Bngcheng Yn ,Ji Tn ,Donghui Wng ,Jijun Qiu ,Xunyong Liu,c,d,*

a State Key Laboratory of High Performance Ceramics and Superfine Microstructure,Shanghai Institute of Ceramics,Chinese Academy of Sciences,Shanghai,200050,China

b Center of Materials Science and Optoelectronics Engineering,University of Chinese Academy of Sciences,Beijing,100049,China

c School of Chemistry and Materials Science,Hangzhou Institute for Advanced Study,University of Chinese Academy of Sciences,1 Sub-lane Xiangshan,Hangzhou,310024,China

d Cixi Center of Biomaterials Surface Engineering,Shanghai Institute of Ceramics,Chinese Academy of Sciences,Ningbo,315300,China

Keywords:Titanium Zirconium Plasma immersion ion implantation Ti–Zr alloy Osteogenesis

ABSTRACT Titanium has been widely used as dental and orthopedic implant,while its corrosion resistance and osteogenic properties need to be further improved.Ti–Zr alloy is considered as one of promising titanium alloy with good corrosion resistance and biocompatibility.In this work,zirconium ions are implanted into the titanium surface by plasma immersion ion implantation (PIII)technology to construct Ti–Zr alloy layer on titanium surface with different proportions of Ti/Zr.The mechanical property,corrosion resistance and cytocompatibility of modified titanium were studied.Results indicated that Ti–Zr alloy layer on titanium exhibited good mechanical property and corrosion resistance.It could promote the cell spreading,proliferation,extracellular matrix (ECM) mineralization,and collagen secretion of rBMSCs.Besides,the zirconium ions released from different Ti–Zr alloy layers could promote the expressions of osteogenesis related genes,including alkaline phosphatase (ALP),type I collagen (Col-I),osteocalcin (OCN),osteopontin (OPN),and runt-related transcription factor 2(Runx2).The study provides reference for the design of Ti–Zr alloys and the surface modification of titanium.

1.Introduction

Titanium and its alloys have been widely used as medical devices due to their good biocompatibility,corrosion resistance,and low elastic modulus [1].Among the various titanium alloys,Ti–6Al–4V and nitinol have been widely used owning to their good mechanical properties[2,3].However,it has been reported that the gradual release of Al,Ni and V from Ti–6Al–4V and nitinol may cause some long-term health problems such as hypersensitivity andinflammatory responses [4–7].Therefore,it has been a continuing interest in exploring new non-toxic titanium alloys.

Zirconium (Zr) is a non-toxic element and usually applied to improve the mechanical and corrosion properties of Ti-based materials [8–13].Zr and Ti belong to the same main group,they possess similar chemical properties and could form a continuous solution according to the Ti–Zr binary phase diagram [14].Recent studies have shown that Ti–Zr alloys have higher corrosion resistance than pure Ti,and it increased with the increase of Zr concentration,which conferred by the combined effect of TiO2and ZrO2that jointly form the protective passive layers [15–18].What's more,Ti–Zr alloys also show better osteogenic ability.The expression of osteogenesis related genes of osteoblast on the Ti–Zr alloys surface was higher than that from pure Ti,and further studies verified their good early osseointegration in vivo[19–22].The composition of Ti–Zr alloys played an important role in regulating their biological properties,a lower concentration of zirconium ions may promote cell adhesion and alkaline phosphatase expression,while the mechanisms of composition on osteoinduction abilities are still a mystery [22].Therefore,a systematic study of the relationship between composition and biological effects of Ti–Zr alloys is the key to reveal its osteogenic mechanism,which is of great significance for the construction of medical Ti–Zr alloys with better biological properties.

At present,most of the biological researches on the effect of composition in Ti–Zr alloys are based on monolithic alloying samples,which has a great impact on the mechanical properties of materials [19–22].Some studies have shown that doping of zirconium could increase the hardness and elastic modulus of Ti–Zr alloys.The mismatched modulus of elasticity may lead to the “stress shielding” phenomenon [23–25].The plasma surface alloying technology may be a solution to this problem,and it has been used to construct Ti–Zr alloy layers on titanium surface,the elastic modulus of the substrate material will not be affected while the surface alloy composition is changed[26,27].Plasma immersion ion implantation(PIII) is a kind of plasma surface alloying technology [28,29].It has the advantages of full-scale implantation,surface reaction,high reactivity of the implanted component,and no distinct interface existing between the substrate and the modified layer [30,31].

In this work,zirconium ions are implanted into the titanium surface by PIII technology to construct Ti–Zr alloy layers with different proportions of Ti/Zr.The effects of Ti–Zr alloys composition on corrosion resistance,mechanical property and osteogenic activity in vitro were investigated systematically.

2.Materials and methods

2.1.Sample preparation

Pure titanium plates(10 mm×10 mm×1 mm)were preprocessed twice in a mixed acid solution (volume ratio of HF:HNO3:H2O was equal to 1:5:4) for 10 min each time,followed by ultrasonic cleaning with ultrapure water.The samples were placed in a target table of PIII vacuum chamber,the vacuum was pulled below 5×10-3Pa,and then the zirconium target (purity ＞ 99.99%) was used to conduct zirconium ion implantation.The detail parameters are displayed in Table 1,and the samples treated by Zr ion implantation for 30,60,and 90 min,were represented by Zr-30,Zr-60,and Zr-90,respectively.

Table 1 Parameters of Zr plasma immersion ion implantation.

2.2.Surface characterization

Surface morphologies of samples were observed with field-emission scanning electron microscope (FE-SEM;S-4800,Hitachi,Japan),the acceleration voltage is 15 kV,and the samples were sprayed with platinum.The elemental types and chemical states were determined by Xray photoelectron spectroscopy (XPS;Physical electronics PHI-5802,PHI,USA).The gamma ray source was a magnesium target Kα series(1253.6 eV);the voltage was 14.0 kV;the power was 250 W;the binding energy was calibrated based on C 1s (284.6 eV).

2.3.Ions release

The Zr-30,Zr-60,and Zr-90 were soaked in 10 mL of phosphate buffered saline (PBS) at 37 °C,and four parallel samples were set for each group.An inductively coupled plasma atomic emission spectroscopy(ICPAES;JY2000-2,Horiba Jobin Yvon,France) was used to determine the amounts of released zirconium ions (1,4,7,14,and 21 days),the Zr 339.198 nm spectral line was selected as the analysis wavelength.

2.4.Surface wettability

A contact angle meter (Automatic Contact Angle Meter Model SL200B,Solon,China) was utilized to analyze the surface wettability.Specifically,2 μL of ultrapure water was dropped vertically on sample surface,and the contact angles of droplets were calculated.

2.5.Surface hardness

Due to the surface of test samples should be smooth in hardness testing,all samples were polished before plasma surface alloying treatment.A G200 nano-indenter was harnessed to measure the micro-hardness value of the test sample's surface.The surface hardness of the material was tested by using the fixed load method.The triangular diamond cone pressure head was pushed into the surface of the material with a load force of 500 mN.The loading speed was 200 μN/s,and the holding time was 10 s.Computer software simultaneously records the corresponding mechanical data,meanwhile the hardness was measured by selecting 7 points on each sample.

2.6.Electrochemical performance evaluation

The AUTOLAB 302 N electrochemical workstation was used to test the corrosion resistance of different samples.The measurement was performed by utilizing a conventional and electrochemical cell with three-electrode.The reference,counter and working electrodes were a saturated calomel electrode,a graphite rod and a test sample,respectively.The electrolyte was a 0.9% NaCl solution.The Tafel curve of each sample was measured at room temperature,and the scanning rate was 0.01 V/s.

2.7.Cytocompatibility evaluation

2.7.1.Cell culture

The rat bone mesenchymal stem cells (rBMSCs;cells were kindly provided by Cell Bank of Chinese Academy of Sciences,Shanghai,China)were used to evaluate the cytocompatibility and osteogenic ability of samples.In brief,rBMSCs were cultured at 37 °C in a 5% CO2incubator with α-minimum essential medium (α-MEM,Gibco,Thermo Fisher Scientific Inc.,USA)containing 10%fetal bovine serum(FBS,Gibco,USA)and 1% antimicrobial of penicillin and streptomycin (Gibco,USA).

2.7.2.Cell adhesion and spreading assay

To investigate initial cell adhesion and spreading,rBMSCs with a density of 5×104cells/mL were seeded on samples and cultured for 1,4,and 24 h.At each time point,the cells were fixed with 4% paraformaldehyde(PFA)solution and permeabilized with 0.1%(v/v)Triton X-100.Subsequently,the cells were stained with Rhodamine BPhalloidin (Sigma-Aldrich,USA) and 4′,6-diamidino-2-phenylindole(DAPI;Thermo Fisher Scientific Inc.,USA) in darkness for 1 h and 5 min,respectively.The corresponding fluorescent images were taken with a fluorescence microscopy (Olympus,Japan).

2.7.3.Cell proliferation and morphology

Cell proliferation of samples was determined by alamarBlue assay.Specifically,1 mL of cell suspensions with a concentration of 5×104cells/mL were seeded on various samples for 1,4,and 7 days.At each time point,0.5 mL of culture medium with 10%alamarBlue(Thermo Fisher Scientific Inc.,USA)was introduced to each sample and cultured for another 2 h.Finally,the fluorescence intensity of the reduced alamarBlue in culture medium was examined with the extinction wavelength at 560 nm and emission wavelength at 590 nm.For cell morphology observation,after culturing for 1,4,and 7 days,cells were fixed with 2.5%glutaraldehyde for 4 h and then dehydrated in a series of ethanol solution,followed by drying in the series of hexamethyl disilazane ethanol solution.Finally,the samples were observed with a scanning electron microscope(SEM;S-3400,Hitachi,Japan) with an accelerating voltage of 15 kV.

2.7.4.Alkaline phosphatase (ALP) activity assay

ALP activity assay was performed on samples.Briefly,rBMSCs with the concentrations of 1×104cells/mL and 0.5×104cells/mL were seeded on the samples and cultured for 7 and 14 days,respectively.Then,the cells were fixed with 4%PFA,followed by staining using the AS-MX phosphate,fast blue RR salt and Mayer's Hematoxylin solution.For quantitative assay of ALP activity,the cells seeded on various samples were cultured with pnitrophenyl phosphate at 37°C for 30 min and corresponding ALP activity was detected by measuring the optical density (OD) at 405 nm.At last,ALP activity levels were normalized against the total protein contents which were analyzed by a BCA protein assay kit.

2.7.5.Extracellular matrix (ECM) mineralization

ECM mineralization was determined with alizarin red staining.Specifically,rBMSCs with the concentrations of 1 × 104cells/mL and 0.5 × 104cells/mL were seeded on samples and incubated for 7 and 14 days,respectively.Then,the cells were fixed with 75% ethanol,stained with alizarin red(40 mM)and observed with fluorescent microscopy.For quantitative assay,the stain was eluted with 10%cetylpyridinium chloride and the corresponding OD value of eluent was detected at 620 nm.

2.7.6.Collagen secretion

Collagen secretion was determined with 0.1% Sirius Red solution.Specifically,rBMSCs with the concentrations of 1 × 104cells/mL and 0.5×104cells/mL were seeded on samples and incubated for 7 and 14 days,respectively.Then,the cells were fixed with 4% paraformaldehyde,stained with 0.1%Sirius Red solution and then rinsed with 0.1 M acetic acid.In terms of quantitative analysis,the stained cells on samples were dissolved by an elution (0.2 M NaOH:methanol=1:1) and the absorbance at 540 nm of the obtained solution was measured.

2.7.7.Quantitative real-time PCR (RT-PCR) analysis

The osteogenesis-related genes expressions of rBMSCs on various samples were evaluated using RT-PCR.To be specific,rBMSCs with the cell density of 1×105cells/mL and 5×104cells/mL were seeded into 6-well plates for 7 days and 14 days respectively.At the end of incubation,the cells were rinsed with PBS twice and the total RNA was extracted with TRIzolTMreagent(Thermo Fisher Scientific Inc.,USA).Afterwards,1.0 μg of the aforementioned RNA was reverse transcribed into complementary DNA(cDNA)by Transcriptor First Strand cDNA Synthesis Kit for RT-PCR.Subsequently,RT-PCR was carried out on the Roche LightCycler480 system using an SYBR Green I Master.Expressions of the osteogenesisrelated genes contained ALP,type I collagen (Col-I),osteocalcin (OCN),runt-related transcription factor 2(Runx2),bone morphogenetic protein-2(BMP2) and osteopontin (OPN).The relative expressions of target genes were normalized to that of reference gene GAPDH and the primers for RTPCR are listed in Table 2.

3.Results and discussion

3.1.Surface characterization

Fig.1 shows the surface morphologies of Ti,Zr-30,Zr-60,and Zr-90.The surface of titanium possessed a gully-shaped structure caused by mixed acid processing.However,as shown in Fig.1a,the surface of titanium became smooth after Zr ion implantation.It may be resulted from the long-term bombardment of Zr ions beam in high-voltage field acceleration.Moreover,there was no obvious new structure appeared on Zr-30,Zr-60,and Zr-90,indicating the zirconium ions were dissolved into titanium substrate [10].

The surface chemical compositions of Ti,Zr-30,Zr-60,and Zr-90,XPS are shown in Fig.1(b-e) and Table 3.According to Table 3,Ti,O,and Zr elements could be detected from Zr-30,Zr-60,and Zr-90,which proves that zirconium was successfully implanted into titanium surface.Exactly,the content of zirconium increased with the increase of implanted time.The relative zirconium atomic percentages of Zr-30,Zr-60,and Zr-90 were about 1.09 at.%,1.77 at.%,and 3.68 at.%,respectively.In comparison with the XPS spectrum of Ti,characteristic peaks of Zr 3d5/2 and Zr 3p3/2 were detected from Zr-30,Zr-60,and Zr-90 as showed in Fig.1b.In the high-resolution spectra of Zr 3d,peaks at 182.11 eV and 184.51 eV corresponding to the typical binding energy of Zr 3d5/2 and Zr 3d3/2 statesin ZrO2can be detected from Zr-30(Fig.1c).Similarly,peaks at positions 182.10 eV and 184.40 eV corresponding to characteristic peaks of Zr 3d5/2 and Zr 3d3/2 states in ZrO2were also found from the Zr-60 surface(Fig.1d).And peaks at positions 182.01 eV and 184.51 eV corresponding to characteristic peaks of Zr 3d5/2 and Zr 3d3/2 states in ZrO2were also found from the Zr-90 surface (Fig.1e),it indicated that ZrO2had been formed on the surface of Zr-PIII treated samples after exposing to an ambient atmosphere[32].

Table 2 Primers for RT-PCR.

Table 3 Atomic concentration of various samples (%).

3.2.Surface physicochemical properties

The wettability of the sample surface could affect the biological behavior of the sample.Water contact angles of different samples are exhibited in Fig.2a.The contact angles of Zr-30,Zr-60,and Zr-90 were all slightly lower than pure titanium,but there was no significant difference among them.Ti–Zr alloys have better wettability than pure titanium,which leads to their different biological properties [18,33].

Fig.2b exhibits the Tafel curves of samples in physiological saline.The corrosion potential (Ecorr) of Zr-30,Zr-60,and Zr-90 showed a shifting to positive direction as compared to that of Ti.This shift suggested the positive influence of Zr ions implantation on the corrosion resistance of Ti.In comparison with Ti,Zr-30,Zr-60,and Zr-90 exhibited reduced corrosion current (Icorr) values,and it reduced with the increase of zirconium content.The decrease in Icorrand increase in Ecorrindicated the increased corrosion resistance of Ti–Zr alloy layers compared to Ti [9].

Fig.2c depicts the hardness value variation versus depth acquired from various surfaces.The hardness values of Zr-30,Zr-60,and Zr-90 are significantly larger than that of Ti.At a depth of 30 nm,the average hardness of all samples reached a peak and then gradually decreased and became stable.The average hardness values of Ti,Zr-30,Zr-60,and Zr-90 surfaces reached the maximum value of 5.31 GPa,7.14 GPa,7.65 GPa and 9.93 GPa,respectively.The results indicate that the hardness of titanium was improved via Zr ion implantation,and it show positive correlation with the implanted Zr content.

Ions release behaviors of Zr-30,Zr-60,and Zr-90 are presented in Fig.2d.During our observation for 1,4,7,14,and 21 days,all samples had a small release rate of zirconium ions.Zr-30 has a weaker corrosion resistance compared with Zr-60 and Zr-90,resulting in its highest ion release amounts all the time.The ion release amount of Zr-60 was a little bit higher than Zr-90.Zr-90 maintained a lower zirconium release due to its stronger corrosion resistance.

3.3.Cell adhesion and spreading

Fig.1.(a) Surface morphology of various samples;(b) XPS full spectra of various samples;Zr 3d high resolution spectra of (c) Zr-30,(d) Zr-60,and (e) Zr-90.

The interaction between materials and cells begins with the spread and adhesion of cells on material surface.The initial cell adhesion and spreading of rBMSCs on various samples are shown in Fig.3.After culturing for 1 h and 4 h,rBMSCs on Ti–Zr alloy layers presented larger spread area than that on Ti,while there was no significant difference among Zr-30,Zr-60,and Zr-90.After culturing for 24 h,rBMSCs on all the sample surfaces had no significant difference and showed a polygonal shape with lots of filopodia and lamellipodia.The wettability could be a major parameter in regulating the adhesion and spreading of cells on the samples and better wettability usually supports better cell adhesion[18,34].In this study,Ti–Zr alloy layer with better wettability were formed on the Zr-PIII treated samples,leading to the better cells adhesion on them compared to Ti.

3.4.Cell viability

Fig.2.(a)Contact angles of various sample surfaces;(b)Tafel curves of different samples in physiological saline;(c)Hardness curves detected by nano-indentation tests for various samples;(d) Ions release behaviors of Zr-30,Zr-60,and Zr-90 after immersing in PBS for 1,4,7,14,and 21 days.

Fig.3.Fluorescent images of rBMSCs cultured on various sample surfaces for 1,4,and 24 h with actin stained with Rhodamine Phalloidin and cell nucleus stained with DAPI.The scale bar is 25 μm.

Better cell adhesion usually results in higher cell proliferation.In this study,alamarBlue assay was performed to investigate the cell proliferation on various samples after culturing for 1,4,and 7 days,and the fluorescence intensity of reduced alamarBlue presented the cell proliferation levels.As presented in Fig.4a,the cell proliferation showed no significant difference among Ti,Zr-30,Zr-60,and Zr-90 after culturing for 1 day.After culturing for 4 days,cells on all Zr-PIII treated samples showed higher cell proliferation rate than that on Ti,and it increases with the increase of zirconium content.After culturing for 7 days,cells on Zr-30 exhibited the highest cell proliferation rate.Cell morphologies on various samples were observed by SEM,and the corresponding results are shown in Fig.4b.For 1 day,rBMSCs spread and grew well on all the sample surfaces.For 4 days,Zr-30,Zr-60,and Zr-90 were almost covered by cells and the spread area are larger than that on Ti.For 7 days,all the sample surfaces were fully covered by cells and multiple layers of cells can be observed on various samples.These results are consistent with the quantitative results (Fig.4a),suggesting that Ti–Zr alloy layers could promote cell proliferation of rBMSCs in the early stage,especially for Zr-30.Such proliferation promotion effect of Ti–Zr alloy layers may be related to the released zirconium ions.Zirconium ions can promote human osteoblasts proliferation by up-regulating the BMP2 expression and activation of BMP signaling [35].In this study,the Zr-30 showed the most amount of released zirconium ions,resulting the best proliferative effect (Fig.2d).

Fig.4.(a) Fluorescent intensity of alamarblue reduced by rBMSCs cultured on sample surfaces for 1,4,and 7 days,＊p ＜0.05,＊＊p ＜0.01,＊＊＊p ＜0.001;(b) SEM morphologies of rBMSCs cultured on sample surfaces for 1,4,and 7 days.The scale bar is 50 μm.

3.5.In vitro osteogenic activity

The ALP is a marker for time-dependent early cell differentiation.The ALP activities of rBMSCs on Ti,Zr-30,Zr-60,and Zr-90 were investigated after culturing for 7 and 14 days.The quantitative analysis results are presented in Fig.5a.After culturing for 7 days,the relative ALP activities of rBMSCs on Ti were larger than that on Zr-30,Zr-60,and Zr-90.However,after culturing for 14 days,ALP activities of cells cultured on all samples had no significant difference.The results of ALP-positive areas are displayed in Fig.5b.The ALP-positive area on Ti was larger than that on Ti–Zr alloy layers after 7 days and it had no significant difference between all of samples after 14 days,it is consistent with the quantitative results.

ECM mineralization of rBMSCs on various samples was determined by alizarin red staining and the results are shown in Fig.6.After culturing for 7 days,the results showed that ECM mineralization levels of rBMSCs on Ti–Zr alloy layers were greatly up-regulated compared to that on Ti,while there was no significant difference among Zr-30,Zr-60,and Zr-90.After culturing for 14 days,there was no significant difference among all of samples.Results of staining images of ECM mineralization are shown in Fig.6b,we could also find that rBMSCs cultured for 7 days on Zr-30,Zr-60,and Zr-90 had the larger ECMpositive areas than those on Ti and there was no significant difference between all of samples culturing for 14 days.

Collagen secretion of rBMSCs cultured on various samples and the corresponding quantitative analysis were displayed in Fig.7.The quantitative analysis results in Fig.7a presented that the collagen secretion of rBMSCs on Ti–Zr alloy layers was significantly enhanced than that on Ti.After culturing for 7 days,there was no significant difference among Zr-30,Zr-60,and Zr-90.After culturing for 14 days,there was no significant difference among all samples.Results of staining images of collagen secretion are shown in Fig.7b,we could also find that the rBMSCs cultured for 7 days on Zr-30,Zr-60,and Zr-90 had the larger collagen secretion-positive areas than those on Ti and there was no significant difference between all of samples culturing for 14 days.

Fig.5.(a) Relative activity of ALP on various samples culturing for 7 and 14 days;(b)ALP positive areas of rBMSCs cultured on various samples for 7 and 14 days.The scale bar is 200 μm ＊p ＜0.05.

Fig.6.(a)Quantitative results of extracellular matrix mineralization on various samples culturing for 7 and 14 days;(b) extracellular matrix mineralization positive areas of rBMSCs cultured on various samples for 7 and 14 days.The scale bar is 200 μm ＊p ＜0.05.

The expression of ALP in cells by Ti–Zr alloy is related to the content of zirconium,but there is no obvious effect in the early stage[22,33].In this work,the lower zirconium content of Ti–Zr alloy layers may not promote the expression of ALP.From the above results,it can be seen that the Ti–Zr alloy layers had a significant influence on the ALP activity,the secretion of ECM and collagen in the early stage,but the influence became negligible in the later stage,compared with Ti.Besides,Zr-30,Zr-60,and Zr-90 showed no significant difference in cell osteogenic differentiation all the time.This may be resulted from the low zirconium content of all the Zr-PIII treated samples,which is not enough to reflect the biological role of zirconium ions,and the cell differentiation behavior is not sensitive enough to small changes in zirconium content in Ti–Zr alloy layers.

The expressions of osteogenesis related genes containing ALP,Col-I,OPN,Runx2,OCN,and BMP2 of rBMSCs cultured on various samples are shown in Fig.8.Ti–Zr alloy layers presented significant higher expression of ALP than that on Ti all the time,and the expressions of ALP increased first and then decreased with the increase of zirconium content.The rBMSCs on Zr-30 and Zr-60 exhibited similar gene expression amounts and were higher than that on Zr-90.The OCN,Col-I,and Runx2 expressions on various samples showed a similar pattern with ALP.Nevertheless,the expression of BMP2 and OPN presented obvious different trends.For the expressions of BMP2,cells on Zr-30 presented the most amounts of gene expressions after culturing for 7 days,but cells on Ti exhibited the highest BMP2 expression when culture time prolong to 14 days.With regard to OPN,no obvious difference could be found in the expression amounts among cells cultured on various samples after 7 days,but 14 days later,cells on Ti–Zr alloy layers showed much higher OPN expression than that on Ti.

Fig.7.(a) Quantitative results of collagen secretion on various samples culturing for 7 and 14 days;(b) collagen secretion positive areas of rBMSCs cultured on various samples for 7 and 14 days.The scale bar is 200 μm＊＊p ＜0.01.

It is known that zirconium ions could promote the expressions of osteogenesis-related genes such as ALP,OCN,OPN,BMP2 and Runx2[35].While the cell gene expression and protein expression may sometimes be inconsistent [36].In this work,cell gene expression appears to be more sensitive to the changes with the zirconium content in Ti–Zr alloy layers.The release of zirconium ions from Ti–Zr alloy layers may results in the expression of osteogenic genes.Zr-30 and Zr-60 have higher ion release than Zr-90(Fig.2d),therefore,the osteogenic gene expressions of rBMSCs on Zr-30 and Zr-60 are larger than that on Zr-90.Besides,the cell gene expression on Zr-90 is sometimes lower than that on pure titanium,which indicates that the release of zirconium ions may not be the only factor affecting cell gene expression,and changes in surface structure may also have an effect.The surface structure of materials,especially the nanostructure,is an essential factor in regulating cell behaviors [34,37,38].Titanium has gully-shaped structure formed by mixed acid processing,which may be more conducive to the expression of osteogenesis-related genes.After Zr-PIII,the titanium surface became smooth and the gully-shaped structure disappeared,which may be the cause of reduced gene expression.For Zr-30 and Zr-60,although their smooth structure is not conducive to osteogenic differentiation,their higher zirconium ion release in early stage lead to higher cell gene expression than pure titanium.From what has been discussed above,the cell gene expression in cells may be affected by both surface structure and the release of zirconium ions.

4.Conclusion

Fig.8.Real-time PCR analysis of(a)ALP,(b)Col-I,(c)OPN,(d)Runx2,(e)OCN,and(f)BMP2 expressions by rBMSCs on various samples after incubation for 7 days and 14 days.＊p ＜0.05,＊＊＊p ＜0.001.

In this work,zirconium ions were implanted into the titanium surface by PIII technology to construct Ti–Zr alloy layers with different proportion of Ti/Zr on pure titanium.Zirconium ion implantation made the surface of titanium smoother and the hydrophilicity of the Ti–Zr alloy layers were slightly higher than that of pure titanium.The surface hardness and corrosion resistance of titanium were improved by Zr-PIII modification,and which showed positive correlation with the zirconium content of Ti–Zr alloy layer.In addition,the Ti–Zr alloy layers exhibited good biocompatibility and could promote cell proliferation,spreading,ECM mineralization and collagen secretion of rBMSCs.The various Ti–Zr alloy layers exhibited different release behaviors of zirconium ions,which was controlled by corrosion rate of Ti–Zr alloy layers.The zirconium ions released from Ti–Zr alloy layers could promote the expressions of osteogenesis-related genes containing ALP,Col-I,OPN,Runx2 and OCN.Compared with other samples,Zr-60(1.77 at.%)has appropriate physicochemical and biological properties.Owing to the titanium-zirconium surface alloying,titanium surface with enhanced mechanical and osteogenic properties were obtained by Zr-PIII.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

Financial support from the National Natural Science Foundation of China(51831011,31670980),National Science Fund for Distinguished Young Scholars of China (51525207),Science and Technology Commission of Shanghai Municipality (19JC1415500,18YF1426900,17441904000) are acknowledged.