鈦表面鈉氫氧鈦納米線的結(jié)構(gòu)、生物活性和MC3T3-E1細(xì)胞響應(yīng)

景紹東 成 夙＊,,2 周 睿 魏大慶 周 玉

(1哈爾濱理工大學(xué)建筑工程學(xué)院，哈爾濱150080)

(2黑龍江省高校材料研究與應(yīng)用重點(diǎn)實(shí)驗(yàn)室，哈爾濱理工大學(xué)，哈爾濱150040)

(3材料科學(xué)與工程學(xué)院，哈爾濱工業(yè)大學(xué)，哈爾濱150001)

鈦表面鈉氫氧鈦納米線的結(jié)構(gòu)、生物活性和MC3T3-E1細(xì)胞響應(yīng)

景紹東1成 夙＊,1,2周 睿3魏大慶3周 玉3

(1哈爾濱理工大學(xué)建筑工程學(xué)院，哈爾濱150080)

(2黑龍江省高校材料研究與應(yīng)用重點(diǎn)實(shí)驗(yàn)室，哈爾濱理工大學(xué)，哈爾濱150040)

(3材料科學(xué)與工程學(xué)院，哈爾濱工業(yè)大學(xué)，哈爾濱150001)

采用化學(xué)方法處理微弧氧化(MAO)制備的含Si、Ca元素的TiO2涂層(SC)，獲得鈦氫氧鈉(Na0.8H1.2Ti3O7)生物活性納米線結(jié)構(gòu)?；瘜W(xué)處理過(guò)程中，SC涂料表面出現(xiàn)了Ca、Na元素溶解，Si元素沉積的現(xiàn)象?；瘜W(xué)處理后的SC涂層比SC涂料具有更好的吸水性和誘導(dǎo)磷灰石形成能力。這與處理后涂層(SHTO)特殊的納米結(jié)構(gòu)有關(guān)，在模擬體液浸泡過(guò)程中更容易形成Ti-OH。同時(shí)，鈉氫氧鈦納米線的表面形貌、相組成、OH基團(tuán)以及良好的濕潤(rùn)能力使其更加適合于MC3T3-E1細(xì)胞的粘附和增值。

納米線；微弧氧化；鈦；化學(xué)處理；MC3T3-E1

Titanium and its alloys have been used widely as load-bearing implants due to their good mechanical properties,biocompatibility and biocorrosion resistance[1].However,they exhibit poor osseointegrationproperty because of no bioactive chemical stimulus to induce the bone formation in vivo[1].Many surface modifying techniques(e.g.,plasma spraying[2-3],sol-gel method[4-5],electrophoresis and electrochemical deposition[6-7])have been developed to deposit bioactive coatings on Ti and its alloys.

Microarc oxidation(MAO)is a relatively convenient and effective technique to prepare coatings on the surfaces of Ti,Al,Mg and their alloys[8].Using MAO technique to deposit bioactive coatings on Ti and its alloys has received much attention in recent years[9-20].We have reported that MAO coatings containing Si and Ca(SC)were fabricated on Ti under different applied voltages[21].Some subsequent treatment methods such as heat treatment,hydrothermal treatment and chemical treatment have been developed to change the surface structure and property of the MAO coatings containing different elements[9,22-31].

In the previous results[9],chemical treatment was used to modify the surfaces of the MAO coatings containing Ca and P(CP).Amorphous calcium titanate hydrogel with a nanoflake-like morphology was formed on the surface[9].The apatite formation ability of the CP coating was improved obviously;however,the cell response to the CP coating decreased after chemical treatment[22].It can be imagined that the element composition and phase composition of the MAO coatings could have important effects on the surface structure and properties of the MAO coatings after subsequent chemical treatments,since different physical and chemical reactions take place during the chemical treatment process.Of course,the chemical treatment procedure such as temperature and solution concentration,could also influence the surface structure and property of the MAO coatings.

Thus,it was attempted to obtain desired surface with good bioactivity and biocompatibility on the MAO coatings by changing the elemental composition of MAO coating and subsequent chemical treatment procedure.In this work,the novel sodium hydrogen titanium oxide(SHTO)nanowire with good bioactivity and biocompatibility was obtained by chemical treatment of the SC coating.The structure,apatite formation ability and MC3T3-E1 cell response of the SHTO nanowire on Ti were investigated.At the same time,the formation process of SHTO nanowire and its apatite inducing mechanism as well as cell response on its surface were discussed.

1 Experimental

1.1 M aterials and m ethods

1.1.1 Microarc oxidation

In the MAO process,Ti plates(10 mm×10 mm× 1.5 mm)were used as anodes and stainless steel plates were used as cathodes in an electrolytic bath.The Ti plates were ground with abrasive papers(according to priority 800#,1500#,3000#),ultrasonically washed with acetone and distilled water,and dried at 40℃. An electrolyte was prepared by the dissolution of reagent-grade chemicals of Ca(CH3COO)2·H2O(8.8 g·L-1),Na2SiO3(14.2 g·L-1),EDTA-2Na(15 g·L-1)and NaOH(20 g·L-1)into deionized water.In the electrolyte,the applied voltage,frequency,duty cycle and oxidizing time were 400 V,600 Hz,8.0%and 5 min,respec-tively.The temperature of the electrolyte was kept at 40℃by a cooling system.In this way, the SC coatings were prepared on Ti.

1.1.2 Chemical treatment of the SC coatings

The samples(10 mm×10 mm×1.5 mm)with SC coating were treated in solutions with 0(distilled water),0.001,0.01,0.1 and 1 mol·L-1NaOH at temperature of 200℃for 24 h,and then gently washed with deionized water(dipping into deionized water 10 times)and dried at 25℃.The sample of the SC coating before and after chemical treatment was labeled as SC(Without treatment),SCH2O(distilled water),SC0.001,SC0.01,SC0.1,SC1,respectively.

1.1.3 Immersion of the samples in a simulated body fluid

The SC coating before and after chemical treatment was soaked in 15 mL simulated body fluid (SBF)[32](Table 1),immersing for different periods of time,and the SBF was refreshed every other day.The SBF was prepared by dissolving reagent-grade chemicalsof NaCl,NaHCO3,KCl,K2HPO4·3H2O,MgCl2·6H2O, CaCl2,and Na2SO4into deionized water and buffering at pH value of 7.40 with tris-hydroxymethylaminomethane((CH2OH)3CNH2)and 1.0 mol·L-1HCl at 37℃.

Table1 Ion concentrations of SBF and human blood p lasma

1.2 Structure characterization

1.2.1 X-ray diffraction(XRD)

The phase compositions of the SC coating before and after chemical treatment were analyzed by X-ray diffraction(XRD,D/max-rB,Japan)using a Cu Kα radiation(λ=0.154 18 nm)with a continuous scanning mode at a rate of 4°·min-1,under an accelerating voltage of 40 kV and a current of 50 mA,sweep range 2θ(10°～90°),graphite curved crystal monochromator.

1.2.2 Scanning electron microscopy(SEM)and

energy dispersive X-ray spectroscopy(EDS) A scanning electron microscopy(SEM,Helios 600i,FEI Co.,USA)was used to observe the surface morphology.In addition,the elemental concentrations of sample surfaces were detected by an energy dispersive X-ray spectrometer(EDS,EDAX,USA) equipped on the SEM system.

1.2.3 Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy(FTIR, Magna-IR 560 E.S.P.,USA)was used to analyze the apatite structure.In the FTIR experiment,the scanning range and resolution were 4 000～400 and 4 cm-1, respectively.Approximately 1 mg of apatite layer on the samples was mixed with about 500 mg of dry KBr powder.The mixed powder was pressed into transparent disks with a diameter of 13 mm for the FTIR work.

1.2.4 X-ray photoelectron spectroscopy

An X-ray photoelectron spectroscopy(XPS,KAlpha,ThermoFisher Scienticfic Co.,USA)was used to determine the chemical compositions of the coating surfaces.In the XPS experiment,an Al Kα(hν= 1 486.6 eV)X-ray source was used with an anode powder of 250 W(12.5 kV,20 mA)for the XPS work under a vacuum of 1.0×10-6Pa and the XPS take-off angle was set at 45°with a scanning step of 0.1 eV. The current of X-ray beam was 6 mA,and the resolution for energy was 0.5 eV.The regions of 400 μm2on the surfaces of samples were analyzed with a hemispherical analyzer.The measured binding energies were calibrated by the C1s(hydrocarbon C-C, C-H)of 285.0 eV.The chemical states of various elements before and after etching by Ar+ions for 60s were analyzed.

1.3 W etting angles

The wetting angles on the SC coating before and after chemical treatment were measured using the liquid drop method on a contact angle goniometer (CAM101,KSV Instruments Ltd.,Finland).

1.4 MC3T3-E1 cell response

1.4.1 MC3T3-E1 cell adhere test

The MC3T3-E1 osteoblast(a mouse calvaria osteoblast-like cell line)was used to the cell test in this work(Shanghai Institute for Biological Sciences, Chinese Academy of Sciences).The SC coatings without and with chemical treatment were placed into a 24-well culture plate and the MC3T3-E1 cells with 5×105mL-1cell density were seeded on the samples. The 24-well culture plate was added by α-modified Eagle′s medium(α-MEM)with penicillin and 10% fetal bovine serum(FBS).The 24-well culture plate was stored in a humidified incubator with 5%CO2at 37℃for 0.5,1,2 and 4 h,and then the culture was terminated.The samples were taken out and carefully washed by a phosphate buffered saline(PBS)for 3 times to remove the MC3T3-E1 cells that did not adhere on the samples.These samples were transferred into a new 24-well culture plate with 0.3m L pancreatic enzyme.After pancreatic enzyme digest for 3 min,the α-MEM was added to terminate the pancreatic enzyme digest.At last,the cell suspension was obtained for detecting the number of the adhered MC3T3-E1 cells.The cell adhesion ratio of Ln was calculated by Eq.(1):

In Eq.(1),Tn is the number of the adhered MC3T3-E1 cells,Jn is the number of the seeded MC3T3-E1 cells,Ss is the area of the bottom of the 24-well culture plate and Sp is the area of the surface of the sample.

1.4.2 MC3T3-E1 cell proliferation test

The SC coatings without and with chemical treatment were put into a 24-well culture plate and the MC3T3-E1 cells after a pancreatic enzyme digest with a cell density of 5×104mL-1were seeded on the samples.After culturing for 1,4,7 and 10 d,the samples were washed by the PBS for 3 times.The washed samples were cultured in a α-MEM with addition of penicillin and 10%FBS,200 μL mediums and 20 μL CCK-8 solution in a humidified incubator with 5%CO2at 37℃for 4 h.Then,the 100 μL cultured solution was taken out to a 96-well culture plate.A microplate reader for enzyme linked immunosorbent assay(ELISA)was used to detect the OD value of the absorbance by 450 nm of wavelength.The MC3T3-E1 cell proliferation ratio of ODawas calculated by Eq.(2):

In Eq.(2),ODsis originated from the culture plate containing mediums,CCK-8 and samples;ODcis originated from the culture plate containing mediums, CCK-8;and ODbis originated from blank controls without any samples.

1.4.3 Statistical analysis

The software of SAS6.0 was used to perform the statistical analysis and the data has statistical difference when P<0.05.In the cell attachment and proliferation numbers tests,each group with eight samples at least was measured.In the elemental concentration of the each kind of coating,five samples were used and measurement was carried out with at least 3 independent repeats.

2 Results and discussion

2.1 Characters of SC coating before and after chem ical treatment

2.1.1 Phase composition

Fig.1 shows the XRD patterns of the SC coating before and after chemical treatment.The main phase compositions of the SC coating are anatase and amorphous phase according to Fig.1(a)and the previous TEM observation[34].On the surface of the SCH2O coating(which is hydrothermally treated SC coating in pure water),the intensity of anatase peaks obviously increases as shown in Fig.1(b).This phenomenon is also observed on the surfaces of the SC0.001 and SC0.01 coatings.

Fig.1 XRD patterns of the SC coatings before and after chemical treatment in different concentrations of NaOH solution for 24 h

In Fig.1(d),(e)and(f),Na0.8H1.2Ti3O7peaks are found on the surfaces of the SC0.01,SC0.1 and SC1 coatings.Meanwhile,the intensity of Na0.8H1.2Ti3O7peak increases with increasing NaOH concentration.In addition,rutile is found on the SC0.1 and SC1 coatings as shown in Fig.1(e)and(f)and the intensity for anatase peaks decreases.Some weak peaks for CaTiO3are also observed on the SC1 coating as shown in Fig.1(f).

2.1.2 Surface morphology

Fig.2 Surface morphology of the SC coatings before and after chem ical treatment in different concentrations of NaOH solution for 24 h

Fig.2 shows the surface morphology of the SC coatings before and after chemical treatment.Both Fig.2(a)and Fig.2(b)show the micropore structure of SC and SCH2O coatings.Very smooth surface is observed at high magnification on the SC coating as shown in Fig.2(a).However,the surface of the SCH2O coating shows too many nano anatase particles according to the above XRD result.With increasing NaOH concentration,Na0.8H1.2Ti3O7nanowires are found on the chemically treated SC coating as shown in Fig.2(e)and(f).

2.1.3 Average roughness

Fig.3 shows the average roughness of Ti and SC coating before and after chemical treatment.The average roughness of SC coating increases significantly (p<0.05)as compared to Ti.The difference in the average roughness between the SC and SCH2Ocoatings is not significant.However,the average roughness of the chemically treated SC coating increases obviously with increasing the NaOH concentration(p<0.05).

Fig.3 Average roughness of Ti,SC coating before and after chemical treatment in different concentrations of NaOH solution,‘*’represents p<0.05

2.1.4 XPS analysis

Fig.4 shows XPS overall spectra of the SC, SCH2O and SC1 coatings.Firstly,Si2p,C1s,Ca2p, Ti2p and O1s are found on the surface of the SC coating as shown in Fig.4(a).On the surface of the SCH2O coating,the Si2p peak disappears and the Ca2p peak decreases sharply.On the SC1 coating, Na1s and Na KLL peaks with strong intensity are observed and Si2p also disappears.At the same time, a weak peak for Ca2p is observed on the surface of the SCH2O and SC1 coating.

Fig.4 XPS survey spectra of the SC,SCH2O and SC1 coatings

Fig.5 shows XPS high resolution spectra of Si2p, Ca2p and Na1s elements on the surfaces of the SC, SCH2O and SC1 coatings.Fig.5(a)and(b)show the Si2p peaks at binding energy of(102.4±0.5)eV suggesting a chemical state of Si4+and no obvious change is observed before and after Ar+ion etching. No Si2p peaks are found on the SCH2O and SC1 coatings,which reveals that Si is released from the surfaces of the SCH2O and SC1 coatings during the chemical treatment.

In Fig.5(c)and(d),the intensity of Ca2p peaks on the surface of the SC coating is obviously higher than that on the SCH2O and SC1 coatings,indicating a great dissolution of Ca during the chemical treatment.The binding energy of Ca2p shows a bimodal structure at(350.7±0.5)eV and(347.2±0.5) eV with a chemical state of Ca2+before and after Ar+ion etching.Moreover,no change in the binding energy of Ca2p is found when the SC coating after chemical treatment in distilled water and NaOH solution.

Figs.5(e)and(f)show the Na1s peak with binding energy at(1 070.8±0.5)eV,suggesting a chemical state of Na+.No change in the binding energy of Na1s is observed before and after Ar+ion etching.In addition,a weak peak for Na1s is found on the SC coating.Na in the SC coating is added during the MAO treatment process.On the surface of the SCH2O coating,no Na1s are found on the surface of the SCH2O coating,indicating that Na is released from the SCH2O coating completely.However,strong peak for Na1s is found on the SC1 coating,revealing that Na in the NaOH solution is added into the surface of SC1 coating during the chemical treatment,forming Na0.8H1.2Ti3O7.

Fig.6 shows XPS high resolution spectra for Ti2p and O1s of the SC,SCH2O and SC1 coatings.Fig.6(a) shows the Ti2p bimodal peaks at binding energy of (464.3±0.5)eV and(458.5±0.5)eV with a chemical state of Ti4+before Ar+ion etching[33-35].The intensity of Ti2p on the SCH2O is obviously higher than that on the SC coating,probably due to the dissolution of Ca and Si and remaining of Ti.In addition,no change in the chemical state of Ti is found before Ar+ion etching.In Fig.6(b),a slight shift of binding energy about 0.9 eV toward high binding energy is found on all surface as compared to Fig.6(a).It could be resulted from the removal of the surface absorption by Ti.At the same time,a peak is found at relative low binding energy of(458.2±0.5)eV,which could correspond to the formation of TiO compound with low chemical state of Ti2+[34].

Fig.5 XPS high resolution spectra for Si2p,Ca2p and Na1s of the SC,SCH2O and SC1 coatings

Figs.6(c)and(d)show the O1s spectra on the surfaces of the SC,SCH2O and SC1 coatings without and with Ar+ion etching.The O1s of the SC coating before and after Ar+ion etching are obvious different. Before Ar+ion etching,the O1s spectra of the SC coating show a symmetric form at(531.5±0.5)eV,indicating the presence of O2-in the coating[18,36].After Ar+ion etching,the O1s spectrum of the SC coating shows asymmetric form,which could be fitted by two components at binding energy(531.1±0.5)eV and(533.5± 0.5)eV,due to the formation of TiO2and TiO phases[33-36]. The O1s of the SCH2O coating is similar before and after Ar+ion etching.Similar results are also found on the SC1 coating before and after Ar+ion etching. However,the SC1 coating shows different O1s curves as compared to the SCH2O coating as shown in Fig.6 (c)and(d).The binding energy at(534.2±0.2)eV could be assigned to the OH structure of Na0.8H1.2Ti3O7[37-38].

2.1.5 Change in the element concentration during chemical treatment

Fig.6 XPS high resolution spectra for Ti2p and O1s of the SC,SCH2O and SC1 coatings

Fig.7 Elemental concentrations of the SC coating before and after chemical treatment in different NaOH solutions

Fig.7 shows the elemental concentrations of the SC coating before and after chemical treatment in solutions with different concentrations of NaOH. Firstly,Ti concentration increases obviously after chemical treatment in the solution without NaOH,and Na,Ca and Si elemental concentrations decrease greatly,suggesting a considerable dissolution from the surfaces.After chemical treatment in the NaOH solutions,Ti concentration decreases and Na concentration increases with increasing NaOH concentration.In addition,the change in O concentration is not obvious.

2.1.6 Wetting ability

Fig.8 shows the contacting angles of the SC coating before and after chemical treatment in different concentrations of NaOH solution.The wetting ability of the SCH2O coating is better than that of theSC coating.Further,the wetting ability of the SC coating is improved obviously(p<0.05)after chemical treatment,especially the SC1 coating,showing a contacting angle about 15°.

Fig.8 Contacting angles of the SC coatings after chemical treatment in different NaOH solutions*represents p<0.05

2.2 Apatite formation on the SC coating before and after chem ical treatm ent

2.2.1 Surface morphology

Fig.9 shows the surface morphology of the SC, SCH2O and SC1 coatings after SBF immersion for 2 and 3 days.After SBF immersion for 2 days,no apatite or other compounds containing Ca and P are found on the surface of the SC coating.However, compounds containing Ca and P elements etc.(as shown by EDS result in Fig.9(c))are found on the surfaces of SCH2O and SC1 coatings.At the same time,the SC1 is more suitable for the deposition of compounds containing Ca and P elements.After SBF immersion for 3 d,a few of compounds containing Ca and P are found on the surface of the SC coating. However,a dense apatite layer is covered on the whole surfaces of the SCH2O and SC1 coatings.According to the observed morphology at high magnification,the apatite shows a nano network-like structure.

Fig.9 Surface morphology of the SC,SCH2O and SC1 coatings after SBF immersion for 2 and 3 d

2.2.2 Phase composition

Fig.10 shows the XRD patterns of the SC and SC1 coatings after SBF immersion for 3 days.The XRD results indicate that the apatite is formed on the surface of the SC1 coating.In addition,anatase,rutile, CaTiO3and Na0.8H1.2Ti3O7are also observed on the surface of the SC1 coating.The SEM and XRD results indidcate that the SC1 coating has good bioactivity, which can induce the formation of apatite in the SBF. 2.2.3 FTIR analysis

Fig.10 XRD patterns of the SC and SC0.1 coatings after SBF

The FT-IR spectrum of the SC1 coatings after SBF incubation for 4 weeks is shown in Fig.11.A broad absorption band at 3 441 cm-1and a bending mode at 1 651 cm-1indicate the presence of bondedwater in the SBF incubated SC1 coating[39].Absorption peaks of PO4bands are observed including the triply degenerated asymmetric stretching mode of ν3PO43-band at1 033 cm-1,triply degenerated bending mode of ν4PO43-band at 602 and 566 cm-1and double degenerated bending mode of ν2PO43-band at 471 cm-1[39].

Fig.11 FTIR spectrum of the SC1 coating after SBF immersion for 28 d

The FTIR spectrum demonstrates the presence of the CO32-absorption bands,including the bending mode of the ν4CO32-group in A-type carbonated HA (CHA)at 1 551 cm-1,the characteristic stretching mode of the ν3CO32-group in CHA at 1 503 cm-1,the characteristic stretching mode of the ν1CO32-group in A-type CHA at 1 461 cm-1,the stretching mode of the ν1CO32-group in B-type CHA at 1 426 cm-1and the bending mode of the(ν3or ν4)CO32-group in CHA at 874 cm-1[39].Also,the HPO42-groups are detected at characteristic peaks of 1 097,955 and 872 cm-1[39]. The FTIR result confirms that the apatite formed on the SC1 coating has a carbonated structure.

2.3 MC3T3-E1 cells response

Fig.12 shows cell adhesion rate to the surfaces of Ti,SC,SCH2O and SC1 coatings.After cell culture for 0.5 h,the cell adhesion rate on the SC coating is lower than that on Ti.However,this change is not significant.In addition,no obvious change is observed between Ti,SCH2O and SC1 coatings.After cell culture for 1 h,the difference in the adhesion rate between Ti and SC coating is observed(p<0.05). However,it is also not evident among Ti,SCH2O and SC1 coatings.After cell culture for 2 h,the cell adhesion rate on the SC coating decreases obviously as compared to Ti(p<0.05).At the same time,it is also lower than that on the surfaces of SCH2O and SC1 coatings(p<0.05).In addition,the cell adhesion rates on Ti,SCH2O and SC1 coatings are at a similar level.However,the adhesion rates on the SCH2O and SC1 coatings are higher than that on the surfaces of Ti and SC coating after cell culture for 4 h.

Fig.12 Adhesion rate of the cells to the surfaces of Ti, SC,SCH2O and SC1

Fig.13 shows the cell proliferation on the surfaces of Ti,SC,SCH2O and SC1 coatings.After cell culturing for 1 d,more cells are observed on the surface of SC coating as compared to Ti(p<0.05), indicating that the SC coating benefits to the cell proliferation.However,the cell number decreases on the surface of the SCH2O coating.With respect to the SC1 coating,it shows higher cell number as compared to the SC coating;however,this change is not significant.Never-theless,the cell number on its surface is obviously higher than that on Ti and SCH2O coating(p<0.05).

Fig.13 Cell proliferation number on the surfaces of Ti, SC,SCH2O and SC1 coatings after cell culture for different time

After cell culturing for 4 d,the cell number on the Ti and SCH2O coating is also lower than that on the SC coating greatly(p<0.05).However,the cell number on the SC1 coating is higher than that on the surfaces of Ti,SC and SCH2O(p<0.05),indicating that the formation of Na0.8H1.2Ti3O7could benefit to the cell proliferation.

After cell culturing for 7 d,the cell number on Ti is greatly lower than that on the surfaces of SC, SCH2O and SC1 coatings(p<0.05).At the same time, the cell number on the SC coating is higher than that on the SCH2O coating.Similar results are observed after cell culturing for 10 d;however,the cell numbers on all surfaces decrease,suggesting a saturation and death of cells on these surfaces.

2.4 Form ation of the SHTO nanowires

In recent years,subsequent treatment methods such as hydrothermal treatment,heat treatment and alkali-and heat-treatment have been developed to modify the MAO coatings for improve the bioactivity and cell response on their surfaces[9,22-31].However,it is difficult to both improve the bioactivity and cell response on their surfaces[9,22-31].Thus,in this work, chemical treatment is performed on the surface of the SC coating to form SHTO nanowire for achieving the objective.

Fig.14 Schematic diagram for the formation process of the sodium hydrogen titanium oxide:(a)the dissolutions of Ca and Si, (b)the attacking of OH-ions to TiO2phase,(c)the formation of the negatively charged HTiO3-and(d)and the deposition of Na+ions to form sodium hydrogen titanium oxide

Fig.14 shows schematic diagram for the possible formation process of the SHTO nanowire.Fig.14(a)shows that the SC coating could highly release Ca and Si during the chemical treatment process,which is demonstrated by XPS and EDS results.As shown in Figs.14(b)and(c),TiO2could be attacked by OH-in NaOH solution.Thus,HTiO3-can be formed by the chemical reaction between OH-and TiO2.The HTiO3-could further react with Na+to form Na0.8H1.2Ti3O7nanowires.

In the previous work[9],chemical treatment was used to modify the surface of CP coating.However, different phase composition and surface morphology of the modified CP coating are obtained as compared to the modified SC coating.No Na0.8H1.2Ti3O7nanowire is found on the surface of CP coating after chemical treatment at low temperature of 60℃with high NaOH concentration of 5 mol·L-1.These changes are caused by different initial elemental compositions of MAO coating.At the same time,different chemical treatment parameters such as solution temperature and concentration can further change the microstructure of chemically treated MAO coating[40].

2.5 Formation of apatite on the chem ically treated SC coating

This work indicates that the SC1 and SCH2O coatings have better apatite induction as compare to the SC coating according to the SEM and XRD results.At the same time,the apatite formation on the SC1 coating is better than that on the SCH2O coating. The mechanism for the apatite formation on the SC coating was demonstrated in the previous work[41].The deposition of apatite on the SC coating could be related with the formation of hydrated silica gel layer on the SC coating surfaces[41],which could provide the chemical stimulus for the apatite deposition.The formation of hydrated silica gel is via dissolution of Na+ion from the SC coating surface[41].

However,Si on the surface of the SCH2O coating is released completely.Thus,the influence by Si on the apatite formation for the SCH2O coating disappears.The other factor could produce the result that the SCH2O coating has better apatite formation ability as compared to the SC coating.In this work, many nano-scale anatase particles are formed on the surface of SCH2O coating after modifying the SC coating.Some references[42-43]indicate thatTi-Ostructure could attract PO43-ions and Ca2+ions etc.In addition, TiO2has good crystallographic matching relation with HA on the specific crystal planes,probably providing good sites for apatite nucleation by the epitaxial deposition process[42-43].In this work,lots of nano protuberance of anatase could be suitable for the absorption of PO43-ions and Ca2+ions on its surface. In addition,the better wetting ability of SCH2O coating as compared to the SC coating also could be suitable for apatite nucleation.

The apatite-formation ability on the SC1 coating is higher than that of the SCH2O coating.This result could be explained as follows.Firstly,the XPS result shows the appearance of OH group in the SHTO nanowires.The OH group could attract Ca2+ions during the SBF immersion process and further attract PO43-ions to cause the apatite nucleation[42-43].Secondly,the release of Na from the SHTO nanowires could take place.An ionic exchange between Na+of the SHTO nanowire and H+in the SBF could occur,which could result in the formation of Ti-OH group[9].Thus,the newly formed Ti-OH group can further improve the apatite formation ability of the SC1 coating.

2.6 Cell response on the surfaces of SC coating before and after chem ical treatment

During the cell culture adhesion process,the cell number on the SC coating is lower than that on Ti. The reason is that the surface factors such as chemical composition,surface roughness and other properties could influence the cell adhesion[44].During the cell attachment process,the chemically treated SC coating is more suitable for cell adhesion as compared to the SC coating and Ti.The higher wetting ability and average roughness of the chemically treated SC coating could benefit the cell attachment as compared to the SC coating and Ti.

During the cell proliferation process,the higher cell proliferation ratio is observed on the SC coating as compared to Ti,which has been explained[44]. However,the cell proliferation number on the SCH2O coating decreases as compared to the SC coating.Themain factors to influence the cell proliferation on the SC coating are the chemical composition and amorphous phase of the SC coating.The releasing of Si and Ca could be a key factor to improve the cell proliferation[44].However,the SCH2O coating does not contain Si and only contains a few of Ca.Si and Ca dissolve into the NaOH solution during the chemical treatment.Thus,the decreased cell proliferation ability on the SCH2O coating is probably due to the change in the elemental composition and phase composition as compared to the SC coating.

With respect to the SC1 coating,the better cell proliferation ability is observed as compared to Ti,SC and SCH2O coating.On the surface of the SC1 coating,no Si is found and a few of Ca is detected, which is similar to that on the SCH2O coating. However,a plenty of Na0.8H1.2Ti3O7nanowires are formed on the surface of the SC1 coating.At the same time,the surface morphology,average roughness and wetting ability of the SC1 coating are greatly changed as compared to the SCH2O coating.These factors could influence the cell proliferation.For example,the higher roughness and better wetting ability could benefit cell proliferation[44].

The chemically treated CP coating shows a lower cell proliferation ability as compared to the CP coating,though this chemically treated CP coating (containing Na and Ca)has higher roughness and wetting ability[22].However,this chemically treated CP coating shows different surface morphologies and phase compositions as compared to the SC1 coating. These could cause the better cell proliferation on the SC1 coating,especially the formation of Na0.8H1.2Ti3O7nanowires.The OH structure of Na0.8H1.2Ti3O7could play an important role for improving the cell proliferation ability.

Fig.15 shows the well cell attachment on the coating.Cell behavior on scaffold surface is very complex,being affected by many factors such as roughness,surface chemistry,and wettability of scaffold[45-56].The early adhesive behavior of MSCs is largely dependent on both the texture and the surface chemistry of the substrate[47,49,54,56-57].However,the surface with appropriate nanotopography favors protein adsorption and may successfully support cell proliferation and differentiation[59-60].

Fig.15 Well cell attachment on the coating SC1

In this work,the chemical treatment is obviously appropriate when attempting to improve the bioactivity of SC coating such as apatite formation ability and cell response such as cell adhesion and proliferation. In addition,other factors such as mechanical property of the materials,also affect cell response[61],which needs to be investigated in the future.

3 Conclusions

The Na0.8H1.2Ti3O7nanowire was obtained by chemical treatment of the SC coating in NaOH solution.The surface morphology,elemental composition,phase composition of the chemically treated SC coatings are highly dependent on the concentration of NaOH solution.The dissolution of Si and Ca appears during the chemical treatment process.With increasing NaOH concentration,the average roughness and wetting ability of chemically treated SC coating are improved evidently.The chemically treated SC coating exhibits good apatite formation ability, especially the SC1 coating due to the formation of Na0.8H1.2Ti3O7nanowire.The SC1 coating has good cell attachment performance and better cell proliferation ability as compared to Ti,SC and SCH2O coatings due to the special surface morphology and phase composition.

Acknow ledgem ents:This work was financially supported by National Basic Science Research Program(2012CB933900),National Natural Science Foundation of China(Grant No. 51002039 and 51021002),the Fundamental Research Funds for the Central Universities(Grant No.HIT.NSRIF.2014002).The partial financial support from Key Laboratory of Materials Research and Application of Provincial Colleges and Universities Open Fund(No.CLJJ2013),Harbin University of Science and Technology is greatly acknow ledged.

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Structure,Bioactivity and MC3T3-E1 Cell Response of Sodium Hydrogen Titanium Oxide Nanow ire on Titanium

JING Shao-Dong1CHENG Su＊,1,2ZHOU Rui3WEI Da-Qing3ZHOU Yu3
(1College of Civil Engineering and Architecture,Harbin University of Science and Technology,Harbin 150080,China)
(2Key Laboratory of Materials Research and Application of Provincial Colleges and Universities, Harbin University of Science and Technology,Harbin 150040,China)
(3Department of Materials Science and Engineering,Harbin Institute of Technology,Harbin 150001,China)

The bioactive nanowire of sodium hydrogen titanium oxide(Na0.8H1.2Ti3O7)was obtained by Chemical treating the surface of TiO2-based coating containing Si and Ca(SC)prepared by microarc oxidation(MAO). During the chemical treatment,the dissolution of Ca and Si,and the deposition of Na appear on the surface of the SC coating.The chemically treated SC coating shows better hydrophilic and apatite-formation ability than those of the SC coating,which could be associated with the special structure such as OH group in the sodium hydrogen titanium oxide(SHTO)as well as the Ti-OH group formation during the simulated body fluid immersion.At the same time,the SHTO nanowire is more suitable for the MC3T3-E1 cell adhesion and proliferation due to surface morphology,phase composition,OH group structure and better wetting ability.

nanowire;m icroarc oxidation;titanium;chemical treatment;MC3T3-E1

O614.41+1

A

1001-4861(2015)04-0824-15

10.11862/CJIC.2015.119

2014-11-10。收修改稿日期：2015-02-25。

國(guó)家基礎(chǔ)研究項(xiàng)目(No.2012CB933900)，國(guó)家自然科學(xué)基金(No.51002039和51021002)，黑龍江省自然科學(xué)基金(No.QC2013C043)；材料研究與應(yīng)用黑龍江省高校重點(diǎn)實(shí)驗(yàn)室基金(No.cljj2013)資助項(xiàng)目。

＊通訊聯(lián)系人。E-mail：chengsu2002@163.com