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Biomaterials 34 (2013) 3184e3195

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Biomaterials

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The synergistic effect of hierarchical micro/nano-topography andbioactive ions for enhanced osseointegration

Wenjie Zhang a,b,1, Guocheng Wang c,1, Yan Liu a,b, Xiaobing Zhao c,d, Duohong Zou e, Chao Zhu e,Yuqin Jin a,b, Qingfeng Huang a, Jian Sun a, Xuanyong Liu f, Xinquan Jiang a,b,c,*, Hala Zreiqat c,**aDepartment of Prosthodontics, Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University, School of Medicine, 639 Zhizaoju Road, Shanghai 200011, ChinabOral Bioengineering and Regenerative Medicine Lab, Shanghai Research Institute of Stomatology, Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University,School of Medicine, Shanghai Key Laboratory of Stomatology, 639 Zhizaoju Road, Shanghai 200011, ChinacBiomaterials and Tissue Engineering Research Unit, School of AMME, The University of Sydney, Sydney, NSW 2006, Australiad School of Materials Science and Engineering, Changzhou University, Changzhou 213164, ChinaeDepartment of Oral and Maxillofacial Surgery, Ninth People’s Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai 200011, Chinaf State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, China

a r t i c l e i n f o

Article history:Received 30 November 2012Accepted 2 January 2013Available online 4 February 2013

Keywords:Strontium-substituted hardystoniteMicro/nano-topographyStrontiumImplantsOsseointegration

* Corresponding author. Department of ProsthodonAffiliated to Shanghai Jiao Tong University, School ofShanghai 200011, China. Tel.: þ86 21 63135412; fax:** Corresponding author. Tel.: þ61 2 93512392; fax:

E-mail addresses: [email protected] (X. Jiang)(H. Zreiqat).

1 These authors contributed equally to this work.

0142-9612/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.biomaterials.2013.01.008

a b s t r a c t

Both surface chemistry and topography have significant influence on good and fast osseointegration ofbiomedical implants; the main goals in orthopeadic, dental and maxillofacial surgeries. A surfacemodification strategy encompassing the use of bioactive trace elements together with surface micron/nano-topographical modifications was employed in this study in an attempt to enhance the osseointe-gration of Ti alloy (Ti-6Al-4V), a commonly used implant. Briefly, we developed strontium-substitutedhardystonite (Sr-HT) ceramic coating with a hierarchical topography where the nanosized grains weresuperimposed in the micron-rough coating structure. Its ability to induce new bone formation wasevaluated by an in vivo animal model (beagle dogs). Hardystonite (HT), classic hydroxyapatite (HAp)coated and uncoated Ti-alloy implants were parallelly investigated for comparison. In addition, weinvestigated the effects of surface topography and the dissolution products from the coatings on thein vitro bioactivity using canine bone marrow mesenchymal stem cells (BMMSCs) cultured on theimplant surface as well as using extracts of the coated implants. Micro-CT evaluation, histological ob-servations, biomechanical test (push-out test) and sequential fluorescent labeling and histomorpho-metrical analysis consistently demonstrated that our developed Sr-HT-coated Ti-alloy implants have thehighest osseointegration, while the uncoated implants had the lowest. The osseointegration ability ofHAp-coated Ti alloy was inferior to that seen for HT- and Sr-HT-coated Ti alloy. We demonstrated that thedissolution products, particularly strontium (Sr) from the Sr-HT-coated implants, enhanced the ALP ac-tivity and in vitro mineralization ability, while the micro/nano-topography was more related to thepromotion of cell adhesion. Those results suggest that our developed Sr-HT coatings have the potentialfor future use as coatings for orthopedic/dental and maxillofacial devices.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Successful integration of orthopedic implants with host bone,not only needs initial stability supported by enough bone stock but

tics, Ninth People’s HospitalMedicine, 639 Zhizaoju Road,þ86 21 63136856.þ61 2 93517060., [email protected]

All rights reserved.

rapid osseointegration. The term “osseointegration” was firstintroduced by Branemark to describe the modality for stable fixa-tion of titanium (Ti) to bone tissue [1,3]. Ti alloy is well-establishedbiomaterial in dental and orthopedic applications due to its goodmechanical properties, biocompatibility and anti-corrosion prop-erty. However, Ti alloy cannot achieve sufficient osseointegrationdue to its suboptimal surface osteoconductivity, thus resulting inthe increased aseptic loosening and subsequent premature failureof the implants. In order to enhance the osseointegration andincrease the successful rate of Ti-alloy implants, various surfacemodification methods have been utilized. And the incorporation ofbioactive trace elements such as calcium (Ca), silicon (Si), zinc (Zn)

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Table 1Plasma spraying parameters for fabrication of HAp, HT and Sr-HT coatings.

Coatings Ar(slpm)

H2(slpm)

Sprayingdistance (mm)

I (A) U (V) Vacuum(mbar)

HAp 40 8 280 550 57 100HT 40 12 100 570 75 N/ASr-HT 40 12 100 570 75 N/A

W. Zhang et al. / Biomaterials 34 (2013) 3184e3195 3185

and strontium (Sr) is the most commonly used and useful approach[4,5].

Ca and Si ions, both of which are essential elements for humanbody, have been proved to promote osteoblast proliferation anddifferentiation [6,7]. They have been utilized to chemically modifybiomaterials for enhanced bioactivity. For example, Ca ion-implantation can alter the surface chemistry of Ti and in turnmodulate the progression of bone cell cycle and enhance theresponse of bone cells to implants [8,9]; Si ions have been widelyused to modify hydroxyapatite (HAp), one of the most popularbiomaterials for bone replacement and regeneration. More rapidremodeling of bone was observed surrounding the Si incorporatedHAp (Si-HAp) granules compared to pure HAp granules [10]. Cal-cium silicates including CaSiO3 and Ca2SiO4 are the most typicalceramic materials capable of releasing Ca and Si ions and theirpotential for use in bone replacement and regeneration applica-tions have been demonstrated in vitro and in vivo [11e13]. However,high degradation rate of CaSiO3 can result in high pH value in thesurrounding environment which is detrimental to cell viability [14].Moreover, if used as coatings for orthopeadic applications, the fastdegradation rate of the CaSiO3 coatings will increase the failure rateof the implants, where high chemical stability is required, leadingto loosening of the interface. These drawbacks were addressed byincorporating ZnO into CaSiO3 resulting in the development ofhardystonite ceramic (Ca2ZnSi2O7, HT) with better chemical sta-bility and bioactivity [15,16].

Using plasma spray techniques, we recently coated nano-structured HTceramics ontoTi-6Al-4V and demonstrated enhancedbioactivity of the coated Ti-6Al-4V [17]. Zn ion was found to inhibitosteoclastic activity and promote bone mineralization [18]. Itsincorporation endowed the new nanostructured ceramic coatings(HT) with an ability to release Zn ions into the surrounding envi-ronment while simultaneously enhancing the chemical stability ofthe coating due to the decreased release rate of Ca, Si from thecoating. Sr is another important trace element found in humanbone and its beneficial effect on bone formation has been welldocumented [19]. Both in vitro and in vivo studies have shown thatSr ions stimulate bone formation and decrease bone resorption[20,21]. We previously demonstrated that the incorporation of Srinto CaSiO3 ceramics improved the physical and biological prop-erties of the pure CaSiO3 ceramics [22,23] and induced in vitro boneformation with enhanced bioactivity and osseointegration prop-erties in vivo [23]. In this study, we used (Ca0.8Sr0.2)2ZnSi2O7 (Sr-HT)ceramic to develop new coatings for commonly used orthopedic/dental implants by substitution of Sr for 20% Ca in HT, with a hy-pothesis that Sr incorporation would further enhance the osteo-conductivity of our previously developed HT coatings.

Topographical modification is another important approach toimprove biological performance of dental and orthopedic implants.The beneficial effects of microscale roughness on the in vitroosteoblast activity and in vivo osteoconductivity have been provedby many studies [24]. Cells in vivo live in a three-dimensional (3D)extracellular matrix (ECM) which is composed of not only micro-scale topographical features but also various nanoscale ones.Others in the field have demonstrated the effects of nanosizedtopographic features on promotion of osteoblast adhesion [25,26]as well as the synergistic effects of micro and nanoscale hybridstructures on osteoblast activity [27e29], implying the promise ofdual-length scale topographical modification of the implants.Considering the significant importance of both micro and nanotopographical features, our developed Sr-HT ceramic coating aswell as the HT coating was designed to have a hybrid micro/nanoscale structure where nanosized grains (less than 100 nm insize) were superimposed in microscale rough structures usingplasma spraying technique.

In this study, the in vivo osseointegration of Sr-HT- and HT-coated Ti-6Al-4V implants was evaluated using a canine femurimplantation model. Pure Ti alloy and HAp-coated Ti-6Al-4V im-plants, currently used in orthopedic applications, were used ascontrols for comparison. Twelve weeks after implant insertion, newbone formation around implants in the bone marrow cavity wasevaluated using Micro-CT, histological and push-out test. In addi-tion, sequential fluorescent labeling was used to compare the newbone formation rate of each group. To validate the beneficial effectsof hybrid micro/nanoscale structures and bioactive trace elements,in vitro experiments were also performed using canine BMMSCs.

2. Materials and methods

2.1. Material synthesis and coating preparation

Sr-HT and HT powders were synthesized by high temperature solid reaction.Briefly, reagent grade CaCO3, ZnO and SiO2 powders were mixed in a 2:1:2 M pro-portions for synthesizing HT ceramic powders; for synthesis of Sr-HT powders,SrCO3 was used to replace 20% (M) CaCO3. The mixed powders were wet-ground inabsolute ethanol for 6 h, and dried at 100 �C, followed by sintering at 1200 �C for 3 h.Then, the as-sintered powders were ground and sieved, powders passing througha sieve with a mesh size of 75 mm were reconstituted using PVA solution to makepowders more flowable for plasma spraying. The procedure for reconstitutingpowders can be found in our previous paper [17]. The reconstituted powders werefinally sieved using 80-mesh sieves. Those below 80-mesh (

W. Zhang et al. / Biomaterials 34 (2013) 3184e31953186

every 2e3 days. When cells reached 80e90% confluence, cells were subcultured.Cells at passage 2e4 were used in the following in vitro experiments.

2.4. Cell seeding on different coatings

Canine BMMSCs were seeded on titanium plates at a density of 5 � 104 cells/ml,cultured with DMEM. For initial adhesion activity assay, samples were washed withPBS for three times after 4 h culture. Remaining cells were fixed in 4% paraf-ormaldehyde for 30 min at 4 �C. Cells were permeabilized with 1% Triton X-100 for30 min and blocked in 10% goat serum for 1 h at room temperature. And then,specific primary antibody targeting integrin b1 (Abcam, USA) was added at 1:400dilutions and co-incubated overnight at 4 �C. DyLight 549-conjugated anti-mouseIgG antibody (Invitrogen, USA), at 1:500 dilutions in blocking buffer, was used for1 h at 37 �C in the dark. Cytoskeleton was stained with FITC-Phalloidin and cellularnuclei were counterstained with DAPI. The specimens were observed using the LeicaCLSM (Leica, Germany).

For osteocalcin (OCN) expression detection, the plates were cultured in DMEMfor 14 days. After washing with PBS for two times, cells were fixed in 4% paraf-ormaldehyde for 30 min at 4 �C, and then treated with 1% Triton X-100 for 30 minand blocked with 10% goat serum for another 1 h at room temperature. Sampleswere incubated in rat specific primary antibodies of OCN (Abcam, USA) at 1:200dilutions overnight at 4 �C. Then samples were incubated with DyLight 549-conjugated anti-mouse IgG antibody (Invitrogen, USA) for 1 h at 37 �C. Cytoskele-ton was stained with FITC-Phalloidin and cellular nuclei were counterstained withDAPI. All specimens were observed using CLSM.

After 14 days of culture, total RNA were extracted with Trizol reagent (Invi-trogen) and cDNA were generated using PrimeScript 1st Strand cDNA Synthesis kit(TaKaRa, Japan). The expression of adhesion-related gene integrin b1 andosteogenic-related gene bone morphogenic protein-2 (BMP-2), alkaline phosphates(ALP) and OCNwere measured using the Bio-Rad Quantitative Real time PCR system(qRT-PCR; Bio-Rad, MyiQ�, USA). Housekeeping gene GAPDH was used for nor-malization and the Ti alloy group was used as control. Specific prime sequences usedin this study were listed in Supplementary Table S1.

2.5. The dissolution products from the coated Ti-6Al-4V implants and their effects onBMMSCs

Ti-6Al-4V rods with different coatings were placed in 15 ml tubes with 0.5 mlDMEM culture medium, and incubated at 37 �C in a humidified atmosphere of 95%air and 5% CO2, The extracts were collected and refreshed with DMEM every 3 days.After 14 days of incubation, the accumulated ion concentrations of Ca, Si, Zn and Srwere measured using an inductively coupled plasma/optical emission spectroscopy(ICP-OES; Varian, USA) [31].

The collected extracts were supplemented with 10% FBS for the following cellculture experiments.

2.5.1. Alkaline phosphates (ALP) activity assayBMMSCs were seeded in 24-well plates at a density of 5.0 � 104 cells/ml in

DMEM [32]. After 4 h of incubation, DMEM was replaced with the extracts of thecoated Ti-6Al-4V implants. After further incubation of 14 days, the BMMSCs of eachgroup were fixed and stained with ALP kit (Beyotime, China). For ALP quantitativeassay, the cells were incubated with p-nitrophenyl phosphate (pNPP) (Sigma) at37 �C for 30 min. ALP activity was measured by testing optical density (OD) values at405 nm. Total protein content was calculated using the Bio-Rad protein assay kit(Bio-Rad, USA) and normalized with a series of BSA (Sigma) standard at 630 nm. ALPactivity was expressed as the OD values at 405 nm per milligram of total proteins.

2.5.2. Calcium deposition assayAfter 14 days of culture in the extracts, BMMSCs were fixed in ice cold 70%

ethanol for 30 min and stained with 40 mM Alizarin Red S for another 15 min [33].Finally, the cells were washed with PBS for five times and observed with opticalmicroscope. For the quantitative assay, the stained wells were eluted using a solu-tion of 10% cetylpyridinium chloride (Sigma) and the OD values were measured at590 nm. Result was normalized and expressed as absorbance OD values at 590 nmper milligram of total proteins.

2.6. In vivo osseointegration evaluation

2.6.1. Surgical proceduresA total of 12 adult male beagle dogs, aged 12e18 months old, were used in this

study. The animal procedures were approved by the Animal Care and ExperimentCommittee of Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University,School of Medicine. The surgical procedures were conducted as previously described[34]. Briefly, under general anesthesia via intramuscular injection of ketamine(10 mg/kg), the mid-shaft of left femur was exposed through a lateral longitudinalskin incision. Four holes with a diameter of 3.2 mm, spaced about 10mm apart, weremade on the shaft of canine femur, as shown in Supplementary Fig. S1a, b. Four kindsof implants were randomly inserted into the four holes (Supplementary Fig. S1c),and then the surgical wound was closed carefully.

2.6.2. Sequential fluorescent labelingThe process of new bone formation and mineralization was assessed using

a polychrome sequential fluorescent labeling method. Different fluorochromes wereadministered intraperitoneally at a sequence of 30 mg/kg Alizarin Red S (Sigma,USA), 25mg/kg Tetracycline Hydrochloride (Sigma) and 20mg/kg Calcein (Sigma) at3, 6 and 9 weeks after the operation, respectively.

2.6.3. Sample preparationAll animals were sacrificed at 12 weeks post-operation. Left femurs with four

groups of implants were harvested and trimmed into smaller ones. Randomlyselected 6 femurs were stored at �80 �C for push-out test. The other 6 specimenswere fixed in 10% buffered formaldehyde (10%) for Micro-CT assay and histo-morphometric observation.

2.6.4. Micro-CT assayThe fixed samples were detected and imaged using Micro-CT (GE eXplore Locus

SP Micro-CT, USA) to determine the newly formed bone around implants. Thescanning parameters were set at 80 kV and 80 mAwith an exposure time of 3000 msand the resolution of 15 mm. After scanning, three-dimensional (3D) images werereconstructed with GEHC MicroView software (GE Healthcare BioSciences, ChalfontSt. Giles, UK). Regions of titanium implants, cortical bone and new formed can-cellous bone were separated and labeled with different colors on the 3D images. Toquantify the new bone formed around the implant, a region with a radius of 1 mmfrom implant surface, within bone marrow cavity, was selected for analysis. Bonemineral density (BMD), bone volume fraction (Bone volume/total volume, BV/TV),trabecular thickness (Tb.Th) and trabecular number (Tb.N) of new bone were ana-lyzed as previously described [35].

2.6.5. Push-out testThe biomechanical test was performed using a universal material testing system

(Instron, High Wycombe, UK). A special custom designed holder was prepared to fixat the test samples to ensure that the test force is along the long axis of implant(Supplementary Fig. S1d) and the specimens were trimmed to fit into it. All testswere performed at a loading rate of 5 mm/min [36]. The load-displacement curvewas recorded during the pushing period. The failure load was defined as the peakload values of the load-displacement curves.

2.6.6. Histomorphometric observationAfter detected by Micro-CT, 6 femur specimens were dehydrated with a graded

series of alcohols from 75% to absolute ethanol, and finally embedded in poly-methylmetacrylate (PMMA) [37]. The embedded specimens were sectioned into150 mm thick sections using a Leica SP1600 saw microtome (Leica, Hamburg, Ger-many). Those sections were subsequently ground and polished to a final thickness ofabout 40 mm for fluorescence labeling observation under confocal laser scanningmicroscope (CLSM, Leica). Excitation/emission wavelengths of chelating fluoro-chromes were used 543/617 nm, 405/580 nm and 488/517 nm for Alizarin Red S(red), Tetracyclin Hydrochloride (yellow) and Calcein (green), respectively.

2.7. Statistical analysis

The data were presented as mean � standard deviation. Statistical comparisonswere carried out via one-way ANOVA and SNK post hoc based on the normal dis-tribution and equal variance assumption test. All statistical analysis was carried outusing an SAS 8.2 statistical software package (Cary, USA). The differences wereconsidered statistically significant at +p < 0.05 and ++p < 0.01.

3. Results

3.1. Crystalline phase of coatings

The XRD pattern of the plasma sprayed Sr-HT and HT coatings isshown in Fig. 1. Sr-HT and HT coatings have similar patterns whereall peaks can be indexed to hardystonite (Ca2ZnSi2O7, PDF No. 39-0235), indicating that the addition of Sr did not lead to the for-mation of new crystal phases. In addition to the sharp peaks, a glassbulge, as indicated by the rectangular frame, is found near thestrongest peaks in both patterns, indicating that both crystallinephase and amorphous phase exist in the Sr-HT and HT coatings.

3.2. Surface topography of plasma sprayed coatings

The surface morphology of Sr-HT and HT coatings was shown inFig. 2. Both Sr-HT and HT coatings have rough surfaces, as shown inFig. 2b, c. The roughness of Sr-HT and HT coatings were

Fig. 1. XRD patterns of the as-sintered HT and Sr-HT coatings.


7.16 � 0.37 mm and 7.69 � 0.49 mm, respectively, while the rough-ness of Ti-6Al-4V and HAp coatings (control groups) were1.40 � 0.63 mm and 5.15 � 0.33 mm, respectively (SupplementaryFig. S2). Under high magnification, nano grains with size less than100 nm can be observed well-imposed in the micron-rough

Fig. 2. SEM photographs of the surface morphology of th

structure, thus forming a nano/micron hierarchical structure. Theformation of the nanostructure on the coating is caused byrecrystallization of the molten powders during the rapid solidifi-cation after plasma spraying [17].

3.3. The thickness and bonding strength of coatings

The cross-sectional of the coating samples is shown in Fig. 3a.The average thickness of each type of coating is in the range of 15e18 mm without significant difference among groups (Fig. 3b). Thebonding strength of each coating with Ti-6Al-4V substrates isshown in Fig. 3c. Compared to HT, Sr-HT coatings have higherbonding strength, and both are higher than the reported values ofpure plasma sprayed HAp coatings without interlayer and post-treatments [38,39].

3.4. The effects of coatings on canine BMMSCs in vitro

In order to qualitatively determine the influence of differentcoatings on BMMSCs adhesion activity, the expression of integrinb1 was detected by cell immunofluorescence staining (Fig. 4). At24 h of culture, red fluorescence stained integrin b1 in BMMSCscultured on HAp coatings was less abundant than that for cellscultured on Sr-HT and HT coatings, while no significant differencecan be observed between cells cultured on the Sr-HTcoating and HT

e as-sprayed HAp (a), HT (b) and Sr-HT (c) coatings.

Fig. 3. Cross-sectional SEM image (a), thickness (b) and bonding strength (c) of HAp (control group), HT and Sr-HT coatings. (+, represents p < 0.05).


coating. After 14 days of incubation on different samples, the pro-tein expression levels of OCN were measured using cellularimmunofluorescence. As shown in Fig. 5, the expression of OCN onSr-HT and HT coatings was stronger than that on the HAp material.And Sr-HT group displayed the strongest expression of OCN.

Fig. 4. The expression of integrin b1 was detected by immunofluorescence assay. “Merge 1”merged images of actin, integrin b1 and nuclei together with the plain confocal laser micr

The expression of adhesion-related gene integrin b1 andosteogenic-related markers for BMP-2, ALP and OCN in cells afterincubation on different coatings for 14 days were detected byquantitative real time polymerase chain reaction (qRT-PCR). Resultswere normalized to GAPDH and expressed as relative expression

represent the merged images of actin, integrin b1 and nuclei. “Merge 2” represent theoscope image.

Fig. 5. The expression of OCN was detected by immunofluorescence assay. “Merge” represent the merged images of actin, OCN and nuclei.


levels to the uncoated Ti-6Al-4V group (Fig. 6). All three coatinggroups promoted the expression of the genes tested. For theadhesion-related gene integrin b1, its expression was significantlyenhanced by BMMSCs cultured on the Sr-HT and HT groups, com-pared to HAp coating, while no significant difference was observedbetween Sr-HTand HTgroups. The expression levels for BMP-2, ALPand OCN in BMMSCs cultured on Sr-HT coatings were highest,followed by HT coatings, both of which were higher than those onHAp coatings. These results indicate that our developed Sr-HT aswell as the HTcoatings not only promoted the adhesion but also thedifferentiation of BMMSCs. Sr-HT was superior to HT in inducingBMMSCs differentiation, but not significantly different in promot-ing BMMSC adhesion.

3.5. The dissolution products from coatings and their effects onBMMSCs

The accumulated amount of Ca, Si, Zn and Sr ions released fromthe coated Ti-6Al-4V after 14 days incubation in cell culture me-dium is displayed in Fig. 7. Calcium (Ca) ions are the only ionsdetected in the extracts from the uncoated and the HAp coating. Siand Zn ions, as expected, were also detected in the extracts of theSr-HT- and HT-coated implants with Sr ions only present in theextracts of the Sr-HT group.

To understand the influence of these released ions on the bio-activity of those coatings, canine BMMSCs were incubated in theextracts for 14 days, and ALP activity and calcium deposition abilitywere measured. As shown in Fig. 8a, ALP-positive area of BMMSCscultured in the extracts from Sr-HT- and HT-coated groups wassignificantly higher than that for the HAp-coated and the uncoatedTi-6Al-4V (p < 0.01, Fig. 8b). Moreover, ALP-positive area for the Sr-HT group was significantly higher than that for the HT group(p < 0.05, Fig. 8b), indicating enhanced ALP activity on the Sr-HTcoating. Similarly, the area of bone nodules formation measuredusing Alizarin Red S staining was the highest for the Sr-HT group

(Fig. 8c), followed by that for HTgroup, while nomineralizationwasevident on the uncoated and the HAp-coated Ti-6Al-4V groups.Quantitative analysis demonstrated significantly higher minerali-zation ability for Sr-HT group (Fig. 8d), than that of Ti alloy group(p < 0.01), HAp- (p < 0.01) and HT-coated groups (p < 0.05).

3.6. In vivo osseointegration of the ceramic coatings

3.6.1. Micro-CT evaluation of in vivo bone formationFig. 9 shows reconstructed Micro-CT images of transverse sec-

tions along the central axis of Ti-alloy implants. The implants weremarked with green color, cortical bone with white color and can-cellous bone with yellow color, respectively (Fig. 9). Bone volumearound implants surface in the bone marrow cavity of the Sr-HTgroup is obviously higher than that for the other groups. BMD,BV/TV, Tb.Th and Tb.N were significantly higher on Sr-HT and HTgroups, compared to those for HAp-coated groups (p < 0.01) withthose of the Sr-HT group being significantly higher than for the HTgroups (p < 0.01) (summarized in Table 2).

3.6.2. Histological observationsHistological stained sections stained with Van Gieson’s picro

fuchsin staining are shown in Fig. 10a. In the cortico-cancellous site,a close apposition of bone to the implant was seenwith the ceramiccoatings. Only small amount of new bone formed around the un-coated Ti-6Al-4V implant within the cortico-cancellous bone.Obvious space between the newly formed bone and the implantsurface can be observed at the interface of bone and Ti-6Al-4Vwhile HAp coatings promoted new bone formation around theimplant, demonstrating enhanced osseointegration. Consistentwith the Micro-CT results, the new bone area and boneeimplantcontact (BIC) for the Sr-HT and HT groups were significantlyhigher than those seen with the HAp group (Fig. 10b, c). Remark-ably, the percent of new bone area around Sr-HT-coated implantswithin the bone marrow cavity was 55.17 � 9.82%, significantly

Fig. 6. The expression of (a) integrin b, (b) BMP-2, (c) ALP and (d) OCN was measured by real time PCR after incubating on different coatings for 14 days.


higher than that of HT-coated implants (40.61 � 5.71%, p < 0.05). Asimilar tendency was also observed in BIC index among the groups.Sr-HT-coated Ti-6Al-4V had the greatest BIC index of 51.20� 9.08%,significantly higher than those of the HT (36.97 � 8.72%, p < 0.05)and HAp (27.72 � 5.48%, p < 0.01) groups.

Fig. 7. Concentrations of (a) Ca, (b) Si, (c) Zn and

3.6.3. Biomechanical push-out testBiomechanical push-out test was used to evaluate the quality

of osseointegration of the implants. As shown in Fig. 11, among thefour investigated groups, Sr-HT group showed the highest failureload (388.84 � 100.51 N) while the uncoated Ti-6Al-4V group had

(d) Sr ions released from different coatings.

Fig. 8. (a) ALP staining of BMSCs after incubating in different extracts for 14 days. (b) ALP activity was measured by colorimetrically quantitative analysis at 405 nm. (c) Alizarin RedS staining of BMSCs after incubating in different extracts for 14 days. (d) Calcium deposition activity was assayed using colorimetrically quantitative analysis at 590 nm. (Forinterpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)


the lowest (45.70 � 21.93 N, p < 0.01). In addition, the load tofailure of HAp group was 160.90 � 42.59 N, which was higher thanthat of Ti-6Al-4V group but less than that of the HT group(229.08 � 58.95 N, p < 0.05).

Fig. 9. Micro-CT images of the transverse sections of cani

3.6.4. Sequential fluorescent labeling histomorphometrical analysisAs shown in Fig. 12e, rectangle region adjacent to the implant

surface, within the bone marrow cavity, was imaged to evaluatednew bone formation rate. New bone formation and mineralization

ne femur with implant 12 weeks after implantation.

Table 2Quantitative results of the micro-CT evaluation.

Parameters Groups

Ti HAp HT Sr-HT

BMD(mgHA/cm3)

23.2 � 8.9 187.1 � 39.4a 353.9 � 43.7a,b 482.8 � 58.9a,b,c

BV/TV (%) 2.9 � 1.6 13.5 � 3.4a 27.0 � 6.2a,b 43.2 � 6.4a,b,cTb.Th (mm) 71.1 � 15.7 121.9 � 17.9a 163.1 � 26.9a,b 225.9 � 34.3a,b,cTb.N (mm�1) 0.2 � 0.1 1.1 � 0.4a 2.1 � 0.4a,b 3.7 � 0.6a,b,c

Data were expressed as mean � SD (n ¼ 6). a, p < 0.01 when compared with groupTi; b, p < 0.01 when compared with group HAp; c, p < 0.01 when compared withgroup HT.

Fig. 11. The graph shows the results of biomechanical test (+, represents p < 0.05;++, represents p < 0.01).


were recorded by three types of fluorochrome at different timepoints and results are shown in Fig. 12. Generally we demonstratedthat our developed Sr-HT- and HT-coated Ti-6Al-4V implants pro-moted new bone formation and mineralization at the boneeimplant interface, compared to the uncoated and HAp-coatedgroups, At 3 weeks, the percent of Alizarin Red S labeling area(red) for Sr-HT group (11.20 � 2.18%) was significantly higher thanthat of HT group (6.14� 1.45%, p< 0.01), both of which were higherthan those of control groups: Ti-6Al-4V group (2.05 � 0.47%,p < 0.01), HAp group (3.44 � 0.46%, p < 0.01). At 6 weeks, thepercent of Tetracycline Hydrochloride labeling areas (yellow) for Ti-6Al-4V, HAp-, HT- and Sr-HT-coated groups were 2.95 � 0.72%,6.21 � 1.38%, 8.52 � 1.86% and 13.66 � 3.40% respectively, havinga similar tendency to that observed at 3 weeks. At 9 weeks, the

Fig. 10. Histological observations and histomorphometric measurements. (a) Undecalcifiedrectangle area are displayed in the lower panel. Results of new bone area (b) and BIC (c) fromin this figure legend, the reader is referred to the web version of this article.)

percent of Calcein labeling area (green) for Ti-6Al-4V group wasonly 1.92 � 0.42%, the lowest among all four groups. By contrast,Sr-HT group demonstrated the highest Calcein labeling area(17.62� 2.14%). The CA labeling area (12.71�1.93%, p< 0.05) for HTgroup was consistently higher than that for HAp control group(9.47 � 1.94%).

sections are stained with Van Gieson’s picro fuchsin. Partial magnifications of the bluethe histomorphometric measurements. (For interpretation of the references to colour

Fig. 12. Sequential fluorescent labeling observations. (aed) Red, yellow and green represent labeling by Alizarin Red S (AL), Tetracycline Hydrochloride (TE) and Calcein (CA),respectively (bar ¼ 500 mm). (e) The image shows the red rectangle region was selected to evaluated new bone formation rate. (f) The graph shows the area of three fluorochromesstained bone. (+, represents p < 0.05; ++, represents p < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of thisarticle.)


4. Discussions

The physico-chemical characteristics of the material play animportant role in the overall bioactivity of the coated implants andplay a significant role in mediating cellular functions. Adhesionstrength between Ti-6Al-4V and the coating layer plays a criticalrole in determining the ultimate success of the implant. Plasmaspray HAp-coated Ti-6Al-4V implants are clinically used in ortho-pedic applications [40] due to their chemical similarity to theinorganic component of human bones. However, the poor bondingstrength of HAp coating to the underlying Ti-6Al-4V remains to bea limiting factor. The weak bonding strength (35 MPa) between the Sr-HT coating and theunderlying Ti-6Al-4V, compared to that reported for the HAp-coated ones. We believe that the addition of Sr into the HT ce-ramics decreased the mismatch of the thermal expansion coeffi-cient between the ceramic coating and the Ti alloy, resulting in thisimproved bonding strength.

Furthermore, when inserted in cortical and cortico-cancellousbone in an in vivo canine femur implantation model, Sr-HT-coated Ti-6Al-4V demonstrated superior osseointegration to thatseen for HT-, HAp coatings and the uncoated alloy, with firmabutments between the implant and the surrounding skeletal tis-sue. Histological analysis confirmed that new bonewas formed andfilled up the gap between the implants and bone for both Sr-HT-and HT-coated Ti-6Al-4V in direct contact with the implant,


compared to that for HAp-coated Ti-6Al-4V. There was no evidenceof any infiltration of fibrous tissue between the bone and implant asevident with the uncoated Ti-6Al-4V implants. The boneeimplantcontact and new bone volume of Sr-HT and HT coatings weresimilar but significantly higher, compared to that for HAp-coatedalloys, reflecting the ability of these coatings to induce osseointe-gration without any infiltration of fibrous tissue between the boneand implant as evident with the uncoated. The uncoated Ti-6Al-4Vimplants had a marked reduction in boneeimplant contact due tothe formation of a wide band of fibrous tissue, particularly notedin the cortico-cancellous implants.

Biomechanical tests (e.g. pull-out/push-out, torque, or tensiletest) are used to characterize the bonding strength at the boneeimplant interface. In this study, push-out test revealed that themechanical fixation of Sr-HT- and HT-coated implants was sig-nificantly stronger than that for HAp-coated implants, furtherconfirming the enhanced osseointegration seen with the Sr-HT-and HT-coated implants.

The surface for both Sr-HT and HT coatings is similar consistingof a hybrid micro/nano-topography, compared to the micro-roughstructures of the HAp-coated implants. Moreover the ion dis-solution products vary significantly between the three ceramicscoatings usedwith Sr ions being the predominant ion released fromthe Sr-HT coatings.

While it is plausible to suggest that the differences in bothtopography and dissolution products contribute to the noted su-perior bioactivity of the Sr-HT-coated Ti-6Al-4V. We believe thatthe released Sr ions from the Sr-HT ceramic coatings married withits hybrid micro/nano topographies contribute to the superiorin vivo bone formation and the improved in vitro biofunctionality.Our study validates that the topographical features of Sr-HT and HTcoatings, not only the ion dissolution products, play a critical role indetermining their biofunctionalities.

Cellebiomaterial interaction is crucial in analyzing the mecha-nisms of osseointegration. Cell adhesion is the first step of cellularinteraction with the underlying substrates. It is a key regulator ofcell proliferation, differentiation, activation and migration, whichmay determine the fate of the biological interfacial interactionwiththe implant.

Similar adhesion of BMMSCs was seen for Sr-HT and HT coatingswithbothbeing superior to that for theuncoatedandtheHAp-coatedgroup. Comparing Sr-HT-coated implant to the HT-coated one, wefound that the prominent difference between them lies in theirdissolution products. However our study confirmed that no differ-ence was observed in the BMMSCs adhesion between Sr-HT and HT-coated implants, while differences were observed in the differenti-ation of these cells on these two coated implants. Therefore webelieve that the topography, instead of the ion released is the con-tributing factor in the promotion of early stages of cellular responses.To validate our notion, we determined the effect of in vitro ionsreleased from coated implants on initial BMMSCs adhesion whencultured for 4 h on the Sr-HT-, HT, and HAP-coated Ti-6Al-4V and onthe uncoated Ti-6Al-4V. No obvious differences were observedamong groups (Supplementary Fig. S3), indicating the validity of ournotion. Synergistic effects of hybrid micro/nanoscale structures onosteoblasts adhesion have been reported by several studies [27e29].

In contrast to the in vitro adhesion results, BMMSCs differenti-ation was superior on the Sr-HT- compared to HT-coated implants,compared to the HAp-coated and the uncoated Ti-6Al-4V. Inagreement with the in vivo studies, the in vitro results confirmedthe enhanced bioactivity of Sr-HT and HT coatings as evident in theenhanced ALP activity and mineralization ability of BMMSCs, overthe HAp coating and the uncoated alloy.

Since the topography (nano/micron hierarchical structure) issimilar for both Sr-HT- and HT-coated implants, it is reasonable to

suggest that the ion dissolution products (Sr), rather than thetopography, is the main contributing factor to the enhancedBMMSCs differentiation on the Sr-HT-coated alloy, emphasizing theimportance of Sr in the osseoconductivity of biomedical implants.

To sum up, in vitro studies demonstrated that hybrid micro/nano-topography played an important role in promoting theadhesion of BMMSCs, whereas dissolution products from ourdeveloped coatings (Sr-HT and HT) are mainly involved in the in-duction of the differentiation of BMMSCs. Moreover, our data alsoemphasize the importance of Sr in the osseoconductivity of bio-medical implants. The in vivo assessment of Sr-HT-coated implantssupports our in vitro observation validating the ability of Sr-HT inpromoting osteogenic cell adhesion, differentiation suggesting thatthe Sr-HT coating supports enhanced osseointegration at theinterface of bone and implant. In total, Sr-HT and HT coatingspossess improved bonding strength and superior ability to bondwith host bone compared with HAp coatings, indicating its po-tential application as orthopedic implants.

5. Conclusion

Strontium-substituted hardystonite (Sr-HT) coatings with hybridmicro/nano-topography were developed for dental and orthopeadicimplants application in this study. The coatinghas improvedbondingat both coating/implant and coating/bone tissue interfaceswhich areof critical importance to the implants. Its fast in vivo bone formationability and in vitro functionality were ascribed to the synergistic ef-fects of the hierarchical hybrid micro/nano-topography and thedissolution products from the coating. In detail, the hybrid micro/nano-topography played a crucial role in promoting adhesion ofcanine BMMSCs on the implant surface while the dissolution prod-ucts were more involved in inducing the differentiation of theBMMSCs. Those results suggest that Sr-HThas thepotential for futureuse as an implant coating in dental and orthopeadic application.

Acknowledgments

This work was jointly supported by the National Basic ResearchProgram of China (973 Program, 2012CB933604), the NationalScience Fund for Distinguished Young Scholars of China (No.81225006), the National Natural Science Foundation of China (No.81170939, 30973342), the National Health and Medical ResearchCouncil (Australia), Rebecca Cooper Foundation (Australia) and theAustralian Orthopaedic Association. We would like to thank ‘KeyLaboratory of Inorganic Coating Materials, Shanghai Institute ofCeramics, China’ for their help in the fabrication of hydroxyapatite-coated Ti alloy samples.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biomaterials.2013.01.008.

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The synergistic effect of hierarchical micro/nano-topography and bioactive ions for enhanced osseointegration1. Introduction2. Materials and methods2.1. Material synthesis and coating preparation2.2. Characterization of coatings2.3. Canine BMMSCs culture2.4. Cell seeding on different coatings2.5. The dissolution products from the coated Ti-6Al-4V implants and their effects on BMMSCs2.5.1. Alkaline phosphates (ALP) activity assay2.5.2. Calcium deposition assay

2.6. In vivo osseointegration evaluation2.6.1. Surgical procedures2.6.2. Sequential fluorescent labeling2.6.3. Sample preparation2.6.4. Micro-CT assay2.6.5. Push-out test2.6.6. Histomorphometric observation

2.7. Statistical analysis

3. Results3.1. Crystalline phase of coatings3.2. Surface topography of plasma sprayed coatings3.3. The thickness and bonding strength of coatings3.4. The effects of coatings on canine BMMSCs in vitro3.5. The dissolution products from coatings and their effects on BMMSCs3.6. In vivo osseointegration of the ceramic coatings3.6.1. Micro-CT evaluation of in vivo bone formation3.6.2. Histological observations3.6.3. Biomechanical push-out test3.6.4. Sequential ﬂuorescent labeling histomorphometrical analysis

4. Discussions5. ConclusionAcknowledgmentsAppendix A. Supplementary dataReferences

the synergistic effect of hierarchical micro/nano-topography and ...€¦ · hardystonite (ht),...

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