample with the layered nano-rough surface were compared with those of the HAp needle crystals in the flake sample with the nano-rough surface, the HAp needle crystals in the flake sample with the layered nano-rough surface showed an average aspect ratio similar to that of the HAp needle crystals in the flake sample with the nano-rough surface, but an average major axis that was about twice as long and an average area that was about five times larger. These results confirmed that the HAp needle crystals in the flake sample with the layered nano-rough surface had a longer and wider shape than the HAp needle crystals in the flake sample with the nano-rough surface.

[0078] From the above evaluations 8 to 10, it was confirmed that the HAp needle crystals in the thin flake sample with the layered nano-rough surface have higher crystallinity, a longer and wider crystal structure, a higher Ca / P ratio, and are more stable and biomimetic than the HAp needle crystals in the thin flake sample with the nano-rough surface.

[0079] Example 2B: Preparation of HAp nanoneedle crystals by immersion of surface-treated titanium disks in simulated body fluid (change in crystal precipitation due to base concentration) The surface-treated titanium disks prepared in Example 1B-1 above were immersed in simulated body fluid at 37°C for 28 days. Scanning electron microscope (SEM) analysis was performed on the surface of each surface-treated titanium disk before immersion in the simulated body fluid, and after 14 and 28 days of immersion. Figure 28 shows SEM images of each surface-treated titanium disk viewed from a vertical direction, with the upper row showing the SEM image before immersion in the simulated body fluid, the middle row showing the SEM image after 14 days of immersion in the simulated body fluid, and the lower row showing the SEM image after 28 days of immersion in the simulated body fluid. Figure 29 also shows SEM images of each surface-treated titanium disk viewed from an oblique direction, with the upper row showing the SEM image after 14 days of immersion in the simulated body fluid and the lower row showing the SEM image after 28 days of immersion in the simulated body fluid. 30 shows the atomic ratio of calcium on the surface of each surface-treated titanium disk obtained by energy dispersive X-ray spectroscopy (EDX) analysis. The atomic ratio of calcium after 14 days of immersion in simulated body fluid is shown on the left, and the atomic ratio of calcium after 28 days of immersion is shown on the right. As shown in Figures 28 to 30, when the hierarchical nano-roughening treatment was performed with a 10.0 M aqueous sodium hydroxide solution, a large amount of needle-shaped crystals containing HAp precipitated on the surface of the surface-treated titanium disk, regardless of whether the nano-roughening treatment was performed with a 5.0 M aqueous sodium hydroxide solution or a 10.0 M aqueous sodium hydroxide solution (Figures 28 and 29), and calcium atoms were contained at a high atomic ratio (Figure 30). Scanning electron microscope (SEM) and energy dispersive X-ray spectroscopy (EDX) analyses of the surface of the layered surface-treated titanium disk after 28 days of immersion in simulated body fluid showed that when the titanium disk surface was subjected to layered nano-roughening treatment with 10.0 M aqueous sodium hydroxide solution, a larger amount of needle-shaped crystals containing HAp precipitated on the surface than when the titanium disk surface was subjected to layered nano-roughening treatment with 5.0 M aqueous sodium hydroxide solution, and that calcium atoms and phosphorus atoms were contained in a higher atomic ratio.

[0080] Example 2C: Preparation of HAp nanoneedle-like crystals by immersion of surface-treated titanium alloy disk in simulated body fluid The surface-treated titanium disk (titanium alloy disk) having a hierarchical nano-roughened surface prepared in Example 1C above was immersed in simulated body fluid for 28 days at 37° C. Fig. 31 shows scanning electron microscope (SEM) images of the surface of the surface-treated Ti-6Al-4V titanium alloy disk having a hierarchical nano-roughened surface before and after 28 days of immersion in the simulated body fluid. 32(a) shows the results of energy dispersive X-ray spectroscopy (EDX) analysis of the surface of a surface-treated Ti-6Al-4V titanium alloy disk with a hierarchical nano-roughened surface after 28 days of immersion in simulated body fluid, and FIG. 32(b) shows the atomic ratios of calcium, phosphorus, and titanium (the proportion of each element among all detected atoms) obtained by EDX analysis of the needle-like structure material precipitated on the surface of the surface-treated Ti-6Al-4V titanium alloy disk with a hierarchical nano-roughened surface after 28 days of immersion in simulated body fluid. The SEM image in FIG. 31 reveals that even when a titanium alloy disk was used instead of a pure titanium disk, a large amount of needle-like structure material precipitated on the surface of the surface-treated titanium disk with a hierarchical nano-roughened surface. 32(a), the needle-like structure material deposited on the surface of the surface-treated titanium disk with a hierarchical nano-roughened surface after immersion in simulated body fluid for 28 days was found to contain calcium and phosphorus. Furthermore, the EDX analysis results shown in Fig. 32(b) revealed that the atomic ratio of calcium and phosphorus in the needle-like structure material deposited on the surface of the surface-treated Ti-6Al-4V titanium alloy disk with a hierarchical nano-roughened surface were as high as 20% and 14%, respectively. As shown in Example 1C above, the surface-treated Ti-6Al-4V titanium alloy disk with a hierarchical nano-roughened surface also contained amorphous titanium oxide, and the average distribution density and distribution anisotropy of the nanoprotrusions constituting the hierarchical nano-roughened surface satisfied the requirements of the present invention. Therefore, it was confirmed that thick, highly crystalline needle-like crystals of calcium phosphate containing HAp were deposited on the hierarchical nano-roughened surface.

[0081] Example 3: In vitro evaluation of the effect of surface-treated titanium with a HAp-coated hierarchical nano-roughened surface on the behavior of BMSCs (bone marrow stromal cells) (3-1) Culturing of BMSCs on surface-treated titanium discs without HAp coating BMSCs collected from the femurs of 8-week-old SD (Sprague-Dawley) rats were cultured on the surface of each of the surface-treated titanium discs prepared in Example 1A above in osteoblast differentiation-inducing medium for 30 days. The titanium discs were placed on polystyrene cell culture plates, and the cells were cultured at a density of 1.0 to 3.0 cells / cm. ２ The cell suspension was adjusted to a cell density of 1000 μg / ml and seeded onto the titanium disks. The cell culture plate was maintained at 37°C and 5% CO ２ The discs were cultured under a 50% CO2 atmosphere. Figure 33 shows scanning electron microscope (SEM) images of the surface of each surface-treated titanium disc before and after culture. The left side of Figure 33 shows an SEM image of the surface of each surface-treated titanium disc before culture, and the right side of Figure 33 shows an SEM image of the surface of each surface-treated titanium disc after 30 days of culture. As shown in Figure 33, no significant changes were observed in the surface of the polished titanium discs and the surface-treated titanium discs with micro-roughened surfaces before and after culture. However, the formation of a mineralized matrix was observed on the surface of the surface-treated titanium discs with nano-roughened surfaces, and in addition to the formation of a mineralized matrix, its enlargement was also observed on the surface of the surface-treated titanium discs with hierarchical nano-roughened surfaces.

[0082] In the evaluation of Example 3, "Machined" refers to a polished titanium disk, "Nano" refers to a surface-treated titanium disk with a nano-roughened surface, "Micro" refers to a surface-treated titanium disk with a micro-roughened surface, "Micro / Nano" refers to a surface-treated titanium disk with a hierarchical nano-roughened surface, "Nano with SBF" refers to a surface-treated titanium disk with a HAp-coated nano-roughened surface, "Micro / Nano with SBF" refers to a surface-treated titanium disk with a HAp-coated hierarchical nano-roughened surface, and "Poly" refers to a polystyrene disk. The evaluations in Example 3 are all results of BMSC culture for 30 days.

[0083] (3-2) Culturing of BMSCs on HAp-coated Surface-Treated Titanium Disks BMSCs collected from the femurs of 8-week-old Sprague-Dawley rats were cultured for 30 days in osteoblast differentiation-inducing medium on the surfaces of the surface-treated titanium disks with nano-roughened surfaces on which needle-like crystals of HAp were precipitated (HAp-coated) and the surface-treated titanium disks with hierarchical nano-roughened surfaces on which needle-like crystals of HAp were precipitated (HAp-coated), which were prepared by immersion in simulated body fluid at 37°C for 14 days in the above Example 2A. The titanium disks were placed on polystyrene cell culture plates, and BMSCs were cultured at a density of 1.0 to 3.0 cells / cm. ２ The cell suspension was adjusted to a cell density of 1000 μg / ml and seeded onto the titanium disks. The cell culture plate was maintained at 37°C and 5% CO ２ The culture was carried out under atmospheric conditions.

[0084] (3-3) Effect of HAp-coated surface-treated titanium discs with a hierarchical nano-roughened surface on BMSC differentiation. The surfaces of the HAp-coated surface-treated titanium discs after BMSC culture were observed using a scanning electron microscope (SEM). The right side of Figure 34 shows SEM images of the surfaces of the HAp-coated surface-treated titanium discs with a nano-roughened surface and the HAp-coated surface-treated titanium discs with a hierarchical nano-roughened surface after 30 days of culture. The left side of Figure 34 shows SEM images of the surfaces of the nano-roughened surface-treated titanium discs and the hierarchical nano-roughened surface-treated titanium discs described in (3-1) above after 30 days of culture. As shown in Figure 34, the surfaces of the HAp-coated surface-treated titanium discs with a nano-roughened or hierarchical nano-roughened surface after immersion in simulated body fluid showed an increased mineralized matrix after 30 days of culture compared to the surface-treated titanium discs with a nano-roughened or hierarchical nano-roughened surface without a HAp coating. In particular, significant thickening of the mineralized matrix formed on the surface of the HAp-coated, hierarchical nano-roughened titanium disks after 30 days of culture was observed. Although this is speculative, it is thought that the HAp coating contributed to the increase and growth of the granular structure of the extracellular matrix. These results suggest that HAp nanocrystals precipitated by immersion in simulated body fluid promote the osteoblast differentiation of rat BMSCs.

[0085] (3-4) Effect of HAp-coated, surface-treated titanium disks with a hierarchical nano-roughened surface on the calcified matrix after BMSC culture—Calcium quantification— Figure 35 shows the atomic ratios (%) of calcium, phosphorus, and titanium, as well as the atomic ratio (%) of calcium to phosphorus, obtained by energy-dispersive X-ray spectroscopy (EDX) analysis of the calcified matrix formed by BMSC culture for 30 days. As shown in Figure 35, when BMSCs were cultured on surface-treated titanium disks with a HAp-coated, hierarchical nano-roughened surface, the atomic ratio of calcium to phosphorus was high, and the atomic ratio of calcium to phosphorus was also high. Figure 36 shows the results of calcium quantification obtained by energy-dispersive X-ray spectroscopy (EDX) analysis of the calcified matrix formed by BMSC culture for 30 days. To eliminate carryover of background HAp precipitated by immersion in simulated body fluid, the quantitative calcium value of the HAp-coated surface-treated titanium disk was subtracted from the EDX analysis of the mineralized matrix obtained by culturing BMSCs on the HAp-coated surface-treated titanium disks described above in (3-2). As shown in Figure 36, the calcium concentration when BMSCs were cultured on the HAp-coated surface-treated titanium disks with a hierarchical nano-roughened surface was 92 mg / dL, indicating a higher calcium concentration than in the other examples. These results suggest that culturing BMSCs on the HAp-coated surface-treated titanium disks with a hierarchical nano-roughened surface increases the mineralized matrix produced and its growth.

[0086] (3-5) Effect of surface-treated titanium disks with HAp-coated hierarchical nano-roughened surfaces on the mineralized matrix after BMSC culture—crystallinity of the mineralized matrix— Figure 37 shows the results of XRD analysis of the mineralized matrix obtained after 30 days of BMSC culture. As shown in Figure 37(a), when BMSCs were cultured on surface-treated titanium disks with HAp-coated hierarchical nano-roughened surfaces, a HAp-derived peak at 2θ = approximately 32° was observed with high intensity and width, and the mineralized matrix produced was not only fertile (large in quantity) but also relatively highly crystallized. In particular, as shown in Figure 37(b), which is an enlarged view (peaks superimposed) of the θ = 25-40° range in Figure 37(a), when BMSCs were cultured on surface-treated titanium disks with a HAp-coated hierarchical nano-roughened surface, the width and intensity of the HAp-derived peak at 2θ = approximately 32° in the resulting mineralized matrix were both larger and more intense, indicating higher crystallinity, compared to when BMSCs were cultured on surface-treated titanium disks with a HAp-coated hierarchical nano-roughened surface. These results demonstrate that high-quality biological HAp was produced by culturing and osteogenic differentiation of BMSCs on surface-treated titanium disks with a HAp-coated hierarchical nano-roughened surface.

[0087] (3-5) Effect of HAp-coated, hierarchical nano-roughened surface-treated titanium disks on cell proliferation in BMSC cultures—BrdU analysis, WST-1 cell proliferation assay—The left side of Figure 38 shows the absorbance at 370 nm after 2 and 4 days of culture using BrdU (bromodeoxyuridine) analysis. Because BrdU is taken up by cells during the S phase of the cell cycle, analysis focusing on cell proliferation can be performed. Therefore, in this analysis, cells were cultured for 2 and 4 days using growth medium alone without osteoblast induction. As shown on the left side of Figure 38, cell proliferation gradually increased in all groups, indicating that the HAp coating on the surface-treated titanium disks did not inhibit cell proliferation. The right side of Figure 38 also shows the cell counts after 1, 5, and 7 days of culture using a WST-1 cell proliferation assay. As shown on the right side of Figure 38, the HAp coating on the surface-treated titanium disks with a HAp-coated hierarchical nano-roughened surface did not inhibit cell proliferation, but it was found that the cells did not proliferate that actively. These results show that, as mentioned above, cell proliferation on the surface-treated titanium disks with a HAp-coated hierarchical nano-roughened surface promoted an increase in the mineralized matrix and growth, but did not have an extreme effect on the reduction of proliferation activity or the suppression of the increase in cell proliferation number.

[0088] (3-5) Effect of surface-treated titanium disks with HAp-coated hierarchical nano-roughened surfaces on differentiation in BMSC cultures - Real-time RT-PCR analysis, ALP quantitative analysis - Figure 39 (a) to (c) show the results of real-time RT-PCR (reverse transcription PCR) analysis, and Figure 39 (d) shows the results of quantitative analysis of ALP (Alkaline Phosphatase). For real-time RT-PCR analysis, total RNA from cells cultured on the surface of each surface-treated titanium disk for 5 days was extracted using TRIzol reagent, and RNA was isolated and purified using the RNAeasy Mini Kit (product name, manufactured by Qiagen). Subsequently, after DNase treatment, complementary DNA was synthesized using the PrimeScript II 1st Strand cDNA Synthesis Kit (product name, manufactured by Takara Bio). Messenger RNA (mRNA) expression was measured using the SYBR Green method with Tunderbird SYBR qPCR Mix (Toyobo). Target gene expression levels were measured using the comparative cycle time (ΔΔCT) method to calculate the fold change in gene expression in cells on each surface-treated titanium disk compared to cells on polystyrene culture dishes (Poly). For ALP quantitative analysis, cells cultured on each surface-treated titanium disk for 5 days were disrupted with 0.05% Triton X and an ultrasonic homogenizer, collected in tubes, and centrifuged. A portion of the resulting supernatant was subjected to absorbance quantification of total protein using Ierce 660 nm Protein Assay Reagent (Thermo Scientific). In a p-nitrophenol production reaction using Labo Assay ALP (trade name, manufactured by Fujifilm Wako Shibayagi Co., Ltd.), the ALP activity in the remaining supernatant was measured by absorbance, and the ALP activity per unit protein amount (Units / μg) was calculated and evaluated.As shown in Figure 39, the expression of mRNA (messenger ribonucleic acid) for early calcification markers Col1a2 (collagen type I alpha 2 chain) and ALP (alkaline phosphatase), a transcription factor essential for osteoblast differentiation, OSX (Osterix), and intermediate-stage marker BSP (bone sialoprotein) was highest when BMSCs were cultured on HAp-coated, surface-treated titanium disks with a hierarchical nano-roughened surface. These results demonstrate that cell proliferation on HAp-coated, surface-treated titanium disks with a hierarchical nano-roughened surface promotes the differentiation of BMSCs into osteoblasts.

[0089] Example 4: Evaluation of bone bonding ability by implantation of HAp-coated surface-treated titanium implants with a hierarchical nano-roughened surface into rats (4-1) Implantation of various implants into rat maxillae and femurs. A mechanically polished grade II titanium miniscrew (manufactured by Nishimura Metals Co., Ltd., Fukui Prefecture) with a diameter of 1.4 mm, a screw length of 3.0 mm, and a length excluding the screw portion and head of 1.5 mm, as shown in FIG. 40(a), was used as the implant base material. The implant base material was subjected to the surface roughening treatments (i) to (iii) described in Example 1A above to prepare surface-treated titanium implants with a hierarchical nano-roughened surface, a nano-roughened surface, or a micro-roughened surface, respectively. Furthermore, the resulting surface-treated titanium implants with a hierarchical nano-roughened surface, a nano-roughened surface, or a micro-roughened surface were immersed in simulated body fluid using the method described in Example 2A above to prepare surface-treated titanium implants with a HAp-coated hierarchical nano-roughened surface, a nano-roughened surface, or a micro-roughened surface, respectively. In the implant base material shown in Figure 40(a), the screw portion enclosed by a square was subjected to the above-mentioned surface roughening treatment and immersion in simulated body fluid. As shown in Figures 40(b) to 40(d), the various implants prepared as described above were implanted into four locations per rat: one in the maxilla immediately after the extraction of the left and right maxillary first molars, and one in the central portion of the left and right femoral shafts. Of the screw portions enclosed by a square in Figure 40(a), 3.0 mm from the screw tip was then implanted one month later, and the following reverse torque test was performed.

[0090] In the following drawings, "Nano" refers to a surface-treated titanium implant having a nano-roughened surface, "Micro" refers to a surface-treated titanium implant having a micro-roughened surface, "Micro / Nano" refers to a surface-treated titanium implant having a hierarchical nano-roughened surface, "Nano with SBF" refers to a surface-treated titanium implant having a HAp-coated nano-roughened surface, "Micro with SBF" refers to a surface-treated titanium implant having a HAp-coated micro-roughened surface, and "Micro / Nano with SBF" refers to a surface-treated titanium implant having a HAp-coated hierarchical nano-roughened surface.

[0091] (4-2) Evaluation of bone bonding ability by counter torque test The counter torque test is an analytical method in which a screwdriver is inserted into the groove of an implanted implant (screw head) and turned, quantitatively evaluating the strength of the force applied to turn the screw. A lower counter torque value indicates stronger osseointegration (bonding between titanium and bone). The screwdriver used was a "DIS-RL05" manufactured by Sugisaki Keiki Co., Ltd. Figure 41 shows the results of the counter torque test on the maxilla, and Figure 42 shows the results of the counter torque test on the femur. As shown in Figure 41, in the implantation experiment into the maxilla, when a surface-treated titanium implant with a HAp-coated hierarchical nano-roughened surface was implanted, a low torque value was observed, indicating a tendency for high osseointegration to be achieved. In particular, only the surface-treated titanium implants with a HAp-coated hierarchical nano-roughened surface were able to fully tighten the screw with the screwdriver in the back-torque test; in several cases, the screw head grooves were stripped before the screw could be fully tightened, making it impossible to complete the back-torque test (note that data for screws that could not be fully tightened are not plotted in Figure 41 ). This indicates that the surface-treated titanium implants with a HAp-coated hierarchical nano-roughened surface were strongly osseointegrated into the maxillary bone. Furthermore, as shown in Figure 42 , in the femoral implantation experiment, implantation of the surface-treated titanium implants with a HAp-coated hierarchical nano-roughened surface showed the lowest average torque value and the highest average osseointegration. These results suggest that the HAp coating on the surface-treated titanium implants with a HAp-coated hierarchical nano-roughened surface stimulates bone-forming cells (differentiation of BMSCs to osteoblasts and proliferation of osteocytes) in vivo, improving the bonding between the implant (titanium) and bone.

Claims

1. A surface-treated titanium substrate having a hierarchical nano-rough surface, wherein the hierarchical nano-rough surface is composed of amorphous titanium oxide, and the average distribution density of nano-protrusions constituting the hierarchical nano-rough surface is 20 to 50 / μm. ２ and a surface-treated titanium substrate, wherein the nanoprotrusions have a distribution anisotropy of 0.70 to 1.

20.

2. The layered nano-rough surface has an average zeta potential of -5.0 to -2.0 mV, and the layered nano-rough surface has an average contact potential difference with a silicone probe of 1.60 to 2.00 V / 10 nm as determined by Kelvin probe force microscopy. ２ 2. The surface-treated titanium substrate according to claim 1, wherein 3. A composite comprising the surface-treated titanium substrate according to claim 1 or 2 and nano-needle crystals of hydroxyapatite formed on the hierarchical nano-roughened surface of the titanium substrate.

4. A culture bed comprising the surface-treated titanium substrate according to claim 1 or 2, or the composite according to claim 3.

5. A method for producing a surface-treated titanium substrate according to claim 1 or 2, comprising: subjecting a titanium substrate having a mechanically polished surface to a heat treatment using an acid to impart micro-roughness to the mechanically polished surface of the titanium substrate; further subjecting the titanium substrate to a heat treatment using a 7.5 to 12.5 M alkali; and then sintering the titanium substrate under atmospheric pressure to form a hierarchical nano-roughened surface on the surface of the titanium substrate.

6. The method for producing a surface-treated titanium base material according to claim 5, wherein the heat treatment with an acid is a treatment with an aqueous sulfuric acid solution at 100 to 150°C, the heat treatment with an alkali is a treatment with a 7.5 to 12.5 M aqueous sodium hydroxide solution at 60 to 100°C, and the sintering treatment is a heat treatment at 500 to 700°C under atmospheric pressure.

7. A method for producing nano-needle crystals of hydroxyapatite, comprising immersing the surface-treated titanium substrate according to claim 1 or 2 in a simulated body fluid containing calcium and phosphorus, thereby precipitating nano-needle crystals of hydroxyapatite.

8. An implant comprising the surface-treated titanium substrate according to claim 1 or 2, or the composite according to claim 3.

9. Nano needle-like crystals of hydroxyapatite in which the ratio of calcium atoms to phosphorus atoms calculated by HAADF-STEM and EDX analysis is 1.00 or more, and the interplanar spacing of the (100) plane determined by electron diffraction structure analysis is 8.25 Å or less.

10. The average area of ​​the nano-needle crystals of hydroxyapatite obtained by analyzing the TEM image using image analysis software is 2000 nm. ２ The above nano needle crystals of hydroxyapatite have an average long diameter of 200 nm or more and an average aspect ratio of 6 to 20.

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