Histological and three-dimensional evaluation of

Acta Biomaterialia xxx (2011) xxx–xxx

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Acta Biomaterialia

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Histological and three-dimensional evaluation of osseointegrationto nanostructured calcium phosphate-coated implants

Ryo Jimbo a,b,⇑, Paulo G. Coelho c, Stefan Vandeweghe a, Humberto Osvaldo Schwartz-Filho a,d,Mariko Hayashi a, Daisuke Ono e, Martin Andersson f, Ann Wennerberg a,b

a Surface Biology Group, Department of Prosthodontics, Faculty of Odontology, Malmö University, Swedenb Department of Biomaterials, Sahlgrenska Academy, University of Gothenburg, Swedenc Department of Biomaterials and Biomimetics, New York University, NY, USAd Division of Periodontology, Department of Oral Diagnosis and Surgery, School of Dentistry, UNESP, São Paulo State University, Araraquara, SP, Brazile Department of Applied Prosthodontics, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japanf Department of Chemical and Biological Engineering, Applied Surface Chemistry, Chalmers University of Technology, Gothenburg, Sweden

a r t i c l e i n f o

Article history:Received 29 May 2011Received in revised form 8 July 2011Accepted 13 July 2011Available online xxxx

Keywords:Nano structuresMicro-CTSurface chemistryBone implant interactions

1742-7061/$ - see front matter � 2011 Acta Materialdoi:10.1016/j.actbio.2011.07.017

⇑ Corresponding author at: Department of ProsthodMalmö University, 205 06 Malmö, Sweden. Tel.: +46 48503.

E-mail address: [email protected] (R. Jimbo).

Please cite this article in press as: Jimbo R etphate-coated implants. Acta Biomater (2011),

a b s t r a c t

Nanostructures on implant surfaces have been shown to enhance osseointegration; however, commonlyused evaluation techniques are probably not sufficiently sensitive to fully determine the effects ofthis process. This study aimed to observe the osseointegration properties of nanostructuredcalcium phosphate (CaP)-coated implants, by using a combination of three-dimensional imaging andconventional histology. Titanium implants were coated with stable CaP nanoparticles using animmersion technique followed by heat treatment. Uncoated implants were used as the control. Aftertopographical and chemical characterizations, implants were inserted into the rabbit femur. After 2and 4 weeks, the samples were retrieved for micro-computed tomography and histomorphometricevaluation. Scanning electron microscopy evaluation indicated that the implant surface was modifiedat the nanoscale by CaP to obtain surface textured with rod-shaped structures. Relative to the control,the bone-to-implant contact for the CaP-coated implant was significantly higher at 4 weeks after theimplant surgery. Further, corresponding 3-D images showed active bone formation surrounding theimplant. 3-D quantification and 2-D histology demonstrated statistical correlation; moreover, 3-Dquantification indicated a statistical decrease in bone density in the non-coated control implant groupbetween 2 and 4 weeks after the surgery. The application of 3-D evaluation further clarified the temporalcharacteristics and biological reaction of implants in bone.

� 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Recent developments in the surface properties of osseointegratedimplants have significantly enhanced both the quality and rateof osseointegration. Current advances enable modifications ofimplants at the macro, micro, as well as nano levels, and severalcommercially available implants possess modifications on all theselevels [1]. Recent studies have reported that the application ofnanostructures to implant surfaces seems to further increase boneapposition [2–4]. Possible explanations for this phenomenon arewidely sought, since it has been suggested that it may be due tothe cumulative effect of various factors. From a topographicalperspective, both in vitro and animal studies have suggested that

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ontics, Faculty of Odontology,0 665 8679; fax: +46 40 665

al. Histological and three-dimdoi:10.1016/j.actbio.2011.07.0

the enhanced surface area seems to create an optimal basis forbone responses. Lamers et al. [5] reported that osteoblastssensitively reacted to nanogrooves (width: 50 nm, diameter:17 nm), resulting in osteogenic gene expression, and suggestedthat nanogrooves on implant surfaces enhanced the bone response.In another study, Puckett et al. [6] reported reduced bacterialattachment on nano-scale rough surfaces than on other surfaces;furthermore, the same nano-scale surface showed higher affinityto fibronectin, which is essential for the initial osseointegrationprocess [7]. Meirelles et al. [8] conducted an animal study withimplants containing nanostructures and polished implantsdeliberately lacking them, and found that the former had higherbone-to-implant contact than the latter implant surfaces. Thesereports suggest that cells, particularly osteoblasts, respond totopographical alterations at the nanometer scale.

In addition to topography, the physical and chemical propertiesof the deposited nano-size materials could enhance theosseointegration cascade. Studies have reported the beneficial

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ensional evaluation of osseointegration to nanostructured calcium phos-17

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2 R. Jimbo et al. / Acta Biomaterialia xxx (2011) xxx–xxx

influences of physical properties such as the wettability of thenanostructured surface [9–11] and this hydrophilicity is one ofthe factors speculated to be closely involved with plasma proteinsand osteogenic cells [12,13].

Nanometer-size calcium phosphate (CaP) coating has drawnconsiderable attention for its chemical composition, as it mimicsthe structure and chemical composition of the surrounding bone.Moreover, the possibility of chemical binding of implants to thebone is of great interest, since reports have indicated that implantsurfaces incorporated with ions such as Ca, Mg, P and Si exhibitsignificant plasma protein adsorption and strong bonding to thebone [14–19]. Biomimetic modification is one of the current trendsin biomaterial development, where the material is preferably‘‘bioactive’’ rather than ‘‘bioinert’’ [20–22].

In this study, we focused on rod-shaped nanometer CaP mate-rial. This unique topography has been reported to accelerate bonemetabolism [23–27]. In our previous study on this nano-CaP coat-ing, we reported enhanced osseointegration to the implant surfaceand higher bioactivity in comparison to implants whose surfaceslacked CaP coating [3].

Although histological evaluations carried out in several studieshave proved that nanometer length scale modification effectivelyenhanced osseointegration, some other studies did not detect theeffects of nano-scale modification; thus, further understanding ofthese delicate alterations would be worthwhile [28,29]. Therefore,we conducted a three-dimensional (3-D) evaluation using micro-computed tomography (micro-CT) in order to investigate theunique surface modifications at the nano-scale. In this study, weaimed to complement 2-D histomorphometry and histologicalobservation with 3-D imaging and quantification in order toobtain further information of bone formation in nanostructuredCaP-coated implants.

2. Materials and methods

2.1. Implant surface preparation and characterization

Thirty-six turned commercially pure titanium implants 8 mm inlength and 3.3 mm in diameter were used (Grade 4, Elos MedtechPinol, Denmark). Half of these implants (test) were coated withnano-sized CaP according to the Promimic HAnano method; detailedinformation regarding the chemical composition can be foundelsewhere [3,30]. In brief, the implants were dipped into a stableparticle suspension containing CaP particles (diameter: 10 nm)followed by heat treatment at 550 �C for 5 min in nitrogenatmosphere. A nanoparticle suspension is considered stable whensedimentation or precipitation of the particles is prevented. Thiswas achieved by electrostatic nanoparticle stabilization at pH 9.Furthermore, surfactants were added to maintain the suspensionstable through steric stabilization. The uncoated implants (control)were subjected to the same heat treatment as the coated testimplants.

2.2. Scanning electron microscopy

The surfaces of the implants were examined by scanningelectron microscopy (SEM) with a LEO Ultra 55 FEG instrument(Zeiss, Oberkochen, Germany) at an accelerating voltage of 5 kV.Three implants from each group were investigated.

2.3. Interferometry

The topography of the implants was characterized using aninterferometer (MicroXam; ADE Phase Shift Technology, Inc.,Tucson, AZ). We randomly selected three implants per group for

Please cite this article in press as: Jimbo R et al. Histological and three-dimphate-coated implants. Acta Biomater (2011), doi:10.1016/j.actbio.2011.07.0

the analysis. Each implant was measured at nine positions (threetop areas, three thread valleys and three flank areas). Theparametric calculation was performed after form errors andwaviness were removed with a 50 lm � 50 lm Gaussian filter. Thefollowing 3-D parameters were selected: Sa (lm) = the arithmeticaverage height deviation from a mean plane, Sds (lm�2) = the densityof summits, and Sdr (%) = the developed surface ratio.

2.4. Animals, implantation and sample preparation

Eighteen lop-eared rabbits (mean body weight 3.9 kg) wereused in this study. Two implants were inserted in each rabbit:one test and one control implant into the left and right femurs,respectively. This planned animal study was approved by theMalmö/Lund regional animal ethics committee (approval no.M282-09).

Before surgery, the rabbits’ hind legs were shaved anddisinfected with 70% ethanol and 70% chlorohexidine. The animalswere anaesthetized by intramuscular injection of a mixture of0.15 ml kg�1 medetomidine (1 mg ml�1 Dormitor; Orion Pharma,Sollentuna, Sweden) and 0.35 ml kg�1 ketamine hydrochloride(50 mg ml�1 Ketalar; Pfizer AB, Sollentuna, Sweden). Lidocainehydrochloride (Xylocaine; AstraZeneca AB, Södertälje, Sweden)was administered as the local anaesthetic at each insertion site ata dose of 1 ml. The implants were inserted using a W&H implantunit (Elcomed, W&H SA310; Burmoos, Austria) at a rotation speedof 20 rpm.

Postoperatively, buprenorphine hydrochloride (0.5 ml Temgesic;Reckitt Benckiser, Slough, UK) was administered as an analgesicfor 3 days.

2.5. Micro-computed tomography and 3-D reconstruction

At 2 and 4 weeks postoperatively, the rabbits were killed withan overdose (60 mg ml�1) of sodium pentobarbital (ApoteksbolagetAB, Stockholm, Sweden). Samples were retrieved and placed in 4%formaldehyde for 24 h, after which they were placed in 70%ethanol. The 3-D bone formations surrounding the implants wereexamined using micro-CT (lCT 40; Scanco Medical, Bassersdorf,Germany) at a slice resolution of 36 lm. A total of 500 micro-CTslices for each bone–implant sample were imaged at an X-rayenergy level of 55 kVp, and a current of 145 lA. The integrationtime was 200 ms with a total scanning time of 36.3 min (128 mA).

Data were exported as DICOM files and reconstructed withcomputer software (MRIcro, Atlanta, USA) for further analyses.First, the 3-D images were oriented and cropped to a rectangularshape that fitted the implant and the surrounding bone. Theimages were then divided into three regions of interest: implant,prosthetic screw-hole and bone.

Thereafter, the images were exported to another softwareprogram (3D Slicer v. 3.6, BSD-style; www.slicer.org) to calculatethe volume of the different sections. The percentage of 3-D bonesurrounding the implant was calculated from the followingformula: (bone volume � 100)/(total volume � implant volume �screw-hole volume).

2.6. Ground section preparation and histological analysis

After the micro-CT analysis, all the samples were processed forundecalcified ground sectioning. In brief, after a series of dehydra-tions and infiltrations in resin, the samples were embedded inlight-curing resin (Technovit 7200 VLC; Heraeus Kulzer Wehrheim,Germany). Thereafter, one central undecalcified cut and groundsection was prepared from each implant by using Exakt sawingand grinding equipment. The sections were ground to a final


http://www.slicer.org


R. Jimbo et al. / Acta Biomaterialia xxx (2011) xxx–xxx 3

thickness of approximately 40 lm and stained with toluidine blueand pyronin.

Histological evaluations were performed using a light micro-scope (Eclipse ME600; Nikon, Japan), and the histomorphometricaldata were analyzed by image analysis software (Image J v. 1.43u;National Institutes of Health). The bone–implant contact (BIC)percentage along the entire implant was calculated at �10objective magnification, and the same amount of bone area definedin the 3-D analysis was calculated.

2.7. Statistical analysis

Statistical analyses were performed using KaleidaGraph soft-ware (Synergy Software; Essex Junction, VT, USA) and SPSS (SPSSInc., Chicago, IL, USA) software. The mean values of surface rough-ness were compared by one-way ANOVA, followed by a post hocTukey–Kramer test with the value of statistical significance set at0.01. The non-parametric Wilcoxon signed-rank test was used forbilaterally inserted implants with the significance level set at 0.01.

3. Results

3.1. Implant surface characterisation

SEM micrographs of pure titanium (control) and CaP-coatedtitanium (test) implants are presented in Fig. 1. At high magnifica-tion, it is evident that the test implant surface (Fig. 1b) was fullycovered with rod-shaped CaP particles approximately 10–15 nmwide and 100–200 nm in length.

Fig. 2a and b present the 3-D optical interferometry images of thecontrol and test surfaces, respectively. The mean Sa value (SD) was1.02 (0.15) and 0.94 (0.12) for the control and test implants,respectively. The mean Sds (SD) was 134806.23 (6982.94) and139358.82 (9366.51) for the control and test implants, respectively.The mean Sdr percentage was 29.85 (5.88) and 27.44 (8.01) for thecontrol and test implants, respectively. There were no significantdifferences between the compared parameters.

Fig. 1. Scanning electron microscopy images (bars: 500 nm) of (a) non-coated (control) ashaped structures. A magnified image (bar: 200 nm) shows further details of the coatin

Fig. 2. Interferometry images of (a) the control and (b) the tes


3.2. Histomorphometry

The postoperative course was uneventful, and there were noclinical signs of infection. All the implants were already immobi-lized by the time the animals were killed.

The histological sections presented newly formed trabeculaewith deeply stained mineralized tissue for both groups after 2and 4 weeks of healing (Fig. 3a–d).

Both cortical and newly formed bone was in close contact withthe implant surface at both time points.

The mean BIC (SD) values for the control and test groups at2 weeks were 24.7% (8.4) and 30.2% (10.4), respectively. Therewere no significant differences between the two groups at thistime point (P = 0.25). At 4 weeks after the surgery, the BIC (SD) ofcontrol and test groups was 24.3% (7.2) and 33.44 (7.94), respec-tively. The BIC was significantly higher in the test implants thanin the control implants at 4 weeks (P = 0.0078, Fig. 3e).

The mean bone area (SD) at 2 weeks was 37.21% (3.58) and32.92% (8.38) for the control and test groups, respectively, and thatat 4 weeks was 30.72% (10.14) and 33.21% (11.28), respectively.There were no significant differences in the mean bone areabetween the test and control between 2 and 4 weeks (control,P = 0.4375 and test, P = 0.5469). In addition, no differences werefound in the test and control implants between 2 and 4 weeks(P = 0.153 and P = 0.87, Fig. 3f).

From the histological observation, there seemed to be lesstrabecular bone and scattered bone formation around the controlimplants after 4 weeks compared to the test implants; however,this histological observation could not be confirmed byhistomorphometry.

3.3. 3-D imaging and 3-D quantification

Fig. 4a–d presents representative 3-D reconstructed imageswith a focus on the interfacial bone formation on the implants inthe control and test groups at 2 and 4 weeks. From the image, sig-nificant bone formation can be observed at the implant interface in

nd (b) CaP-coated (test) implant surfaces. The test surface is fully covered with rod-g.

t implant surfaces. Measurement area: 200 lm � 260 lm.



Fig. 3. Histological observations of (a) control 2 weeks, (b) test 2 weeks, (c) control 4 weeks, (d) test 4 weeks (toluidine blue and pyronin staining; original magnification �20,bars: 100 lm). (e) Bone-to-implant contact (BIC, %) for control and test after 2 and 4 weeks of healing (⁄P = 0.0078). (f) Bone area (%) for control and test implants after 2 and4 weeks of healing.


the test group at 4 weeks; these results are in accordance with theBIC measurements.

The mean 3-D bone density (SD) for the control and test groupsat 2 weeks was 38.89% (5.59) and 33.88% (10.25), respectively, and30.38% (4.79) and 30.25% (6.27) at 4 weeks, respectively (Fig. 4e).No significant differences between the test and control groupswere observed at both 2 and 4 weeks (P = 0.085 and P = 0.965).However, there was a significant decrease in the 3-D bone densityof the control implants at 4 weeks (P = 0.007), while the bone den-sity of the test implants did not significantly change between 2 and4 weeks (P = 0.491).

Additionally, there was no significant difference between thebone area determined by histomorphometry and bone densitydetermined by micro-CT (P = 0.820), and there was a significantcorrelation coefficient value of 0.350 (P = 0.05).

4. Discussion

Our results showed that the rod-shaped CaP-coated implantssignificantly influenced osseointegration. Evaluation of thetopography evaluation by inteferometry revealed no significantdifferences, indicating that the topography was only altered at


the nanometer scale below the possible detection level (theanalysis affords a lateral resolution of 0.3 lm and verticalresolution of 0.05 nm). Since the coating is a monolayer withapproximately 10–15 nm developed crystal structures, wecharacterized its structural characteristics by high-resolution SEM.

The rod-shaped crystal structure of CaP has been reported toenhance the osteogenic activity surrounding it. Ono et al. [27]reported that CaP blocks with rod-shaped structures enhanced thebone metabolism cycle, exhibiting higher levels of osteoclastactivity and progressive bone formation. In their study, increasedTRAP-positive multinucleated cells existed on the surface of theCaP blocks, and this process was accompanied by cuboidal activeosteoblasts with potent alkaline phosphatase (ALP) activity in thenewly formed bone. Okuda et al. [25] demonstrated that rod-shapedCaP showed superior bone formation as compared to globular CaPstructures. It is speculated that cells or their signalling pathwaysrespond to the rod-shaped structure by upregulating and balancingthe osteoclastic and osteoblastic activity for enhanced boneformation.

In the current study, the rod-shaped CaP-coated implantpresented significantly higher bone-to-implant contact than thecontrol at 4 weeks. Although no significant differences could be



Fig. 4. Descriptive 3-D reconstructed image from the micro-CT evaluation for (a) control 2 weeks, (b) test 2 weeks, (c) control 4 weeks, (d) test 4 weeks. (e) Corresponding 3-Dbone density quantification (%) for control and test implants after 2 and 4 weeks of healing. No significant differences were seen for the test and control at both time points,however, the control group showed a significant decrease from 2 to 4 weeks (⁄P = 0.007).

R. Jimbo et al. / Acta Biomaterialia xxx (2011) xxx–xxx 5

observed, the mean values were higher for 2 weeks and active boneapposition to the implant surface appears to have continued in thecase of the test implant, whereas that of the control implantreached a plateau.

The bone area measurements and the 3-D bone density evalua-tion did not differ between the non-coated and CaP-coated implantgroups. However, the bone density in the control implantssignificantly reduced over time, although this difference was onlysignificant when evaluated using micro-CT. Although a high rateof bone formation at an earlier time point that reduces over timeis a part of the biological process due to periosteal reaction[31,32], and the bone reduction is compensated by mineralizedbone formation, i.e. maturation, 2-D histology may not havecaptured the actual process due to its limited scope of evaluation,since it may depend on where the section was taken from. On theother hand, micro-CT is advantageous in that it enables evaluationof the total circumferential space, while in histomorphometry theevaluation is limited to one or a few 2-D slices. Therefore, the3-D evaluation is a reliable method and is more accurate thanhistological analysis, since the evaluation includes all the acquireddata. However, one must keep in mind that it is important tounderstand the biological responses by combining informationfrom 2-D histomorphometry and from 3-D evaluation. Since thereexist certain biological events that cannot be observed bymicro-CT, e.g. cell alignment, cell shape, localization of proteinsor enzyme detection, the biological information that can beobtained by 2-D histology can never be neglected. For example,the enzyme histochemical findings of Ono et al. [27] would neverhave been possible without histology, and it would have beendifficult to interpret the results without this technique. It must beemphasized that the intention of the current study was not toexplore for a possible alternative to 2-D histology, but to possiblycomplement and further clarify biology from a different perspective.


Three-dimensional evaluation with micro-CT has received con-siderable attention in recent years because it facilitates the obser-vation and quantification of bone formation surrounding implantsor bone substitutes in a 3-D plane [33–35]. Although the amountof bone surrounding the implant did not differ across differentimplant treatments, bone formation at the implant interfacewas found to be higher (i.e. direct bone contact to the implant)in the test implants at 4 weeks. Along with image reconstruction,it was easy to observe the state of trabecular bone formation tothe implant surface, which provides additional information fromboth qualitative and quantitative perspectives. However, in thisstudy, quantifying the amount of BIC three-dimensionally wasconsidered a challenge due to metallic halation artifacts and thepotentially significant effect of these on the evaluation [36].Although the quantification may still be possible by the applica-tion of various filtering techniques [37], it remains to be knownwhether the actual bone or artifact has been fully differentiated.In order to improve the technique and obtain clearer results,determination of the optimal conditions such as power voltageor the focus size to control the X-ray absorption is necessary atthe time of scanning; moreover, optimal software that can effi-ciently filter out artifacts during the reconstruction is necessary.Hence, in future studies, we intend to focus on improving thenovel 3-D evaluation technique to further clarify the effect ofvarious surface modifications on osseointegration.

5. Conclusion

Implants coated with rod-shaped nanostructured CaP showedenhanced osseointegration compared to non-coated implants.Three-dimensional analysis using micro-CT proved to be an effec-tive method to further understand the temporal characteristics ofimplants in bone.




Acknowledgements

We gratefully acknowledge Dr. Ryuta Muratomi, Dr. Kenji Miya-hara, Dr. Kazuhiro Nakashima and Dr. Kotaro Tsuiki for the groundsection preparation. The current study was supported by theKnowledge Foundation (KK stiftelsen, Biofilms-research centrefor biointerfaces) and the Swedish Research Council (VR).

Appendix A. Figures with essential colour discrimination

Certain figures in this article, particularly Figures 2–4, are diffi-cult to interpret in black and white. The full colour images can befound in the on-line version, at doi:10.1016/j.actbio.2011.07.017.

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Histological and three-dimensional evaluation of