Analysis of Titania Nanosheet Adsorption Behavior Using a Quartz Crystal Microbalance Sensor

We investigated the adsorption of albumin and fibronectin on a titania nanosheet(TNS-) modified quartz crystal microbalance (QCM) sensor. A Ti QCM sensor was fabricated by reactive magnetron sputtering. A thin layer of Ti was deposited on the QCM sensor. ,is sensor was then alkali-modified by treatment with NaOH at room temperature to fabricate the titania nanosheets. Scanning probe microscopy, X-ray photoelectron spectroscopy, and scanning electron microscopy were performed to investigate the surface topology and chemical components of each sensor. ,e TNS had a titanium oxide film exhibiting a nodular structure and a thickness of 13 nm on the QCM sensor. Furthermore, QCMmeasurements showed significantly greater amounts of albumin and fibronectin adsorbed on the TNS than on titanium. ,e NaOH treatment of titanium modified the sensor surface and improved the adsorption behaviors of proteins related to the initial adhesion of bone marrow cells. ,erefore, we concluded that TNS improves the initial adhesion between the implant materials and the surrounding tissues.


Introduction
Titanium is a biocompatible material that is commonly used in dentistry and orthopedic reconstruction.e surface features of implant materials have important functions in cell or extracellular matrix interrelationships and eventual osseointegration.
erefore, interrelationships between cells and microtopography have been intensively studied.
An important contemporary advance in dental implant research is the ability to modify implant surface materials at the nanoscale [1,2].Materials with an expanded surface region and a better surface roughness may yield better mechanical interlocking between tissues and titanium [3].However, more importantly, such nanoscale features are also believed to directly affect osteogenic cell behaviors around implant fixtures with nonconventional surfaces, creating a biomimetic relationship between alloplastic surfaces and host tissues by the replication of the natural cellular environment at the nanometer level [1,2,4].
Low-dimensional TiO 2 nanostructures have attracted recent attention because these materials can take the forms of nanotubes [5], nanofibers [6], and nanowires [7].Compared with mass materials or nanoparticles, TiO 2 nanotubes have high particular surface zones accessible for the adsorption of color sensitizers, and they provide channels to improve electron exchange, thus expanding the effectiveness of solar cells [5].Titania nanosheets (TNSs) are similar to TiO 2 nanotubes, created by titania deposition using the sputtering process [5].
In our previous study [8], we showed that TNSs created by compound processing enhance the osteogenic separation of rodent bone marrow (RBM) cells.e surface properties and structural characteristics of materials play an important role in protein and cell adsorption behaviors.
e initial amount adsorbed and the conformation of proteins contained in the serum could alter the bioactivity of stem cells on TNSs.However, the bioactivities of TNS materials, including their roles in osteogenic differentiation and the biointegration of dental implants into the alveolar bone, have not been elucidated.
A quartz crystal microbalance (QCM) sensor is a profoundly delicate and handy device that is used to observe protein adsorption and cell behavior in situ.A QCM-based sensor comprises a quartz crystal and a detection material.A 27 MHz QCM can provide highly sensitive measurements of mass in aqueous solutions; the resonance frequency decreases in relation to the mass of the protein bound on the QCM electrode surface.We previously fabricated several types of QCM sensors by coating the gold electrode of the quartz crystal with a thin film of a biomaterial [9]; this QCM sensor achieved an increase in sensitivity approximately 24 times that of a conventional 5 MHz QCM.PMMA (poly (methyl methacrylate)), Au, and Ti have been used as QCM electrode surface materials to imitate denture materials and evaluate the adsorption behaviors of various bovine salivary proteins [10].ese previous findings support the potential utility of the QCM method for the evaluation of protein adsorption behaviors on implant surfaces.
In this study, we evaluated the effects of modified surfaces on the adsorption of albumin and fibronectin in RBM cells and simulated body fluid (SBF) in QCM analyses.

Sputtering Procedure.
A thin layer of Ti was deposited on quartz discs (diameter: 8 mm, area: 4.9 mm 2 ) by reactive magnetron sputtering using a radio-frequency magnetron sputtering system (CFS-4ES-231; Shibaura Mechatronics Co., Ltd., Kanagawa, Japan).e QCM crystal was cleaned using piranha solution (H 2 SO 4 /30% H 2 O 2 (v/v): 7 : 3).Before deposition was conducted, the quartz surfaces were ultrasonically cleaned in high-purity acetone (99.999%).Pure Ti powder was used to prepare the target, which was formed by pressing the powder into a disc with a diameter of 75 mm.e quartz substrates were positioned 85 mm above the target, and the magnetron sputtering chamber was evacuated to a pressure of 3 × 10 −3 Pa.Argon was used as the working gas, and its pressure was kept constant at 6.7 × 10 −1 Pa.All of the films were fabricated using a constant radio-frequency discharge power of 480 W, and the Ti thin films were deposited at room temperature at a deposition rate of 20.0 nm/min, yielding a film thickness of approximately 240 nm. e crystals were washed and cleaned with both sodium dodecyl sulfate and UV-Ozone Cleaner (PC450; Meiwafosis Co., Ltd., Osaka, Japan) prior to QCM measurements.

Sample Preparation.
In the TNS group, Ti sensors were treated to produce TNS on their surfaces.An unprocessed QCM sensor was used as the Ti sensor.ese sensors were immersed in 10 M NaOH (aq) and were then placed in an oil bath, which was kept at a temperature of 30 °C for 24 h.e solution in each flask was replaced and treated with distilled water (200 mL), and this procedure was repeated until the point that a conductivity of 5 μS/cm 3 was reached.e specimens were then dried at room temperature.

Characterization of Materials.
Scanning electron microscopy (S-4800; Shimadzu, Kyoto, Japan) and scanning probe microscopy (SPM-9600; Shimadzu) over a surface area of 2.0 μm × 2.0 μm were conducted to observe the surface topology and roughness of the fabricated TNS and Ti sensors.e composition of the coating was analyzed by X-ray photoelectron spectroscopy (XPS; ESCA 5600, Ulvac-Phi Inc., Kanagawa, Japan) using surface etching with ionized argon.In addition, the surfaces of the fabricated Ti sensors were subjected to XPS analysis with an Al Kα line (15 kV, 300 W) as an X-ray source.During XPS, argon ion sputtering was applied to determine the thickness and structure of the surface layers.

Contact Angle
Measurements.Contact angles were measured for the TNS and Ti sensors using a video contact angle measurement system (model VSA 2500 XE; AST Products Inc., Billerica, MA, USA).A small droplet of a deionized water solution with Hanks' Balanced Salt Solution and bovine serum albumin (BSA; approximately 3 mg) was placed on the TNS to measure the static contact angle.Estimation of the contact edge is a straightforward strategy for breaking down the vitality and hydrophilic nature of a surface.
2.5.Proteins.BSA (Wako Pure Chemical Industries Ltd., Osaka, Japan) was dissolved in phosphate-buffered saline (PBS; pH 7.4) at 200 μg/mL.Human plasma fibronectin (HFN; Nacalai Tesque, Inc., Kyoto, Japan) was dissolved in PBS (pH 7.4) at 500 μg/mL.2.6.Cell Culture.Since most bone embed materials are embedded in adult bone that is in direct contact with bone marrow tissue, the effects and success of new embed materials can be investigated by examining bone marrow cell cultures from adult rats.RBM cells multiply and separate into a phenotype that expresses bone cell markers in vitro.RBM cells were extracted from the femurs of 7-week-old Sprague-Dawley rats.
e rats were humanely sacrificed utilizing 4% isoflurane, and the bones were aseptically extracted from the hind limbs.e external soft tissues were discarded, and the extracted bone samples were immersed in 50 mL of Eagle's minimal essential medium (EMEM; Wako) supplemented with 20% fetal bovine serum (lot number 1,412,447; Invitrogen, Life Technologies Corp., Carlsbad, CA, USA) and penicillin (850 U/mL) for approximately 15 min.e proximal end of the femur and the distal end of the tibia were cut.An 18-gauge needle (TERMO, Japan) was introduced into the opening at the knee-joint end of each bone, and the marrow was washed out of the bone shaft by EMEM.
e obtained marrow pellet was separated by trituration, and the cell suspensions obtained from all the bones were combined by centrifugation.RBM cells were cultured in 75 cm 2 culture flasks (TD75; Falcon) in EMEM.At confluence, the cells were removed by trypsinization, washed twice in EMEM, resuspended in culture medium, 2 Advances in Materials Science and Engineering and seeded on test and control titanium disks at a concentration of 4 × 10 4 cells/cm 2 in 24-well tissue culture plates.e cells were incubated for 3 days in a CO 2 incubator at 37 °C. is investigation was conducted in accordance with the Guidelines for Animal Experimentation of Osaka Dental University (Approval no.16-08001).

QCM Measurements.
e amounts of proteins (BSA and HFN) and RBM cells were determined by QCM measurements (Affinix QN μ; Initium Co., Ltd., Tokyo, Japan).Affinix QN μ had a 550 µL cell outfitted with a 27 MHz QCM plate at the base of the cell.e diameter of the quartz plate was 8 mm, and the area of the gold-plated quartz was 4.9 mm 2 .e unit also included a mixing bar and a temperature controller.
e adjustment in recurrence was checked utilizing a universal frequency counter connected to a microcomputer.
e Ti QCM sensors and TNS were immersed in 500 μL of PBS (0.01 M PBS at pH 7.4).Changes in the QCM frequency were measured as a function of time; recording started immediately after the infusion of 5 μL (20 μg/mL) of BSA, HFN, and RBM cells.e solution was mixed to avoid any influence of protein dispersion on the measured results.Stirring did not influence the soundness of the frequency or the degree of frequency adjustment.e frequency change relied upon the adsorbed mass in accordance with the Sauerbrey equation: As per this condition, at 27 MHz, a frequency shift of 1 Hz relates to a mass difference in roughly 0.62 ng•cm −2 .In the Sauerbrey equation, F 0 is the fundamental frequency of the quartz crystal (27 × 10 6 Hz), ΔF is the measured frequency shift (Hz), ρ q is the density of quartz (2.65 g•cm −3 ), Δm is the mass change (g), A is the electrode area (0.049 cm 2 ), and μ q is the shear modulus of quartz (2.95 × 1011 dyn•cm −2 ).QCM observation was performed at 25 °C, and the test was repeated four times.Results are depicted as mean ± standard deviation.

XPS Analysis after Measuring the Adsorption of BSA and HFN Using Ti and TNS QCM Sensors.
e biochemical constituents of the adsorbed protein films on the QCM sensors were investigated by XPS on an AXIS Ultra DLD spectrometer (Kratos Instruments, Manchester, UK) equipped with a monochromated Al Kα X-ray source (hv � 1486.6 eV) operated at 75 W. XPS was utilized to investigate the proteinaceous carbon (C1s) and nitrogen (N1s) signals produced by the protein.Evaluation of the C1s and N1s signals emerging from the peptide bonds of the protein was conducted to determine the relative amount of protein adsorbed on various surfaces.

Preparation of SBF.
Since SBF is supersaturated with respect to apatite, improper planning can prompt the precipitation of apatite in the solution.erefore, the solution should remain colorless and transparent, and there should not be any deposition on the inner surface of the container.If precipitation is observed, the preparation of SBF should be halted, and the procedure should be restarted at the step of washing the apparatus.
For preparation of 1 L of SBF, 700 mL of ion-exchanged and distilled water was added to a 1 L plastic beaker with a stir bar.e beaker was covered with plastic wrap, and the water was heated to 36.5 ± 1.5 °C under stirring.Reagents 1-8 were then dissolved into the solution in order (given in Table 1) at 36.5 ± 1.5 °C, and reagents 9 (Tris) and 10 (HCl) were added after pH adjustment.
Note that during this procedure, instead of glass containers, plastic containers with smooth, unscratched surfaces were used because apatite nucleation can be induced on the surfaces of glass containers or the edges of scratches.Additionally, reagents were dissolved completely before addition of the next reagent.
e volume of 1 M HCl was measured using a cylinder after the cylinder had been washed with 1 M HCl.Finally, hygroscopic reagents, such as KCl, K 2 HPO 4 •3H 2 O, MgCl 2 •6H 2 O, CaCl 2 , and Na 2 SO 4 , were measured as quickly as possible.
e temperature of the solution was set to 36.5 ± 1.5 °C.If the amount of solution was less than 900 mL, ion-exchanged and distilled water was added to increase the volume to 900 mL. e pH of the solution was then determined.Before Tris was added to the solution and dissolved, the pH of the solution was 2.0 ± 1.0.e solution temperature was kept within the range of 35-38 °C (optimal: 36.5 ± 0.5 °C), and the added Tris was dissolved slowly, while changes in pH were noted.Tris was added until the pH reached approximately 7.45.

Scanning Electron Microscopy (SEM) and SPM
Analysis.SEM images are shown in Figure 1.After modification in NaOH at 30 °C, the TNS sensor surfaces showed a nanoscale network structure.SPM was utilized to gauge the depth of the surface characteristics of the specimens, and the surface morphologies of the TNS and Ti sensors are shown in Figure 2.Many nanonodules were detected on the TNS surface; these formations had horizontal dimensions of approximately 300 nm. e surface roughness (Ra) values were 3.9 and 18.9 nm for the Ti and TNS sensors, respectively.

XPS Analysis.
Figure 3 shows the results of wide-scan XPS surface chemical analyses of Ti and TNS QCM sensors.e presence of Ti, O, C, and N was confirmed on the surfaces of Ti and TNS QCM sensors.In addition, the presence of Na was confirmed on the surface of the TNS QCM sensor.Moreover, the Ti and O concentrations of TNS QCM sensors were higher than those on the surface of TNS QCM sensors.

Surface Wettability.
e contact angle for a water drop on the Ti sensor was 42.5 °.However, the water drops spread very rapidly as they reached the test sensor, and the contact angle could not be determined.However, the behavior on Advances in Materials Science and Engineering the TNS sensor indicated that the sensor was superhydrophilic (contact angle of less than 5 °).

QCM Measurement of Proteins.
Figure 4 shows the adsorption of albumin and fibronectin based on QCM measurements.An immediate decrease in frequency was observed after the injection of albumin and fibronectin.is decrease in frequency was identified with the adsorption of albumin and fibronectin.
e adsorption of albumin and fibronectin on the TNS sensor produced a decrease in frequency that was greater than that measured for the Ti sensor.According to Sauerbrey's equation, at 27 MHz, a frequency decrease of 1 Hz compares to a mass difference of around 0.62 ng/cm 2 [10].After 30 min, the amounts of albumin adsorbed on the TNS and Ti sensors were 187.8 and  4 Advances in Materials Science and Engineering 117.5 ng/cm 2 , respectively, and the amounts of bronectin adsorbed on the TNS and Ti sensors were 827.6 and 533.7 ng/cm 2 , respectively.

QCM Measurement of RBM Cells.
Figure 5 shows the adsorption of rat bone marrow cells based on QCM measurements.An immediate decrease in frequency was observed after the injection of RBM cells.e adsorption of RBM cells on the TNS sensor produced a decrease in frequency that was greater than that measured for the Ti sensor.Using Sauerbrey's equation, after 30 min, the amounts of albumin adsorbed on the TNS and Ti sensors were 1491.1 and 872.5 ng/cm 2 , respectively.

XPS Analysis after Adsorption of Proteins.
e XPS spectra of the Ti and TNS QCM sensors after immersion in BSA are summarized in Figures 6 and 7, and those of the Ti and TNS QCM sensors after immersion in HFN are shown in Figures 8 and 9.For XPS analysis, the N1s and C1s spectra were measured.e coupling energies (BEs) of the C1s range for adsorbed BSA were 284.Advances in Materials Science and Engineering

Characterization after Immersion in SBF.
Figure 10 shows the adsorption of apatite after exposure to the SBF solution based on QCM measurements.A quick frequency reduction was seen after the infusion of SBF. e adsorption of SBF on the TNS sensor produced a decrease in the frequency that was greater than that measured for the Ti sensor.Advances in Materials Science and Engineering Figure 11 shows the surface morphology of the Ti and TNS QCM sensors after immersion in SBF for 24 h.After immersion, a recently framed layer was seen on the Ti and TNS surfaces.e surface was covered by single and grouped ball-like particles with a size of around 1.5 μm. e number of particles on the surface of the TNS QCM sensor was clearly greater than that on the surface of the Ti QCM sensor.

Discussion
In this study, Ti sensors were realized by depositing a thin Ti lm on a QCM electrode using a reactive DC magnetron sputtering technique; this sensor was further alkali modi ed using NaOH treatment to fabricate the TNS.SPM and XPS analyses were carried out to characterize the nanostructure of the TNSs.We evaluated the quality of the TNSs and the potential application of the high-frequency TNS sensors by investigating the in situ binding behaviors for TNS sensors, two proteins, RBM cells, and SBF to determine the e ects of these parameters on biological reactions in solution.
Several studies have demonstrated that implant surfaces a ect nanoscale topography and thereby alter cell behaviors or change the nanofeatures of structures to improve the osseointegration process [4,11,12].e embed surface can be adjusted by various approaches to add nanoscale features to the surfaces in speci c combinations.
e most wellknown techniques are chemical processes, such as alkaline hydrothermal [13,14] or acid [15,16] oxidation, on titanium surfaces to produce diverse nanoscale topographies.Kasuga et al. [5] demonstrated that TiO 2 nanotubes with a diameter of about 8 nm and a length of about 100 nm could be formed by Ti treatment with a 10 M NaOH aqueous solution for 20 h at 110 °C, without the need for templates or replication.Essential factors in regulating cell reactions at the implanttissue interface can dramatically a ect tissue coordination [17].In a recent work, we demonstrated that TiO 2 nanotubes and TNSs could be formed on titanium metal surfaces by treatment with a 10 M NaOH aqueous solution at 30 °C, and we used this method to prepare TNS-modi ed disks.Komasa et al. [8] suggested that TNSs on titanium surfaces can be applied to control the osteogenic di erentiation of bone marrow cells and enhance mineralization.Our results demonstrated that TNS-modi ed titanium disks were more hydrophilic and showed uniquely enhanced wettability in comparison with unmodi ed disks.Further studies of the surface roughness and topography of modi ed titanium alloy surfaces are needed to assess their wettability.Ra is a commonly used height parameter to describe implant surface roughness; Ra of the TNS-modi ed titanium surface was 19 nm, which was greater than that of the untreated titanium surface.
e contact angles of the alkali-treated titanium disks gradually decreased in comparison with those of the control group, indicating that the wettability of the surface of the test group was reduced by NaOH treatment.In previous studies, a surface roughness of between 13 and 16 nm was found to be optimal for RBM cell culture [18,19].
e nanonetwork structure framed on the titanium disks here was like the hierarchical structure outlined by Zhao et al. [12].In their work, hierarchical nanotextured titanium surface topographies with TNS structures that mirrored the hierarchical structures of bone tissues were created by etching followed by anodization.Natural tissues are hierarchical structures of nanoscale building blocks organized in a structured way.Hierarchical structures composed of nanocomponents may give a more reasonable surface topography for bone marrow cell functions than simpler structures because they can better copy the structures of natural tissues.Our research revealed that NaOH treatment prompts the development of a Ti-O-Na titanate layer on the titanium surface.
us, we expect that NaOH treatment Advances in Materials Science and Engineering results in the formation of a thick oxide film on the TiO 2 layer on the titanium surface.e deconvolution procedure suggested that this may have been due to surface contamination resulting from the binding of O to C [20].Before argon ion etching was performed, the XPS depth profiles perpendicular to the surface of the sample showed a carbon contamination overlayer on the surface.is carbon contaminant was removed after the initial etching cycles.
e oxygen and Ti concentrations gradually decreased and increased, respectively, as the number of etching cycles increased.Kim et al. [21] demonstrated that NaOH and heat treatment resulted in the covering of the Ti substrate with a titanium oxide layer with a thickness of approximately 1000 nm; AES depth profiles of the Ti oxide layer indicated the presence of an amorphous sodium titanate hydrogel layer.Kasuga et al. [5] likewise showed that NaOH treatment prompted the formation of a Ti-O-Na titanate layer on the Ti surface.At a greater depth of approximately 250 nm, the oxygen concentrations remained almost constant at 5 atom%.In addition, the remaining oxygen content was approximately 5 atom% inside the titanium film, which was attributed to the presence of residual oxygen in the chamber after preparation of the Ti sample.
All embed surfaces are promptly covered with a layer of protein from the in vitro culture medium or in vivo biological fluids, and this interface regulates the course of cell reactions and behaviors [22].To elucidate the connection between the Ti implant surface properties, opsonization, and phagocytosis under in vivo conditions, phagocytic experiments were previously conducted using a cell culture medium supplemented with serum albumin and human opsonizing serum factors [23].Fibronectin plays a crucial role in the progressive differentiation of osteoblasts [24].Additionally, fibronectin has RGD sequences and is a large extracellular matrix dimer glycoprotein [25] with a molecular weight of approximately 440 kDa [26].Albumin is the most abundant plasma protein; it suppresses the adsorption of other proteins that may empower aggravation and bacterial colonization [27].
e molecular weight of BSA is approximately 6-7 kDa [28].In this study, the amount of fibronectin adsorbed was greater than the amount of albumin adsorbed, suggesting that adsorption quantity was related to the molecular weight of the injection solutions.
After adsorption of BSA and BSF, N1s core-level spectra were obtained for both QCM sensors.Endo [29] detected C, O, and N obtained from the organic material or metal oxide on the titanium surface by XPS investigation.In the present study, based on XPS, the C1s and N1s peaks derived from BSA and BSF were detected on both QCM sensor surfaces tested.Evaluation of the N1s peak emerging from the peptide bonds of the implant-bone bonding protein may be a successful measure of the relative amount of protein adsorbed onto implant surfaces on the TNS QCM sensor.
Our results are the first comparison of RBM cell proliferation on TNS-modified titanium surfaces and unprocessed controls.Surfaces with nanostructures have higher surface areas than those of surfaces without such features [11,30].
is expanded surface territory permits increased adhesion of cells, such as osteoblasts and fibroblasts.us, the altered surface energies of materials with nanostructures may promote tissue growth via increased adsorption of select proteins compared with materials with microscale features [31].e adsorption of select proteins can in this manner guide the adhesion of cells on the implant material surface, among other capabilities.Various investigations have exhibited improved cell adhesion and multiplication on nanostructured surfaces, with various potential tissue applications, including applications in the bladder, bone, vasculature, and nervous system [32][33][34].A prior study suggested that TNSs on titanium surfaces facilitate the regulation of osteoblastic differentiation of bone marrow cells and enhance mineralization.e current study showed that the TNSs formed nanonodules with a diameter of about 19 nm on the titanium surfaces, and these structures promoted the adhesion and/or multiplication of cells.e network structure of TNSs on titanium alloy facilitated rapid cell adhesion, spreading, and multiplication due to the mechanics of the TNS structure and chemical nature of the Ti-O-Na layer.
To enhance bone-titanium bonding, Kokubo et al. recently demonstrated that a blend of alkalis resulted in the formation of bone-like apatite on the surface of titanium in SBF with an ion concentration almost equivalent to that of human blood [35].Apatite development on the material surface is accepted to be essential for bioactivity, that is, direct bone bonding.In our study, there was an apatite layer on the TNS QCM sensor after 24 h.us, the increased adhesion of RBM cells and SBF on the TNS QCM sensor suggested that TNS induced bone differentiation.
In the TNS sensor, the concentration of Ti exceeded that of oxygen at a depth of approximately 170 nm.Subsequently, the NaOH treatment was thought to produce a thick oxide film.Nonetheless, the chemical structure of the treated surfaces did not vary fundamentally; Ti oxides (mostly TiO 2 ) formed at the surface.Albumin and fibronectin were readily adsorbed, to a greater extent on the TNS sensor than on the reference Ti sensor.e TNS surface seemed to adsorb more protein for a given geometric surface area than the reference Ti sensor; this may have been a result of its rough morphology, as evidenced by the SEM and SPM results.Several studies [36,37] have shown that nanostructured topographies can act as good mimics of natural extracellular matrixes.Advancement of the surface topography could be indirect; the adsorption of proteins or ions may function as an extension between the nanosurface structure and cells [38].Webster et al. [39] observed increases in the adsorption of vitronectin on nanostructured surfaces (compared with conventional surfaces), which resulted in preferable adhesion of osteoblasts.In addition, here, the protein adsorption rate on the TNSs was correlated with the contact angle, suggesting that the hydrophilicity of titanium greatly affected its protein adsorption ability.e contact angle of the TNS and Ti sensors fluctuated in the hydrophilic range; the hydrophilicity of the surfaces expanded after treatment, corresponding to the formation of the TNSs.us, the growth of the titanium oxide layer increased the surface energy, resulting in a more hydrophilic surface [40].

Conclusions
In conclusion, TNS structures were obtained on a titanium surface via treatment with a NaOH aqueous solution at room temperature.Nanoscale network structures and a large number of nanoscale nodules were observed by SEM and SPM.In addition, the chemical composition of the TNS structures was estimated by XPS. e results confirmed the presence of a combined titanium and oxide titanate layer, which induced the adsorption of albumin and fibronectin.In the fields of tissue engineering and biomaterials, nanostructuring technologies are expected to yield novel, biologically optimized surfaces.

Figure 6 :
Figure 6: C1s XPS spectra of (a) Ti and (b) TNS QCM sensors after immersion in BSA.

Figure 7 :
Figure 7: N1s XPS spectra of (a) Ti and (b) TNS QCM sensors after immersion in BSA.

Figure 11 :
Figure 11: SEM images of exposure to SBF solution on (a) Ti and (b) TNS QCM sensors.

Table 1 :
Order, amounts, weighing containers, purities, and formula weights of reagents for the preparation of 1 L of SBF.