in vivo gene expression in response to anodically oxidized versus machined titanium implants

In vivo gene expression in response to anodically oxidizedversus machined titanium implants

Omar Omar,1 Sara Svensson,1 Neven Zoric,2 Maria Lenneras,2 Felicia Suska,1 Stina Wigren,3

Jan Hall,3 Ulf Nannmark,4 Peter Thomsen1,5,6

1Department of Biomaterials, Sahlgrenska Academy at University of Gothenburg, Sweden2TATAA Biocenter AB, Goteborg, Sweden3Nobel Biocare AB, Goteborg, Sweden4Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine at University of Gothenburg, Sweden5Institute of Biomaterials and Cell Therapy, Goteborg, Sweden6BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Goteborg, Sweden

Received 2 September 2008; revised 5 February 2009; accepted 6 February 2009Published online 8 May 2009 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32475

Abstract: A quantitative polymerase chain reaction tech-nique (qPCR) in combination with scanning electron mi-croscopy was applied for the evaluation of early geneexpression response and cellular reactions close to tita-nium implants. Anodically oxidized and machined tita-nium miniscrews were inserted in rat tibiae. After 1, 3,and 6 days the implants were unscrewed and the sur-rounding bone was retrieved using trephines. Both theimplants and bone were analyzed with qPCR. A greateramount of cells, as indicated with higher expression of18S, was detected on the oxidized surface after 1 and 6days. Significantly higher osteocalcin (at day 6), alkalinephosphatase (at days 3 and 6), and cathepsin K (at day 3)expression was demonstrated for the oxidized surface.

Higher expression of tumor necrosis factor-a (at day 1)and interleukin-1b (at days 1 and 6) was detected on themachined surfaces. SEM revealed a higher amount of mes-enchymal-like cells on the oxidized surface. The resultsshow that the rapid recruitment of mesenchymal cells, therapid triggering of gene expression crucial for boneremodeling and the transient nature of inflammation, con-stitute biological mechanisms for osseointegration, andhigh implant stability associated with anodically oxidizedimplants. � 2009 Wiley Periodicals, Inc. J Biomed MaterRes 92A: 1552–1566, 2010

Key words: bone; gene expression; interface; osseointegra-tion; titanium; animal model

INTRODUCTION

Events leading to integration of an implant inbone, and hence determining the performance ofthe device, mainly take place at the tissue-implantinterface.1 Molecular and cellular responses towarda foreign material begin directly after insertion.2,3

Few studies have examined the bone healing andremodeling processes taking place around implants

at very early time points.4–6 However, in order toreveal the mechanisms of osseointegration, there isa need to examine the function of the interfacialcells in addition to histological observations. Severaltechniques have been used to analyze the molecularactivities of biological markers in response to tita-nium, for example ELISA7–10 and fluorescencemicroscopy,2 but due to the inaccessibility of thebone-implant interface zone as well as the insuffi-cient resolution of many techniques, the functionalperformance of the cells in vivo is not resolved. Inrecent years, PCR and qPCR have been employedto explore the genetic alterations in implant sur-rounding tissue.11–15 qPCR has the advantage ofquantifying genes through the addition of a fluores-cent probe or dye, giving the opportunity to com-pare gene expressions more accurately. The tech-nique has the ability to detect as small amounts asa single RNA-copy and represents a promisingnew tool to monitor early and decisive interfacial

Correspondence to:O. Omar; e-mail: [email protected] grant sponsor: Swedish Research Council;

contract grant number: K2006-73X-09495-16-3Contract grant sponsors: Institute of Biomaterials and

Cell Therapy (IBCT), the VINNOVA VinnVaxt ProgramBiomedical Development in Western Sweden, and VINNExcellence Center of Biomaterials and Cell Therapy

� 2009 Wiley Periodicals, Inc.

processes, which precede clinically manifest condi-tions (i.e., integration or failure).

The anodically oxidized implant (TiUnite) wasintroduced in 200016 and has been shown to enhanceosseointegration and high implant stability at earlytime points compared to the traditionally usedmachined implants.17,18 High-resolution microscopicanalysis of retrieved implants has shown a stronginterlock between the bone and the anodically oxi-dized titanium implants.19 Oxidized implants havealso shown higher bone-to-metal contact and re-moval torque values than other surfaces in experi-mental studies.20–22 However, the underlying cellularand molecular mechanisms responsible for the favor-able results seen with oxidized implants areunknown. We emphasize that the first events takingplace in response to an implant are critical for thefollowing healing and osseointegration.

The aims of the present in vivo study were as fol-lows: first, to develop a method for the analysis ofgene expression of cells around implants, using asampling procedure and subsequent qPCR; second,to determine whether gene expression denotinginflammation and bone remodeling is differentlymodulated around implants with different surfaceproperties; and third, to examine the role of distanceof cells to the implant by comparing the geneexpression of cells at the immediate vicinity of theimplant surface to that detected in the peri-implantbone. The study addressed the expression of selectedmarkers of inflammation (tumor necrosis factor-alpha (TNF-a) and interleukin-1beta (IL-1b)), boneformation (osteocalcin (OC) and alkaline phospha-tase (ALP)), and bone resorption (tartrate-resistantacid phosphatase (TRAP) and cathepsin K (CATK)).On the basis of the results, the changes in theexpression of selected growth (transforming growthfactor-beta 1 (TGF-b1), platelet derived growth fac-tor-B (PDGF-B), and bone morphogenic protein-2(BMP-2)) and transcription (runt related transcriptionfactor-2 (Runx2) and peroxisome proliferator-acti-vated receptor-gamma (PPAR-g)) factors were ana-lysed at early time points by qPCR. The morphologyof cells associated with the implant surfaces wasevaluated by histology, immunohistochemistry, andscanning electron microscopy.

MATERIALS AND METHODS

Implants

Screw-shaped titanium implants, 2 mm in diameter and2.3 mm in length, were used. Two types of surfaces wereselected: a machined surface and anodically oxidized(TiUnite) surface (Nobel Biocare, Goteborg, Sweden), withsurface roughness (Ra) of 0.3 and 1.2 lm, respectively, as

measured by light interferometry (MicroXAM Interfero-metric Profiler, ADE Phase Shift, Tucson, AZ). The testimplants were manufactured and sterilised by Nobel Bio-care, Goteborg, Sweden.

The animal model

Thirty female Spraque–Dawley rats (200–250 g), fed on astandard pellet diet and water were anesthetized using aUniventor 400 anesthesia unit (Univentor, Zejtun, Malta)under isoflurane (Isoba Vet, Schering-Plough Uxbridge,England) inhalation (4% with an air flow of 650 mL/min).Anesthesia was maintained by continuous administrationof isoflurane (2.7% with an air flow of 450 mL/min) via amask. Each rat received analgesic (Temgesic 0.03 mg/kg,Reckitt & Coleman, Hull, Great Britain) subcutaneouslyprior to the implantation, and daily postoperatively. Aftershaving and cleaning (5 mg/mL chlorhexidine in 70%ethanol) the medial aspect of the proximal tibial metaphy-sis was exposed through an anteromedial skin incision, fol-lowed by skin and periosteum reflection with blunt instru-ment. After bone preparation with Ø1.4- and Ø1.8-mmburs under profuse irrigation with NaCl 0.9%, one oxi-dized and one machined implant were inserted in eachtibia with a hexagonal screw driver. The locations ofimplants were decided using a predetermined schedule,ensuring a rotation of sites. The distances from the epiphy-sis to the center of the proximal and distal implants were4 and 8 mm, respectively. The subcutaneous layer of thewound was closed with resorbable polyglactin sutures(5–0, Vicryl, Ethicon, Johnson & Johnson, Brussels, Bel-gium) and the skin was closed with transcutaneouslyplaced nonresorbable nylon sutures (5–0, Ethilon, Ethicon,Johnson & Johnson, Brussels, Belgium). The animals wereallowed free postoperative movements with food andwater ad libitum. The retrieval procedure was done at 1, 3,and 6 days (10 rats at each time point). The rats were sac-rificed by an intraperitoneal overdose of sodium pentobar-bital (60 mg/mL; ATL Apoteket Production & Laborato-ries, Sweden) under anesthesia, with 0.5 mL mixture ofpentobarbital (60 mg/mL), sodium chloride, and diazepam(1:1:2) and cleaned with 5 mg/mL chlorhexidine in 70%ethanol. The retrieval procedure was performed accordingto RNA preserving protocol established by the researchgroup. After cutting the sutures, incision, if needed, of theskin and reflection of the periosteum were done. Becauseof the early retrieval time points, the implants were notyet strongly locked in bone, and could therefore beunscrewed with adherent biological material by a hexago-nal screw driver and placed immediately in specific pre-serving solutions depending on the subsequent analysis(qPCR or SEM). For qPCR analysis, peri-implant bone wasexplanted from the tibia, using a trephine with internal Ø2.3 mm. In addition, to verify that implants were locatedunicortically in the metaphysis and to exclude any grossdisruption of the interface in conjunction with theunscrewing, a histological sample from each time point (1,3, and 6 days; n 5 3) was prepared from additional rats.The implant was unscrewed and the site fixated in glutar-aldehyde, dehydrated and embedded in paraffin, and sec-tioned and stained with hematoxylin and eosin (H and E)

GENE EXPRESSION IN RESPONSE TO OXIDIZED VS. MACHINED TITANIUM IMPLANTS 1553

Journal of Biomedical Materials Research Part A

stain. The animal experiments were approved by theUniversity of Gothenburg Local Ethical Committee forLaboratory Animals (Dnr 306-2006).

Quantitative PCR

After 1, 3, and 6 days, unscrewed implants and thecorresponding surrounding bone were placed in RNAlatersolution (QIAGEN GmbH, Hilden, Germany) and storedat 2808C until analysis. The bone samples were homoge-nized in phenol/guanidine-based Qiazol lysis reagent,using 5-mm stainless steel beads (QIAGEN GmbH, Hilden,Germany) and TissueLyser (QIAGEN GmbH, Hilden, Ger-many). After addition of chloroform, the samples werecentrifuged at 12,000g for 15 min and the aqueous phasewas used for subsequent RNA extraction. Total RNA fromthe screw and from the surrounding bone was extractedusing RNeasy Micro kit and Mini kit (QIAGEN GmbH,Hilden, Germany), respectively, according to the manufac-turer’s instructions. DNAse treatment was performed inorder to eliminate any contamination from genomic DNA.Reverse transcription was carried out using iScript cDNASynthesis Kit (Bio-Rad, Hercules) in a 10 lL reaction,according to the manufacturer’s instructions. Design of pri-mers for OC, ALP, TRAP, CATK, TNF-a, IL-1b, TGF-b1,BMP-2, PDGF-B, Runx2, PPAR-g, and 18S was performedusing the Primer3 web-based software.23 Assays were pur-chased from TATAA Biocenter AB, Goteborg, Sweden.Design parameters were adjusted to minimize formation ofartifact products and to be able to use an annealing tem-perature in the PCR at about 608C. Primers were designedto yield short amplicons (preferably shorter than 200 bp)and to function well with SYBR Green I fluorescent dyefor detection of the PCR products in real-time. Real-timePCR was performed in duplicates, using the Mastercyclerep realplex (Eppendorf, Hamburg, Germany) in 20 lLreactions. Cycling conditions were 958C for 10 min fol-lowed by 45 cycles of 958C for 20 sec, 608C for 20 sec, and728C for 20 sec. The fluorescence was read at the end ofthe 728C step. Melting curves were recorded after the runby stepwise temperature increase (18C/5 sec) from 65 to958C. Quantities of target genes were normalized using theexpression of 18S ribosomal subunit. The normalized rela-tive quantities were calculated using the delta Ct methodand 90% PCR efficiency (k*1.9Dct).24

Scanning electron microscopy

Retrieved screws from 1, 3, and 6 days (three speci-mens/surface/time point) were fixed with a modifiedKarnovsky solution (2% paraformaldehyde, 2.5% glutaral-dehyde in 0.05M sodium cacodylate) (pH 7.4) for 4 h.Specimens were then rinsed with sodium cacodylate bufferand subsequently impregnated with a conductive, metalliclayer of osmium, using a modified osmium-thiocarbohy-drazide-osmium technique (OTOTO).25 Specimens werethen dehydrated in graded series of ethanol and driedwith hexamethyldizilasane for 2 3 5 min. Specimens weremounted on stubs by means of carbon-coated adhesivetape. In case of reduced conductivity, specimens were sub-

jected to an additional sputter coat with palladium. Allspecimens were examined in a Zeiss DSM 982 Geminiscanning electron microscope.

Histology and immunohistochemistry

For immunostaining rats were fixated after 3 days byperfusion of modified Karnovsky media (2% paraformalde-hyde, 2.5% glutaraldehyde in 0.05M sodium cacodylate)(pH 7.4) after anesthetization with 0.5 mL mixture of pen-tobarbital (60 mg/mL), sodium chloride, and diazepam(1:1:2). The implant-bone specimens were postfixated inmodified Karnovsky media for 2 h. Specimens were decal-cified in 10% EDTA for 10–12 days. The specimens werethen dehydrated in ascending series of ethanol, clearedwith xylene, and embedded in paraffin. While the paraffinwas still in melting stage, the implants were unscrewedand the embedding procedure was continued. The ideabehind removing the implant at this stage was to preservethe implant-bone interface as intact as possible. Subse-quently, 10 lm sections were produced, mounted on glassslides and stained with H and E, for light microscopic ob-servation. For the immunostaining, 4 lm sections wereproduced, mounted on polylysine slides (Menzel GmbHand Co KG, Braunschweig, Germany) and then maintainedat 608C in an oven for 1 h. The sections were deparaffi-nised and hydrated in descending series of ethanol. Theintrinsic peroxide activity was removed by reacting with3% H2O2 in distilled water. Slides were incubated with pri-mary antibodies, CD163 (sc-58965, Santa Cruz Biotechnol-ogy) and periostin (ab14041, Abcam, Cambridge, UK) for60 min at room temperature. The immunoreactivity ofCD163 labeled sections was detected and visualized usingLSAB2 System-HRP kit (K0609; DAKO, Sweden), diamino-benzidine (Victor’s kit, Immunokemi, Sweden), and coun-terstained with Mayer’s hematoxylin. Periostin stainingwas detected with PK6101 kit, diaminobenzidine (Victor’skit, Immunokemi, Sweden), and counterstained withMayer’s hematoxylin. The primary antibodies were dilutedby using 1% bovine serum albumin (Sigma A7638 fromSigma Aldrich, Sweden) in phosphate-buffered saline.Negative control slides were prepared by omission of theprimary antibody and incubation with 1% BSA in PBS.

Statistics

Analysis of the gene expression data was based on com-paring the 18S-normalized relative expression of each geneat the two surfaces. For statistical comparisons, Studentt-test was used to analyze the differences in the geneexpression levels between the two compared implanttypes, at each specific time point. One-way ANOVA fol-lowed by Dunnett T3 test was used to compare geneexpression levels between the three different time pointsfor a specific implant type. For analysis of gene expressionat the implant surface (N 5 15) and in the surrounding tis-sue (N 5 10). The level of confidence for either test wasset to 95% (i.e., p < 0.05 is significant). The data presentedin the graphs are mean 6 SEM.


1554 OMAR ET AL.

RESULTS

Implant surfaces

The machined surface had a smooth appearance,characterized by ordered grooves and ridges, due tothe manufacturing process [Fig. 1(a)]. The oxidizedsurface was characterized by a porous surface tex-ture with open pores in micrometer range [Fig. 1(b)].

Implant model

The implants were inserted unicortically in theproximal tibial metaphysis, characterized by mainlycortical bone (Fig. 2). No disruption of the interfacewas detected.

Gene expression analysis

On the basis of temporal expression at the selectedthree time points, the panel of gene markers was

divided into three groups; namely, bone formation,bone resorption and proinflammatory markers.Additional two groups of genes, one representingcellular growth factors and the other representingtranscription factors were analyzed for both surfacesat the 1 and 3 days time points.

18S ribosomal RNA expression at the screw surface

The expression of 18S ribosomal subunits is indica-tive for the total number of cells at the screw surface.Significantly higher 18S level was associated with theoxidized surface at days 1 and 6, after implantation.At day 3, the 18S expression was higher at machined

Figure 1. SEM images of the machined (a) and oxidized (b) titanium implants.

Figure 2. Decalcified paraffin-embedded and H&Estained section of tibial metaphysis after unscrewing. Nodisruption of the interface was detected and the implantwas inserted unicortically (Magnification 310).

Figure 3. The expression of 18S ribosomal subunits at theoxidized and machined surfaces. Statistically significantdifferences between the two surfaces are indicated in stars.The temporal changes were 1?3*, 3?6***, and 1?6*** foreither implant type. The differences between the twotested implants at specific time points were analyzed witht-test. One way ANOVA was used to test the differencesamong the different time points at specific implant surface.(*p < 0.05; **p < 0.005; ***p < 0.0001) n 5 15 Mean 6 SEM.



surfaces (Fig. 3). The 18S expression decreased at theoxidized surface between days 1 and 3, and there-after increased dramatically to the day 6 time point.The machined surface showed a continuous increaseof 18S expression level with implantation time.

Gene expression of bone formation markersat the screw surface

A statistically significant difference was demon-strated between oxidized and machined surfaces

when comparing the expression of bone formationgenes. In comparison with machined surfaces, theexpression of ALP at oxidized surfaces was abouttwo-fold, five-fold, and two-fold higher after 1, 3,and 6 days, respectively [Fig. 4(a), Table I]. A similarpattern was observed for the expression of OC after3 and 6 days [Fig. 4(b), Table I]. For both ALP andOC, a peak of expression was observed after 3 days,during the early phase.

Figure 4. Gene expression of bone formation markers atthe oxidized and machined surfaces. Statistically signifi-cant differences between the two surfaces are indicated instars. a: Alkaline phosphatase (ALP) gene expression. Thetemporal changes were: 1?3*, 3?6*, and 1?6* for the oxi-dized implants, whereas the temporal changes at themachined implants were not significant. b: Osteocalcin(OC) gene expression. The temporal changes were: 1?3*and 1?6*** for oxidized implants and 1?6* for machinedones. The differences between the two tested implants atspecific time points were analyzed with t-test. One wayANOVA was used to test the differences among the differ-ent time points at specific implant surface. (*p < 0.05; **p< 0.005; ***p < 0.0001) n 5 15 Mean 6 SEM.

Figure 5. Gene expression of bone resorption markers atthe oxidized and machined surfaces. Statistically signifi-cant differences between the two surfaces are indicated instars. a: Tartrate resistant acid phosphatase (TRAP) geneexpression. The temporal changes were: 1?3* and 3?6*for the oxidized, whereas the temporal changes at themachined implants were not significant. b: Cathepsin K(CATK) gene expression. The temporal changes were: 1?3 *,3?6*, and 1?6* for oxidized implants and 1?6** formachined ones. The differences between the two testedimplants at specific time points were analyzed with t-test.One way ANOVA was used to test the differences amongthe different time points at specific implant surface. (*p <0.05; **p< 0.005) n5 15 Mean6 SEM.

1556 OMAR ET AL.


Gene expression of bone resorption markersat the screw surface

Although no statistically significant differencesbetween the surfaces were found when TRAP genelevels [Fig. 5(a), Table I] were compared at any ofthe three time points, CATK expression level [Fig.5(b), Table I] was significantly higher after 3 days atthe oxidized surfaces. Similarly to bone formationmarkers, the expression level of both bone resorptionmarkers was increased from days 1 to 3 and thendecreased at the day 6.

Gene expression of proinflammatory markersat the screw surface

Higher expression levels of TNF-a (at day 1) andIL-1b (at days 1 and 6) were seen at the machinedsurfaces compared to the oxidized ones [Fig. 6(a,b),Table I]. At both implant surfaces peak TNF-amRNA was detected after 3 days of implantation. Incontrast, IL-1b had its peak expression after 1 day atthe machined surface and thereafter decreased.

Gene expression in the surrounding bone

No significant differences were seen in the expres-sion of 18S or any other gene between the surround-ing bone samples corresponding to the two implanttypes at any specific time point [Fig. 7(a–g), TableII]. Nevertheless, the temporal pattern of the osteo-genic markers was different in the surrounding bonefrom that seen at the implant surface during thetime course of implantation. 18S, OC, ALP, TRAP,

and CATK expression levels increased steadily withtime, with a peak at day 6. This temporal increasewas statistically significant for all of the markersdenoting bone formation and bone resorption sur-rounding the machined implants, and only for 18Sand OC in the bone surrounding oxidized ones. Onthe other hand, the proinflammatory marker expres-sion in the surrounding bone showed similar patternto that occurring at the implant surface. This patternwas characterized by a peak of TNF-a expression3 days after implantation and subsequent decrease

TABLE IResults Presented as the Ratio Between RNA Expressionin Cells Attached to Oxidized Implants (n 5 15) and

RNA Expression in Cells Attached to Machined Implants(n 5 15) After Normalization to 18S

1 Day 3 Days 6 Days

18S 6.980* 0.355** 1.680*ALP 1.628 5.118** 1.847**OC 0.470 4.794 2.317*TRAP 0.966 2.282 0.846CATK 1.039 3.988** 0.862TNF-a 0.565** 1.841 0.607IL-1b 0.221** 1.902 0.339**Runx2 1.83 6.086** –PPAR-g 0.39** 5.715 –BMP-2 1.06 2.918 –PDGF-B 0.602 3.27 –TGF-b 0.277** 1.76 –

Values above 1 indicate more gene expression at the oxi-dized surfaces, whereas values below 1 indicate more geneexpression at the machined surfaces.

*p < 0.05; **p < 0.005.

Figure 6. Gene expression of pro-inflammatory markersat the oxidized and machined surfaces. Statistically signifi-cant differences between the two surfaces are indicated instars. a: Tumor necrosis factor-a (TNF-a) gene expression.The temporal changes were: 1?3*, 3?6*, and 1?6*** foroxidized implants and 1?6** for machined ones. b: Inter-luekin-1b (IL-1b) gene expression. The temporal changeswere: 3?6* and 1?6** for oxidized implants and 1?6* formachined ones. The differences between the two testedimplants at specific time points were analyzed with t-test.One way ANOVA was used to test the differences amongthe different time points at specific implant surface. (*p <0.05; **p < 0.005; ***p < 0.0001) n 5 15 Mean 6 SEM.



Figure 7. Gene expression in the surrounding bone. a: The expression of 18S ribosomal subunits. The temporal changeswere: 3?6* and 1?6** for the bone surrounding the oxidized implants and: 3?6** and 1?6*** for the bone surroundingthe machined ones. b: Alkaline phosphatase (ALP) gene expression. The temporal changes were: 1?6* for the bone sur-rounding the machined implants, whereas the temporal changes in the bone surrounding the oxidized implants were notsignificant. c: Osteocalcin (OC) gene expression. The temporal changes were: 1?6** for the bone surrounding the oxidizedimplants, whereas the temporal changes in the bone surrounding the machined implants were not significant. d: Tartrateresistant acid phosphatase (TRAP) gene expression. The temporal changes were: 1?3* and 1?6* for the bone surroundingthe machined implants, whereas the temporal changes for the bone surrounding the oxidized implants were not signifi-cant. e: Cathepsin K (CATK) gene expression. The temporal changes were 1?6* for the bone surrounding the machinedimplants, whereas the temporal changes for the bone surrounding the oxidized implants were not significant. f: Tumor ne-crosis factor-a (TNF-a) gene expression. The temporal changes were: 1?3*, 3?6* for the bone surrounding the oxidizedimplants and: 1?3**, 3?6** for the bone surrounding the machined ones. g: Interleukin-1b (IL-1b) gene expression. Thetemporal changes were: 1?3**, 1?6** for the bone surrounding the oxidized implants and: 1?3*, 1?6* for the bone sur-rounding the machined ones. The differences between the two tested implants at specific time points were analyzed witht-test. One way ANOVA was used to test the differences among the different time points at specific implant surface. (*p <0.05; **p < 0.005; ***p < 0.0001) n 5 15 Mean 6 SEM.

TABLE IIResults Presented as the Ratio Between Normalized

RNA Expression in Tissue Corresponding to OxidizedImplants (n 5 10) and Normalized RNA Expression in

the Tissue Corresponding to Machined Implants (n 5 10)

1 Day 3 Days 6 Days

18S 1.145 1.102 0.728ALP 0.971 0.641 0.824OC 1.277 0.602 0.516TRAP 1.462 0.581 0.950CATK 1.621 0.720 1.018TNF-a 0.781 0.611 1.009IL-1b 0.253 0.729 0.986

Values above 1 indicate more gene expression at the oxi-dized surfaces, whereas values below 1 indicate more geneexpression at the machined surfaces.

Figure 8. Gene expression of transcriptional factors at theoxidized and machined surfaces. Statistically significantdifferences between the two surfaces are indicated in stars.a: Runt-related transcription factor2 (Runx2) gene expres-sion. b: Peroxisome proliferator-activated receptor-gamma(PPAR-g) gene expression. The differences between thetwo tested implants at specific time points were analyzedwith t-test (*p <0.05). n 5 15 Mean 6 SEM.

Figure 9. Gene expression of growth factors at the oxi-dized and machined surfaces. Statistically significant differ-ences between the two surfaces are indicated in stars. a: Pla-telet derived growth factor-B (PDGF-B) gene expression.b: Bone morphogenetic protein-2 (BMP-2) gene expression.c: Transforming growth factor-beta1 (TGF-b1) gene expres-sion. The differences between the two tested implants atspecific time points were analyzed with t-test (*p < 0.05). n515 Mean6 SEM.



thereafter, while the expression of IL-1b was maxi-mum at 1 day for both implant types.

Temporal changes in gene expression oftranscriptional factors at the screw surface

Runx2 is an important transcription factorinvolved in the differentiation of osteogenic cells.The expression of Runx2 was significantly higher(6-fold) at the oxidized compared to the machinedsurfaces 3 days after implantation [Fig. 8(a)]. Fromdays 1 to 3, significant 38- and 15-fold increaseswere detected at the oxidized and machined surfa-ces, respectively (Tables III and IV). Another impor-tant transcription factor is PPAR-g, implicated inthe proliferation and differentiation of several celltypes including osteogenic cells and macrophages.The expression of PPAR-g was significantly higherby 3-fold at the machined surface compared to oxi-dized surface 1 day after implantation. At day 3,the expression of PPAR-g was 6-fold higher atthe oxidized surface [Fig. 8(b)]. Temporally, theexpression of PPAR-g showed a significant 77-fold

increase from days 1 to 3 at the oxidized surface(Table III).

Temporal changes in the expression of growthfactors with chemotactic effects at the screw surface

Both, PDGF-B and BMP-2 genes showed signifi-cant increase by 56- and 43-fold, respectively, fromdays 1 to 3 at the oxidized surfaces (Table III). Theexpressions of PDGF-B and BMP-2 did not show anystatistically significant differences between the twoimplant types neither at day 1 nor at day 3 of im-plantation [Fig. 9(a,b)]. TGF-b1 expression was sig-nificantly higher by 4-fold at the machined surfacescompared to the oxidized ones at day 1 of implanta-tion [Fig. 9(c)]. A 71-fold increase in the temporalexpression of TGF-b was seen from days 1 to 3 atthe oxidized surfaces (Table III).

SEM Analysis

At every time point, the oxidized surface showedbetter organization of tissue attached to the implants.More tissues were left on the oxidized than on the

TABLE IIIResults Presented as the Ratio Between Day 3 and Day 1

RNA Expression at Oxidized Implants (n 5 15)

Oxidized Day3/Day1

Runx2 38.12*PPAR-g 76.98*BMP-2 43.38*PDGF-B 56.16*TGF-b 71.06*

All the values are above 1, indicating more gene expres-sion at the oxidized surfaces at the day 3 compared withday 1. The statistically significant difference between thetwo time points are indicted in stars *p < 0.05.

TABLE IVResults Presented as the Ratio Between Day 3 and Day 1

RNA Expression at Machined Implants (n 5 15)

Machined Day3/Day1

Runx2 14.59*PPAR-g 5.27BMP-2 11.17PDGF-B 16.87TGF-b 15.78

All the values are above 1, indicating more gene expres-sion at the machined surfaces at the day 3 compared withday 1. The statistically significant difference between thetwo time points are indicted in stars *p < 0.05.

Figure 10. SEM image of machined implant retrieved after 6 days. a: Tissue surrounding the implant was looselyattached to the surface and less organized than that seen at oxidized surface at the same time point. Captured within thisfibrin-like tissue were numerous erythrocytes and WBCs (Magnification 3500). b: A mesenchymal-like cell is shown hav-ing a flat shape spreading out on the surface. The cellular processes do not show firm anchorage on the smooth surface(Magnification 33800).

1560 OMAR ET AL.


machined surface. At all the time points, the oxidizedsurface showed more attached mesenchymal-likecells scattered all over the surfaces; and this wasmore pronounced from day 3 onwards. At day 6,[Fig. 10(a)] tissue surrounding the machined implantwas loosely attached to the surface and less organizedthan that seen at oxidized surface at the same timepoint. Captured within the fibrin-like tissue werenumerous erythrocytes and white blood cells. Imag-ing in high magnification of the machined surface[Fig. 10(b)] showed mesenchymal-like cells assuminga flat shape spreading out on the surface. The cellularprocesses did not show firm anchorage on the smoothsurface. On the other hand, SEM imaging of oxidizedimplants retrieved after 6 days [Fig. 11(a)] showedfairly organized tissue with small bundles of collagenforming a mesh on most parts of the surface. Mesen-chymal-like cells were scattered over the surface.High magnification SEM imaging of the same implant[Fig. 11(b)] showed the mesenchymal-like cells onthis surface assuming a more rounded shape. Cellularprocesses appeared to be firmly anchored in the pores(<1 lm) of the surface.

Histological and immunohistochemical analysis

The histological H and E stained sections showsthat implants were well positioned unicortically inthe compact bone and penetrating into the medullarycanal. An observation was that the implant-tissueinterface was more intact after unscrewing themachined implants compared to the oxidized ones.This might reflect a higher degree of interlocking ofoxidized implants in newly regenerated tissue at thisearly stage of healing. For both types of implants,the tissue located inside the threads was well-organ-ized, and different cellular populations could bedistinguished [Fig. 12(a,b)]. The oxidized implantsappeared to show a higher degree of vascularity and

organization compared to machined implants. Pre-liminary immunohistochemical studies revealed thatCD163 (a marker for tissue macrophages) positivecells were scattered in the newly organized tissuewithin the threads and at some locations very close tothe implant surface [Fig 13(a–d)]. The reactivity of theperiostin (a marker for osteogenic cells and bone for-mation) was detected and distributed throughout theregenerated tissue [Fig. 13(e)]. Osteoblasts lining bonetrabeculae stained strongly for periostin. Positivelystained cells were also localized at the interface.

DISCUSSION

In the present study, a procedure for the analysisof gene expression of implant-adherent cells in bonewas described. This procedure allowed the detectionof early gene expression which was differently trig-gered by implant surface properties. Further, it wasdetermined that the analysis of cells in the immedi-ate interface to the implant surface providesadditional information about the early peri-implantcellular response than that obtained by analyzing theperi-implant bone collar.

The possibility to analyze the gene expression ofcells in peri-implant tissues was achieved by the sep-arate sampling of implant-adherent cells and theperi-implant bone. This approach allowed a distinc-tion of gene expression of cells at the surface of theimplant and those occurring further out in the tissue.The temporal change of ALP, OC, TRAP, and CATKgene expression in the surrounding bone was differ-ent than that at the surface of the implants. In con-trast, a similar time course was observed for theexpression of proinflammatory TNF-a and IL-1bclose to and distant to the implant. No differencewas observed between the implants at any specifictime point in the surrounding bone, whereas signifi-

Figure 11. SEM image of oxidized implant retrieved after 6 days. a: Tissue looks fairly organized with small bundles ofcollagen forming a mesh on most parts of the surface. Possible mesenchymal cells are scattered over the surface (Magnifi-cation 3500). b: On this surface, the mesenchymal-like cell assume a more rounded shape. Cellular processes show a firmanchorage into the pores (<1 lm) of the surface (Magnification 33000).



cant differences were observed at the surface of theimplant.

Previous observations indicate that the surgicaltrauma in association with implant insertion is astrong stimulus for inflammation.26–30 The observa-tions that machined surfaces elicited a strongerTNF-a and IL-1b expression than oxidized surfacessuggest that the material surface properties is an

additional stimulus and modulating factor for theinflammatory response close to the implant surfacein bone. Although this has not previously beendemonstrated in bone, the findings are in agree-ment with previous observations on early inflam-mation in soft tissue induced by different implantmaterials.3 Macrophages are present on machinedtitanium implants4,5 and hydroxyapatite coatedimplants,31,32 during early stage after implantationand prior to bone formation at these surfaces, asjudged by transmission electron microscopy of thedeveloping bone-implant interface. The histologi-cal, immunohistochemical, and SEM observationsin the present study support the assumption thatdifferent cell types are recruited and become ad-herent to the oxidized and machined surfaces invivo, revealing a predominance of mesenchymal-like cells on the oxidized surfaces. Nevertheless,distinct proof and quantification of specific cellularsubsets on the surfaces in vivo will require theapplication of critical techniques, such as immuno-histochemistry and flow cytometry. Such combina-tion of antibody labeling techniques and site-spe-cific determination of gene expression would cre-ate important and powerful tools for furtherstudies of events in the interface.

The present results strongly suggest that theproperties of a titanium implant modulate the ini-tial inflammatory and osteogenic cell response invivo. Major differences were shown between oxi-dized and machined titanium implants. A majorobservation was that these differences were spa-tially confined to the interface between the implantsurface and the surrounding tissue. The cellularactivation was remarkably fast and strong, bothwith respect to the recruitment of different celltypes and the expression of different genes. Geneexpression in this study was influenced at threedifferent levels. Machined implants induced a 2-and 5-fold higher level of TNF-a and IL-1 b,respectively, after 1 day. OC and ALP expressionshowed 5- and 2-fold increase after 3 and 5 days,respectively, at oxidized surfaces in comparisonwith machined surfaces. In addition, CATK wasupregulated 4-fold after 3 days. Taken together,these observations are providing first line in vivoevidence of the role of the material surface proper-ties for bone formation and bone resorption. Thisis substantiated by the finding that the geneexpression of markers of inflammation and coupledbone formation and bone resorption is simultane-ously initiated but differently regulated by theimplant surface properties.

The mechanisms responsible for the proinflam-matory gene expression at machined surfaces andthe stimulation of gene expression of bone remod-eling at oxidized surfaces in vivo are hitherto

Figure 12. Decalcified paraffin-embedded and H&Estained section of tissue-implant interface after 3 days ofimplantation. a: Machined implant (Magnification 340).The implant (Ti) is removed; Mesenchymal-like cells(MLC), cells of macrophage linage (MØ), and newlyformed blood vessels (BV) are present within the thread(some of which are indicated by arrows). b: Oxidizedimplant (Magnification 340). The implant (Ti) is removed;Mesenchymal-like cells (MLC), osteoblast-like cells (OLC),and newly formed blood vessels (BV) are detected (someof which are indicated by arrows).

1562 OMAR ET AL.


unknown. A plausible explanation is that the sur-face chemistry and/or the microtopography of thesurface oxide influence protein adsorption, the gen-eration of chemotactic signals and cell adhesion tothe surfaces, subsequently influencing the cellshape and the expression and secretion of factors

important for bone regeneration. The present obser-vations are corroborated by morphological studiesshowing that anodic oxidation of electropolished ti-tanium surfaces, which produced areas ofincreased roughness on the submicrometer scaleand a thicker surface oxide, had an enhancing

Figure 13. Immunolocalization of cells in the tissue-implant interface after 3 days of implantation. a: Immunolocalizationof CD163-positive macrophages (MØ) in the tissue-implant interface at the removed machined implant (Ti) (Magnification320). This magnification of the interface shows aggregates of macrophages (arrows) outside and within the thread of theimplant. b: Immunolocalization of CD163-positive macrophages (MØ) in the tissue-implant interface at the removedmachined implant (Ti) (Magnification 340). This higher magnification shows randomly distributed round, oval, or dendri-tic macrophages (some of which are indicated by arrows) close to the implant surface and the blood vessels. c: Immuno-localization of CD163-positive macrophages (MØ) in the tissue-implant interface at the removed oxidized implant (Ti)(Magnification 320). This view of the interface shows few macrophages (arrows) outside and within the thread of theimplant. d: Immunolocalization of CD163-positive macrophages (MØ) in the tissue-implant interface at the removed oxi-dized implant (Ti) (Magnification 340). The view in higher magnification shows a few number of round, oval, or dendriticmacrophages close to the implant surface and the blood vessels (arrows). e: Immunolocalization of periostin in the tissue-implant interface at the removed machined implant (Ti) (Magnification 340). The reactivity of this protein is distributedwithin the tissue. Osteoblasts (OB) (arrows) lining bone trabeculae stained strongly for periostin. Labeled cells were alsolocalized at the interface.



effect on the rate of bone formation.33 Nevertheless,no data is available in the literature on geneexpression in relation to systematically altered ox-ide thicknesses.

The surface topography of the oxidized implantsis different from that characterizing the machinedones.16 Oxidized surfaces are considered moder-ately rough (having a Sa value of 1.55) and exhib-iting pores on the micro- and nanoscale.34 In con-trast, machined implants are relatively smoother.The present results showed that the oxidizedimplant surface had influenced the adhesion andcell shape in relation to the extracellular matrix ina manner different than that observed on themachined surface. Mesenchymal-like cells were pre-dominantly located on the oxidized surface. Theextensions of cellular processes into the pores inthe micron-size range reveal an interaction betweenthese surface features and cells during the initialevents. The implant surface microtopography haspreviously been implicated as an important factorfor gene expression of mesenchymal cells invitro.35,36 The expression of Runx2, a regulator ofosteoblast differentiation, increased 2-fold, and OCshowed 5- and 2.5-fold increase, after 3 weeks inhuman mesenchymal cells on rough and groovedtitanium surfaces, respectively, compared to tissueculture polystyrene.35 The present observations of a6-fold increase of Runx2 and 5-fold increase in OCafter 3 days between the oxidized and themachined implant suggest that the in vivoresponses are triggered differently at implants withdifferent surface topographies and as early as 3days after implantation in the rat model.

Anodic oxidation does not only change the topog-raphy and the oxide layer thickness but also thecrystallinity of the TiO2 surface layer. Previous stud-ies have shown that machined titanium has acrystalline form corresponding to rutile, whereasthe thicker oxide layer produced by anodic oxida-tion mainly consists of the anatase phase ofTiO2.

22,34,37,38 The role of the crystalline phase forearly bone formation is not known at the moment.In vitro studies showed that titanium discs withanatase coating increased the transcription of somemRNAs, which enhance osteoblast activity ascompared to control uncoated titanium discs.39

Another material surface property, which couldbe crucial in explaining the pro-osteogenicresponse and gene expression, is the hydrophilic-ity.38 This factor could be crucial for the early pro-tein adsorption and the initial recruitment and ad-hesion of different cell populations. At this stage,however, no systematic studies in vivo have yetbeen performed and no available data exist ongene expression as well as quantitative morphome-try.

The mechanism for implant-induced bone forma-tion at the immediate interface to the surface in vivomost likely involves the generation of signalingmolecules, which promote the recruitment, adhe-sion, and activation of critical cell types, includingmesenchymal stem cells and osteoblast progenitors.On the other hand, these events are coinciding withthe recruitment of cells belonging to the defensesystem. In an effort to pin-point some of the chemo-tactic/growth factors and transcription factors,which may be particularly important during theearly stage and differently expressed depending onthe two model surfaces oxidized and machined tita-nium, a selection of genes were investigated duringthe early time stage (1–3 days). The expression ofPDGF-B and BMP-2, two growth factors known tohave chemotactic effects on different cells includingmesenchymal cells with osteogenic potential,40 didnot differ at the compared surfaces. TGF-b, agrowth factor with chemotactic properties, showedhigher level of expression at the machined surfaceat day 1 after implantation compared to the oxi-dized implants. TGF-b1 has dual effects and is con-sidered to possess both proinflammatory and anti-inflammatory effects. In this experiment, the higherexpression of TGF-b1 at the machined surface wascoupled with higher expression of the other twoproinflammatory markers TNF-a and IL-1b. Theprocesses of cellular proliferation and differentiationare controlled at several levels. The expression ofseveral markers is controlled also with differentmechanisms. Transcription factors play importantroles in switching on or off the expression of differ-ent genes. Runx2 is a major transcription factor thathas been shown to influence the differentiation ofboth osteoblasts and osteoclasts from their progeni-tors. In this study, the higher expression of osteo-blast (ALP at days 3 and 6) and osteoclast markers(CATK at day 3) were parallel to a higher expres-sion of Runx2 at the oxidized surfaces compared tomachined ones at day 3 after implantation. Anothertranscription factors is PPAR-g, which has been sug-gested to control the proliferation, differentiation,and survival of various cell types41 including osteo-genic cells42 and macrophages.43,44 We used thisgene for the first time as a potential in vivo geneexpression marker in relation to implants. PPAR-gwas significantly higher at the machined surface atday 1, implicating that macrophages and cells frommonocytic lineage could be the major source. Fur-ther, at day 3, PPAR-g showed a higher level ofexpression at the oxidized surface coupled withhigh expression of osteogenic markers, tentativelyindicating a role in the activation of osteoprogeni-tors. Further studies are required to establish therole of this transcription factor in cells localized atthe implant surface in vivo.

1564 OMAR ET AL.


CONCLUSIONS

The results demonstrate that the combination ofthis in vivo experimental model and qPCR providesunique possibilities to analyze in detail the mecha-nisms of osseointegration. Early differences in geneexpression in cells associated with different implantmaterials can be detected as early as 1 day after im-plantation. Gene expression analyzed on the screwlevel appears to provide additional information com-pared with that of surrounding bone. The rapidrecruitment of mesenchymal cells, the rapid trigger-ing of gene expression crucial for bone remodeling,and the transient nature of inflammation, constitutebiological mechanisms for the osseointegration andhigh implant stability associated with oxidizedimplants.

References

1. Puleo DA, Nanci A. Understanding and controlling the bone-implant interface. Biomaterials 1999;20:2311–2321.

2. Eriksson C, Broberg M, Nygren H, Oster L. Novel in vivomethod for evaluation of healing around implants in bone.J Biomed Mater Res A 2003;66:662–668.

3. Suska F, Esposito M, Gretzer C, Kalltorp M, Tengvall P,Thomsen P. IL-1alpha, IL-1beta and TNF-alpha secretion dur-ing in vivo/ex vivo cellular interactions with titanium andcopper. Biomaterials 2003;24:461–468.

4. Sennerby L, Thomsen P, Ericson LE. Early tissue response totitanium implants inserted in rabbit cortical bone. Part I Lightmicroscopic observations. J Mater Sci Mater Med 1993;4:494–502.

5. Sennerby L, Thomsen P, Ericson LE. Early tissue response to ti-tanium implants inserted in rabbit cortical bone. Part II Ultra-structural observations. J Mater Sci Mater Med 1993;4:240–250.

6. Masuda T, Salvi GE, Offenbacher S, Felton DA, Cooper LF.Cell and matrix reactions at titanium implants in surgicallyprepared rat tibiae. Int J Oral Maxillofac Implants 1997;12:472–485.

7. Ataoglu H, Alptekin NO, Haliloglu S, Gursel M, Ataoglu T,Serpek B, Durmus E. Interleukin-1beta, tumor necrosis factor-alpha levels and neutrophil elastase activity in peri-implantcrevicular fluid. Clin Oral Implants Res 2002;13:470–476.

8. Nomura T, Ishii A, Shimizu H, Taguchi N, Yoshie H,Kusakari H, Hara K. Tissue inhibitor of metalloproteinases-1,matrix metalloproteinases-1 and 28, and collagenase activitylevels in peri-implant crevicular fluid after implantation. ClinOral Implants Res 2000;11:430–440.

9. Johansson CB, Roser K, Bolind P, Donath K, Albrektsson T.Bone-tissue formation and integration of titanium implants:An evaluation with newly developed enzyme and immuno-histochemical techniques. Clin Implant Dent Relat Res1999;1:33–40.

10. Shirakura M, Fujii N, Ohnishi H, Taguchi Y, Ohshima H,Nomura S, Maeda T. Tissue response to titanium implanta-tion in the rat maxilla, with special reference to the effects ofsurface conditions on bone formation. Clin Oral Implants Res2003;14:687–696.

11. Ozawa S, Ogawa T, Iida K, Sukotjo C, Hasegawa H, Nishi-mura RD, Nishimura I. Ovariectomy hinders the early stageof bone-implant integration: Histomorphometric, biomechani-cal, and molecular analyses. Bone 2002;30:137–143.

12. Schierano G, Canuto RA, Navone R, Peirone B, Martinasso G,

Pagano M, Maggiora M, Manzella C, Easton M, Davit A,

Trombetta A, Amedeo S, Biolatti B, Carossa S, Preti G. Bio-

logical factors involved in the osseointegration of oral tita-

nium implants with different surfaces: A pilot study in

minipigs. J Periodontol 2005;76:1710–1720.

13. Saeed S, Revell PA. Production and distribution of interleu-kin 15 and its receptors (IL-15Ralpha and IL-R2beta) in theimplant interface tissues obtained during revision of failedtotal joint replacement. Int J Exp Pathol 2001;82:201–209.

14. Guo J, Padilla RJ, Ambrose W, De Kok IJ, Cooper LF. Theeffect of hydrofluoric acid treatment of TiO2 grit blasted tita-nium implants on adherent osteoblast gene expression invitro and in vivo. Biomaterials 2007;28:5418–5425.

15. Preti G, Martinasso G, Peirone B, Navone R, Manzella C,Muzio G, Russo C, Canuto RA, Schierano G. Cytokines andgrowth factors involved in the osseointegration of oral tita-nium implants positioned using piezoelectric bone surgeryversus a drill technique: A pilot study in minipigs. J Peri-odontol 2007;78:716–722.

16. Hall J, Lausmaa J. Properties of a new porous oxide surfaceon titanium implants. Appl Osseointegration Res 2000;1:5–8.

17. Rocci A, Martignoni M, Gottlow J. Immediate loading of Bra-nemark System TiUnite and machined-surface implants inthe posterior mandible: A randomized open-ended clinicaltrial. Clin Implant Dent Relat Res 2003;5(Suppl 1):57–63.

18. Zechner W, Tangl S, Furst G, Tepper G, Thams U, Mailath G,Watzek G. Osseous healing characteristics of three differentimplant types. Clin Oral Implants Res 2003;14:150–157.

19. Schupbach P, Glauser R, Rocci A, Martignoni M, Sennerby L,Lundgren A, Gottlow J. The human bone-oxidized titaniumimplant interface: A light microscopic, scanning electron mi-croscopic, back-scatter scanning electron microscopic, andenergy-dispersive x-ray study of clinically retrieved dentalimplants. Clin Implant Dent Relat Res 2005;7(Suppl 1):S36–S43.

20. Albrektsson T, Johansson C, Lundgren A-K, Sul Y-T, GottlowJ. Experimental studies on oxidized implants. A histomorpho-metrical and biomechanical analysis. Appl OsseointegrationRes 2000;1:21–24.

21. Gottlow J, Johansson C, Albrektsson T, Lundgren A-K. Bio-mechanical and histologic evaluation of the TiUnite andOsseotite implant surfaces in rabbits after 6 weeks of healing.Appl Osseointegration Res 2000;1:25–30.

22. Sul YT, Johansson C, Albrektsson T. Which surface propertiesenhance bone response to implants? Comparison of oxidizedmagnesium, TiUnite, and Osseotite implant surfaces. Int JProsthodont 2006;19:319–328.

23. Rozen S, Skaletsky H. Primer3 on the WWW for generalusers and for biologist programmers. Methods Mol Biol2000;132:365–386.

24. Pfaffl MW. A new mathematical model for relative quantifi-cation in real-time RT-PCR. Nucleic Acids Res 2001;29:e45.

25. Reiss G. Atypical cells in the normal guinea pig organ ofCorti as revealed by micromanipulation in SEM. Microsc ResTech 1992;20:288–297.

26. Rostlund T, Thomsen P, Bjursten LM, Ericson LE. Differencein tissue response to nitrogen-ion-implanted titanium andc.p. titanium in the abdominal wall of the rat. J BiomedMater Res 1990;24:847–860.

27. Thomsen P, Ericson LE. Inflammatory cell response to boneimplant surfaces. In: Davies JE, editor. The Bone-BiomaterialInterface. Toronto: University of Toronto; 1991. p 153–164.

28. Gretzer C, Emanuelsson L, Liljensten E, Thomsen P. Theinflammatory cell influx and cytokines changes during transi-tion from acute inflammation to fibrous repair aroundimplanted materials. J Biomater Sci Polym Ed 2006;17:669–687.



29. Suska F, Gretzer C, Esposito M, Emanuelsson L, WennerbergA, Tengvall P, Thomsen P. In vivo cytokine secretion andNF-kappaB activation around titanium and copper implants.Biomaterials 2005;26:519–527.

30. Fujii N, Kusakari H, Maeda T. A histological study on tissueresponses to titanium implantation in rat maxilla: The pro-cess of epithelial regeneration and bone reaction. J Periodon-tol 1998;69:485–495.

31. van Blitterswijk CA, Grote JJ, Kuypers W, Blok-van Hoek CJ,Daems WT. Bioreactions at the tissue/hydroxyapatite inter-face. Biomaterials 1985;6:243–251.

32. Muller-Mai CM, Voigt C, Gross U. Incorporation and degra-dation of hydroxyapatite implants of different surface rough-ness and surface structure in bone. Scanning Microsc1990;4:613–622; discussion 622–624.

33. Larsson C, Thomsen P, Aronsson BO, Rodahl M, Lausmaa J,Kasemo B, Ericson LE. Bone response to surface-modifiedtitanium implants: Studies on the early tissue response tomachined and electropolished implants with different oxidethicknesses. Biomaterials 1996;17:605–616.

34. Jarmar T, Palmquist A, Branemark R, Hermansson L,Engqvist H, Thomsen P. Characterization of the surface prop-erties of commercially available dental implants using scan-ning electron microscopy, focused ion beam, and high-resolu-tion transmission electron microscopy. Clin Implant DentRelat Res 2008;10:11–22.

35. Schneider GB, Zaharias R, Seabold D, Keller J, Stanford C. Dif-ferentiation of preosteoblasts is affected by implant surfacemicrotopographies. J Biomed Mater Res A 2004;69:462–468.

36. Masaki C, Schneider GB, Zaharias R, Seabold D, Stanford C.Effects of implant surface microtopography on osteoblastgene expression. Clin Oral Implants Res 2005;16:650–656.

37. Sul YT, Johansson CB, Petronis S, Krozer A, Jeong Y,Wennerberg A, Albrektsson T. Characteristics of the surfaceoxides on turned and electrochemically oxidized pure tita-nium implants up to dielectric breakdown: The oxide thick-ness, micropore configurations, surface roughness, crystalstructure and chemical composition. Biomaterials 2002;23:491–501.

38. Sawase T, Jimbo R, Wennerberg A, Suketa N, Tanaka Y,Atsuta M. A novel characteristic of porous titanium oxideimplants. Clin Oral Implants Res 2007;18:680–685.

39. Sollazzo V, Palmieri A, Pezzetti F, Scarano A, Martinelli M,Scapoli L, Massari L, Brunelli G, Caramelli E, Carinci F.Genetic effect of anatase on osteoblast-like cells. J BiomedMater Res B Appl Biomater 2008;85:29–36.

40. Ozaki Y, Nishimura M, Sekiya K, Suehiro F, Kanawa M,Nikawa H, Hamada T, Kato Y. Comprehensive analysis ofchemotactic factors for bone marrow mesenchymal stem cells.Stem Cells Dev 2007;16:119–129.

41. Heikkinen S, Auwerx J, Argmann CA. PPARgamma inhuman and mouse physiology. Biochim Biophys Acta 2007;1771:999–1013.

42. Bruedigam C, Koedam M, Chiba H, Eijken M, van LeeuwenJP. Evidence for multiple peroxisome proliferator-activated re-ceptor gamma transcripts in bone: Fine-tuning by hormonalregulation and mRNA stability. FEBS Lett 2008;582:1618–1624.

43. Szanto A, Roszer T. Nuclear receptors in macrophages: Alink between metabolism and inflammation. FEBS Lett 2008;582:106–116.

44. Rigamonti E, Chinetti-Gbaguidi G, Staels B. Regulation ofmacrophage functions by PPAR-alpha, PPAR-gamma, andLXRs in mice and men. Arterioscler Thromb Vasc Biol 2008;28:1050–1059.

1566 OMAR ET AL.


in vivo gene expression in response to anodically oxidized versus machined titanium implants

Documents