Plasma chemical oxidation (PCO ®) enhances implant fixation and bone-implant contact in a rat model

C. Schrader 1, J. Schmidt 1, M. Diefenbeck 2, T. Mückley 2, S. Zankovych 3, J. Bossert 3, K. D. Jandt 3, M. Fau-con 4 and U. Finger 4

1 INNOVENT e.V. Technologieentwicklung Jena, Prüssingstraße 27B, D-07745 Jena, Germany 2 Klinik für Unfall-, Hand- und Wiederherstellungschirurgie, Universitätsklinikum Jena, Erlanger Allee 101,

D-07747 Jena, Germany 3 Institut für Materialwissenschaft und Werkstofftechnologie, Löbdergraben 32, 07743 Jena, Germany

4 Königsee Implantate GmbH, Am Sand 4 / OT Aschau, D-07426 Allendorf, Germany

Abstract: Metal surface properties can be modified by various methods like plasma-spraying, micro-spheres or µ-metal scaffolds to generate nano-, micro- and macro-porous structures to enhance osseointegration and thus implant fixation. This paper describes an electrochemical routine which covers the whole range of surface modifi-cations from the nm- to the µm-scale. The plasma assisted process produces bioinert and bioactive properties under typical plasma conditions in an electrolyte.

Keywords: plasma chemical oxidation, bioinert, bioactive, antibacterial

1. Introduction

Orthopaedic and dental implants rely on early ridged fixation to the host bone, which is a prerequi-site for good clinical outcome [1-2]. The implant anchorage apparatus has two structural components. The bone-implant bonding (osseointegration [OI]) and peri-implant trabecular bone [PIB]. OI is formed with trabecular struts that are integrated into the PIB, which bridges the implant to the bony cortex, thus forming a structural unit between implant and skele-ton [3-5]. In the past, different approaches have been used to enhance osseointegration and peri-implant bone formation. Hydroxyapatite coatings [6], often in combination with growth factors [7-8] or bisphos-phonates [9] have been shown to improve osseointe-gration of titanium implants. Some of the stimuli used to enhance implant fixation might bear the risk of complications [10-13]. Titanium and its alloys were utilised as biomaterials in orthopaedics since the end of the nineteen-seventies because of their excellent corrosion resistance, advantageous me-chanical properties and biocompatible interaction with the human body [14-15]. Certain disadvantages from biomaterials made of titanium and its alloys take into account that cytotoxic elements can possi-bly diffuse into the surrounding body tissues and cause irritations. Additionally, temporary implants for short-time repositioning purposes, especially in sophisticated bone geometries stabilised by self-drilling screws in angle-stable plate combinations,

tend to increased removal torques. This leads on the one hand to tissues irritations and on the other hand to an escalated bone fracture risk in implant removal resulting in extended hospitalisation. Our approach to face these challenges is to modify the titanium surface from the implant itself by plasma chemical oxidation [PCO®] to a ceramic coating. A selected element contribution of this coating can be combined and varied with topographical properties for useful applications [16]. For permanent implants porous structured coatings are predestined. They deliver an appropriate element contribution for fast OI with an interlocking surface topography [17] for PIB. Tem-porary implants with an optimised surface topogra-phy to prevent OI and PIB can be removed after its bone reposition duty. The structural and chemical properties of the PCO®-coating can be varied over quite a wide range by altering the process parame-ters, such as anode potential, electrolyte composi-tion, temperature and current density [18].

2. Anodic oxidation (AO)

Under atmospheric conditions the high affinity of titanium towards oxygen establishes a natural insu-lating titanium oxide film of only a few nanometres on all titanium work pieces. This passivity oxide film works as a dielectric barrier and inhibits e.g. corro-sive charge carrier to pass the boundary layer. In an ordinary electrolysis cell, it is possible to enlarge the barrier thickness by connecting the chosen work

piece as anode and to deposit under galvanostatic conditions (anodic oxidation).

Fig. 1: Products by courtesy of Königsee Implantate GmbH after anodic oxidation

With a constant current density of about 100 A·m-2, it can be observed, that the voltage increases and layer forming mechanisms take place at the same time. The process ends with a layer thickness of several hundred nanometres. This layer thickness range shows typical interference effects demonstrating the interaction with the electromagnetic spectrum of visible light by the impression of a coloured surface. This is appreciated by the surgeon to differentiate more easily between implants and their suggested place of implantation or screw and plate combina-tions translated in colour codes. Different diluted acids (H2SO4, H3PO4, acetic acid and others) can be used as electrolyte for this process. The main techno-logical advantage of these converted titanium sur-faces is improved adhesion and bonding (> 20 MPa), which is particularly relevant in the aerospace indus-try. It can also be used to increase the oxide thick-ness for corrosion protection, for decreased ion re-lease, coloration and for dielectric coatings on elec-trode materials e.g. dielectric barrier discharges [DBD].

3. Plasma chemical oxidation (PCO ®)

In this paper a special electrolyte composition is presented that enlarges the deposition range from the nm- to the µm-scale in combination with selected ion implantation and finishing methods for evidentially improved OI and PIB under typical clinical condi-tions. This is carried out by a process called Plasma Chemical Oxidation (PCO®).

Ti / Ti oxide interface:

−+ +⇔ eTiTi 440 (1.1)

Ti oxide / electrolyte interface:

+− +⇔ HOOH 422 22 (TiO2-formation) (1.2)

−+ ++⇔ eHOOH 442 22 (O2 formation) (1.3)

Both interfaces:

224 2 TiOOTi ⇔+ −+ (1.4)

The titanium and oxygen ions formed in these electrochemical reactions have to be driven through the oxide film. This is carried out by the externally

Fig. 2: Schemes of anodic oxidation (0 V – 100 V) and Plasma chemical oxidation (200 V – 300 V)

applied electric field and linearly increases the oxide film thickness with approximately 1.5 nm·V-1 to 3.0 nm·V-1 [18] in an anodic oxidation process.

Fig. 3: Cross section of a PCO® coating (300 V)

The applied voltage drop mainly occurs across the oxide film because of its high resistivity compared to the electrolyte or metallic parts of the electric circuit. With a suitable electrolyte further layer growth be-yond the coloration regime of the anodic oxidation can be induced.

U / V

0 V

Steel cathode (-) Titanium anode (+) Elektrolyte

Power Supply

U / V

100 V

Plasma Ignition voltage

Steel cathode (-) Titanium anode (+) + coating

Elektrolyte

Power Supply

Steel cathode (-) Titanium anode (+) + coating

U / V

200 V

Plasma Ignition voltage

Elektrolyte

Power Supply

U / V

300 V

Plasma Ignition voltage

Power Supply

Steel cathode (-) Titanium anode (+) + coating

Elektrolyte

Fig. 4: PCO®-process on a distal palmar radius plate

Above a certain voltage, mainly determined by the electrolyte, work piece and power supply settings, the oxide will be no longer resistive enough to pre-vent further current flow. The result is an increased oxygen formation (see equation 1.3) with local field strengths above 106 V·m-1 - 109 V·m-1 that ignite a thermal arc discharge in pure oxygen surrounded by an aqueous electrolyte.The additional layer forma-tion by a thermal arc discharge increases the deposi-tion rate and leads to the storage of electrolyte com-pounds into the coating when the melted plasma channels and - pores (Fig. 3) solidify again. There-fore, it is possible to create coatings doped with use-ful elements for osseointegration, antibacterial prop-erties, etc. Furthermore, the longer the discharge regime of the deposition process occurs the more electrolyte material is incorporated into the ceramic coating. In-vitro adhesion- and proliferation assays of MC3T3 cells on these PCO® modified titanium surfaces show enhanced growth. The purpose of the present study is to determine, if surface modifica-tions by PCO® have an effect on peri-implant bone volume, bone-implant contact and implant fixation.

Fig. 5: Surface of a PCO®-bioactive coating (280 V)

Fig. 6: Surface of a PCO®-bioinert coating (220 V, blasted)

A modified rat tibial implantation model, described by Gao et al. [9] with bilateral implantation of tita-nium rods is used. Pure titanium, typ-III anodization (blue) titanium implants and implants with two dif-ferent surfaces modifications by PCO® (300 V [bio-active] and 200 V [bioinert]) were evaluated in his-tomorphometric analysis and mechanical pull-out testing.

4. Materials and methods

4.1 Preparation of implants

The power supply in the experimental setup in Fig. 2 consists of a pulsed rectifier set D400 G500/50 WRG-TFKX from Munk Ltd. in Hamm, Germany. The electric supply is realised by three feed cables (3 x 400 V / 50 Hz / 100 A) and delivers a positively pulsed directed current of 50 A and a negatively pulsed directed current of 30 A with voltages from 0 V to 500 V. The system control with a digital out-put provides constant voltages and currents with an accuracy of ±1% (± 1.5%) and a ripple content of w = 0.5 %. The adjustable pulse frequency ranges from 10 Hz to 2 kHz.

The implants were divided into four groups:

(1) Ti: Grinded and ceramic-blasted pure titanium implants

(2) Ti-blue: Grinded and ceramic-blasted pure tita-nium implants coated by type III anodisation (blue)

(3) bioinert TiOB®: Grinded and ceramic-blasted pure titanium implants with PCO®-bioinert coat-ing (220 V, 1000 Hz, blasted)

(4) bioactive TiOB®: Grinded and ceramic-blasted pure titanium implants with PCO®-bioactive coat-ing (280 V, 1000 Hz)

4.2 Cell biological investigation

To monitor cytotoxic-, proliferation- or any other growth inhibiting effects a very useful in-vitro cyto-toxicity assay for biomaterials, the live/dead assay, is performed. Therefore, direct inoculation with culture medium (25.000 cells·cm-2 MC3T3-E1) on auto-claved specimen (HiClave HV-50, HMC Europe GmbH, Tüssling, Germany), incubation for 24h and 96h at 37°C (Revco Ultima, Revco Technologies, NA-Ashville, U.S.A) and examination with a fluo-rescence microscope (Axiovert 25 / filter 44 with beam splitter FT 500 / filter 14 with beam splitter FT 580, Carl Zeiss GmbH, Jena, Germany) after staining with a diluted solution of Fluoresceindiacetate (FDA) and Ethidiumbromide (EtBr) in phosphate buffer solution (PBS) is carried out. The amount of green fluorescent cells indicates vital, FDA metabo-lising organisms in contrast to orange-red fluorescent cells indicating dead organisms with perforated cell membranes and subsequent diffusion of EtBr into the nuclei.

4.3 Animal model and implantation

Animals

All experiments were approved by the Animal Care Committee of Thuringia (Reg. No. G 02-008/10). Sixty-four, 3-month-old male Sprague Dawley rats (Harlan Laboratories GmbH, Eystrup, Germany) weighing 300 g - 389 g were used. All were given free access to standard rat-chow and water and were raised in relative steady temperature and humidity in an air-conditioned environment with lighting con-trolled in a cycle of light 12h / dark 12 h. Institu-tional guidelines for the care and treatment of labora-tory animals were followed.

Implants

128 custom Ti6Al4V rods (Königsee Implantate GmbH, Aschau, Germany), measuring 0.8 mm in diameter and 10 mm in length were used. All im-plants were pre-treated with aluminium oxide abra-sives and finished with spherical ceramic particles. Next, surface modifications were performed as men-

tioned above. Before implantation, the implants were sterilised in a steam autoclave (Vacuklav 44B, Me-lag, Berlin, Germany) for 35 minutes at 134°C - 138°C and 2.16 · 105 Pa.

Implantation procedure

Surgery was performed under general anaesthesia by weight-adopted intraperitoneal injection of Domi-tor® (Meditomidin) 0.15mg / kg BW(Pfizer, Berlin, Germany), Dormicum® (Midazolam) 2.0 mg / kg BW (Ratiopharm, Ulm, Germany) and Fentanyl® (Fentanyl) 0.005 mg / kg BW (Janssen-Cilag, Neuss, Germany). Animals were prepared for surgery as follows: Both hind legs were shaved and disinfected with alcohol. To provide sterile conditions during surgery animals were placed on sterile drapes and bodies were covered with sterile sheets. Both hind legs were draped with a sterile incision foil (Rau-codrape, Lohmann & Rauscher, Rengsdorf, Ger-many). A medial incision to expose the knee joint in both hind limbs was made over 5 mm longitudinally, a pilot hole marked at the intercondylar eminence. A custom-made awl with a tip in the size of 0.9 mm diameter and 10 mm length was gradually twisted to make a channel from the proximal tibia epiphysis into the medullary canal. The implants were inserted into this channel and positioned 2 mm beyond the articulating cartilage. Soft tissue was irrigated with sterile saline and fascia and skin incisions were closed in single-knot technique (Vicryl 5/0 and Prolene 5/0, Ethicon, Norderstedt, Germany). Pro-phylactic i.m. antibiotic (Terramycin, Pfizer GmbH, Berlin, Germany) and analgesics (Buprenovet, Bayer, Leverkusen, Germany) was administered once at the time of surgery. Correct position of implants was controlled by x-ray. After x-ray control general anaesthesia was antagonised by Antisedan® (Ati-pamezol) 0.75mg / kg BW (Pfizer, Berlin, Germany), Flumazenil-hameln® (Flumazenil) 0.2mg / kg BW (Invera Arzneimittel GmbH, Freiburg, Germany) and Naloxon (Naloxon) 0.12mg / kg BW (Deltaselect GmbH, Dreieich, Germany).

Explantation and bone processing

The animals were sacrificed after three weeks and eight weeks. Tibiae were harvested and denuded of soft tissues prior to further analyses. From eight ani-mals per group, five tibiae were fixed in formalin

(5%) for histological and ten frozen at – 20°C for biomechanical examination (all in a blinded manner).

4.4 Biomechanical testing

The biomechanical property of bone–implant inter-face was assessed using a pull-out test. The tibia epi- and metaphysis was trimmed with a bur (Minimot 40IE, Proxxon, Niersbach / Eifel, Germany) to ex-pose the proximal tip of the implant (2 mm - 3 mm). Afterwards the samples were again stored at -20°C. The distal part of the tibia was embedded in a special polyester resin (CEM 2000, Cloeren Technology GmbH, Wegberg, Germany) with a comparable low polymerisation enthalpy. For embedding a custom made fixture was used to permit coaxial alignment of the implant in the direction of force. The tibial im-plant-bone interface were tested in a commercial material testing system (Tiratest 2710, Tira GmbH, Schalkau, Germany) after thawing in air at room temperature. The distraction speed was set at 1 mm·min-1 throughout the test, and the load-displacement curve was recorded simultaneously. From these curves, maximum force was determined and interfacial shear strength was calculated by di-viding the force (N) at the point of failure by the surface area of the implant in contact with tissue (mm2).

4.5 Histological examination

Proximal tibiae with implant in situ (n=5/group) were fixed in 5% neutral buffered formalin for 10 days, dehydrated with increasing concentrations of alcohol, then impregnated with a mixture of alco-hol/Technovit 7200VLC = 1:1, followed by infiltra-tion with pure Technovit 7200VLC (Heraeus Kulzer, Wehrheim / Ts, Germany) and embedded into this methacrylate-based resin without decalcification. 200 µm thick cross-sections were performed using an EXAKT 300 diamon band saw.

Fig. 7: X-ray image of the tibia (a) cross-section of the implant Masson-Goldner stain, 40-fold magnification (b)

Next, the slices were grinded with the EXAKT 400CS grinding system and special grinding papers down to a thickness of 10 µm – 20 µm. Four sections (proximal (1), median I (2), median II (3) and distal implant (4)) were selected and stained with modified Masson-Goldner without removing the polyme-thacrylate (Fig. 7). Histomorphometric analysis of the percentages of bone contact and bone area were performed with a semi-automated digitizing image analyzer system, consisting of a Nikon ECLIPSE E600 stereomicroscope, a computer-coupled Nikon Digital Camera DXM1200 and NIS-Elements F 2.20 image software. Bone contact (BC) was calculated as a length percentage of the direct bone-implant inter-face to total implant surface. Bone area (BA) was defined as the area percentage of the newly formed bone within a circle of 0.1 mm around the implant to the whole area.

4.6 Statistical analysis

All data are presented as box- and whisker plots in-dicating median, quartiles, whiskers and outliers. StatGraphics Centurion (Statpoint Technologies, Inc. Warrenton, Virginia, USA) was used for statistical analysis. One-way analysis of variance (ANOVA) following multiple comparisons with Fishers´s least significant difference (LSD) procedure at the 95.0 % confidence level was performed to determine which means were significantly different from each other.

5. Results

5.1 In vitro testing

Fig. 8 - Fig. 11 present the results of the vi-tal / dead-assay for biomaterials via cell staining (Fluoresceindicacetate/Ethidiumbromide). In com-parison with a referenced petridish (Fig. 10 - Fig. 11) the bioinert TiOB®-surface (Fig. 8 - Fig. 9) shows neither cytotoxic nor any other proliferation- and growth inhibiting effects.

Fig. 8: Image of dead cells on bioinert TiOB® after 24 h (l) and 96 h (r) of incubation

Fig. 9: Image of vital cells on bioinert TiOB® after 24 h (l) and 96 h (r) of incubation

Fig. 10: Image of dead cells on a reference after 24 h (l) and 96 h (r) of incubation

Fig. 11: Image of vital cells on a reference after 24 h (l) and 96 h (r) of incubation

5.2 Clinical outcome of osseointegration

During explantation and sample preparation for mechanical testing, some implants were not inte-grated in the bone and could be remove by a forceps without using any force. Others fell out of the bone during embedding of the tibiae. Theses implants were defined as not osseous integrated (non-integrated). In the three week group, only four of eleven pure titanium implants were fixed, whereas all of the bioactive modified implants were inte-grated (Tab. 1). After eight weeks eight of ten pure titanium, nine of ten bioactive and all of the bioinert modified implants were integrated.

Tab. 1: Status of implants during explantation and sample preparation. Osseointegration OI / %: Number of integrated implants vs. total num-ber of implants

Ti Ti blue bioinert TiOB®

bioactive TiOB®

3 weeks 4/11

(36.4 %) 4/10

(40.0 %) 7/11

(63.6 %) 10/10

(100 %)

8 weeks 8/10

(80%) 9/11

(81.8 %) 10/10

(100 %) 9/10

(90 %)

5.3 Biomechanical analysis

A total of four to ten tibiae in the three week groups and eight to ten samples in the eight week

groups have been included in this analysis. The bio-mechanical data revealed significant influences of the bioinert and bioactive coating either on maxi-mum force required to pull out the implant or on the corresponding interfacial shear strength after three and eight weeks (Fig. 12 and Fig. 13). Bioactive TiOB® surface modification induced a marked in-crease in the maximum force and the interfacial shear strength, whereas bioinert modification showed a trend towards a higher interfacial shear strength.

Fig. 12: Shear strength after 3 weeks

Fig. 13: Shear strength after 8 weeks

5.4 Histology evaluation

Light microscope images of implant–bone inter-faces eight weeks after implantation are shown in Fig. 14. Histomorphometric analysis revealed the results of bone contact (BC) and bone area (BA) (Fig. 15 and Tab. 2).

Tab. 2: Histomorphometric analyses: Bone area (BA) after three and eight weeks in percent (± SEM)

Ti Ti blue bioinert TiOB®

bioactive TiOB®

3 weeks

27,5 (±2,55)

39,9 (±1,65)

34,3 (±3,42)

47,4 (±4,07)

8 weeks

69,0 (±2,67)

67,5 (±3,23)

60,4 (±4,77)

60,8 (±3,59)

One is the length ratio of direct bone–implant inter-face to total implant surface, the other is the area ratio of newly formed bone area to the whole area within a circle of 0.1 mm width [6]. BA showed a dynamic increase in all groups from three to eight

weeks, but no significant difference within the groups after eight weeks (Tab. 2).

Fig. 14 Implant-bone interfaces after 8 weeks

However, BC showed significant increased values for the bioactive TiOB® group after eight weeks, followed by bioinert TiOB® surfaces (Fig. 15).

Fig. 15 Bone contact after 8 weeks of implantation

6. Discussion

To our knowledge, this is the first time that bioac-tive- and bioinert TiOB® surfaces have been tested in an animal model. The bioactive TiOB® surface dem-onstrates a significantly stronger implant fixation in mechanical testing and a significant increased bone contact. The modification of titanium surfaces by PCO® provides three features, which might act in a synergistic way for a stable implant / bone- interface. First, the natural passive titaniumoxide film on a titanium implant of a few nanometers is increased to a TiOB® layer up to several micrometers. This con-version layer is defined by its compactness, uniform-

ity and excellent anchoring properties between im-plant material and surface. It provides a diffusion barrier for metallic ions and body fluids. Second, a finishing porous cover layer with a specific pore density is processed by a temporary thermal arc dis-charge. Depending on the dielectric barrier charac-teristics of the TiOB® coating and the applied volt-age, these discharges generate temperatures of 1.000 K to 50.000 K in an electrolyte at room tem-perature. Where these oxygen plasmas occur, the previously built TiOB® layer melts and creates a nano-porous surface. Third, the electrolyte itself can be modified by adding different electrolyte com-pounds. These compounds are embedded in the po-rous surface by PCO®. In the bio-active TiOB® layer a high concentration of phosphorous and calcium was integrated. Both elements are important for syn-ergetic bone growth mechanism with osteoblastic cells [13]. Thus, PCO® has a different mechanism of action than the commonly used coatings of implants with hydroxyapatite. PCO® is solely a modification of a titanium surface, compared to the local applica-tion of growth factors, bisphosphonats or other medication by different other coatings. To show the effects of TiOB® layers, a modified rat tibial implan-tation model, described by Gao et al. [9] with bilat-eral implantation of titanium rods was used. Since we present the first in-vivo results of TiOB® sur-faces, we used animals with a normal bone structure [7] and not osteoporotic animals after bilateral ova-riectomization [5, 9, 19]. We plan those experiments at a later stage. In the literature, different time inter-vals of the mechanical testing of implants are dis-cussed; animals are sacrificed between two weeks [20], four weeks [7], six weeks [3] and three months [9, 13]. We decided to elevate implant fixation at three weeks and eight weeks, which was well chosen to assess the dynamic process of the formation of the bone / implant-interface. Implant fixation was ana-lysed by mechanical pull out tests. Compared to the Ti control, the bioactive TiOB® layer increased maximum pull-out force 7.3-fold after 3 weeks and 12.1-fold after 8 weeks. Gao Y et al. reported in their study of different hydroxyapatit-bisphosphonate coatings in an osteoporotic rat model an increase of maximum force between 2.9- and 6.8-fold [9]. From our point of view, it is remarkable that the TiOB® surface modification revealed results in a comparable range than the local treatment with hydroxyapatite

plus bisphosphonates. De Raniery et al. could not show significant differences in mechanical pull out tests for the local application of rhTGF-β2 growth factor compared to titanium implants in his animal model [7]. The bioinert TiOB® layer showed a 0.9-fold reduced maximum pull-out force after 3 weeks and a 3.2-fold increase after 8 weeks com-pared to Ti. Since the bio-inert layer has a smooth surface and almost no phosphorus and calcium com-ponents, this may show a time-dependent effect: Probably the nano-porous structure of the bioactive layer is responsible for the earlier implant fixation after three weeks. For the histomorphometric meas-urements an established method was used [9, 19]. We found for all groups a dynamic increase of the bone area (BA) (e.g. Titanium: 27.5 % after three weeks vs. 69.0 % after eight weeks) from three to eight weeks, but no significant difference within the groups after eight weeks. BA is a parameter for the amount of the de-novo bone formation. Our interpre-tation of the BA results is that the de-nove bone for-mation is not influenced by PCO®. Bone contact (BC) was significantly increased by bioactive TiOB® surfaces about 2.5 fold and by bioinert TiOB® sur-faces 1.5-fold compared to titanium after eight weeks. This correlates well with the enhanced shear strength in both groups. From our point of view, BC is the more important parameter for implant ancorage than BA. Gao et al. report a stronger correlation be-tween BC and sheer force than for BA and sheer force [19], which is in general agreement with our findings.

7. Conclusion

The results of our study demonstrate that the sur-face modification of titanium implants by PCO® can promote osseointegration and implant fixation. Bio-active TiOB® surfaces have a nano-porous structure, are enriched with phosphorus and calcium and show comparable results in biomechanical test to the local treatment with hydroxyapatite, bisphosphonates and growth factors without their known disadvantages.

8. Acknowlegement

This work was supported by a grant from the “Eu-ropäischer Fonds für regionale Entwicklung” [EFRE] (2006 FE 0183).

9. References

[1] Bauer TW, Schils J. The pathology of total joint athroplasty: 1. Mechanisms of implant fixation. Skeletal Radiol 1999; 28: 423-432.

[2] Mjöberg B. The theory of early loosening of hip prostheses. Orthope-dics 1997; 20: 1169-1175.

[3] Gabet Y, Kohavi D, Kohler T, Baras M, Müller R, Bab I. Trabecular bone gradient in rat long bone ketaphyses: mathematical modelling and application to morphometric measurements and correction of implant positioning. J Bone Miner Res 2008; 23: 48-57.

[4] Davies JE. Understanding peri-implant endosseous helaing. J Dent Educ 2003; 57:932-949.

[5] Gabet Y, Kohavi D, Voide R, Mueller TL, Müller R, Bab I. En-dosseus implant anchorage is critically dependent on mechanostructural determinants of peri-implant bone trabeculae. J Bone Miner Res 2010; 25: 575–583

[6] Wermelin K, Suska F, Tengvall P, Thomsen P, Aspenberg P. Stainless steel screws coated with bisphosphonates gave stronger fixation and more surrounding bone. Histomorphometry in rats. Bone 2008; 42: 365 – 371

[7] De Ranieri A, Virdi AS, Kuroda S, Shott S, Leven RM, Hallab NJ, Sumner DR. Local application of rhTGF-ß2 enhances peri-implant bone volume and bone-implant contact in a rat model. Bone 2005; 37: 55 – 62

[8] Sachse A, Wagner A, Keller M, Wagner O, Wetzel WD, Layher F et al. Osseointegration of hydroxyapatite-titanium implants coated with nonglycosylated recombinant human bone morphogenetic protein-2 (BMP-2) in aged sheep. Bone 2005; 37:699-710

[9] Gao Y, Zou S, Liu X, Bao C, Hu J. The effect of surface immobilized bisphosphonates on the fixation of hydroxyapatite-coated titanium implants in ovariectomized rats. Biomaterials 2009; 30: 1790 – 1796

[10] Silverman SL, Landesberg R. Osteonecrosis of the jaw and the role of bisphosphonates: a critical review. Am J Med 2009; 122(2 Suppl): S33-45.

[11] Mroz TE, Wang JC, Hashimoto R, Norvell DC. Complications related to osteobiologics use in spine surgery: a systematic review. Spine 2010; 35 (9 Suppl): S86-104

[12] Vaidya R, Sethi A, Bartol S, Jacobson M, Coe C, Craig JG. Complications in the use of rhBMP-2 in PEEK cages for interbody spinal fusions. J Spinal Disord Tech. 2008; 21: 557-62

[13] Li LH, Kong YM, Kim HW, Kim YW, Kim HE, Heo SJ, Koak JY. Improved biological performance of Ti implants due to surface modifica-tion by micro-arc oxidation. Biomaterials. 2004 Jun;25(14):2867-75.

[14] Williams DF, Brunette DM, Tengvall P,Textor M, Thomsen P. Titanium in Medicine 2001; 13.

[15] Steinemann SG, Müsle PA, Lacombe P, Trocit R, Beranger G. Proceedings of the 6th World Conference on Titanium, Les Editions de Physique 1988; 535.

[16] Nebe B, Lüthen F, Lange R, Becker P, Beck U and Rychly J. Mate-rials Science and Engineering: C 2004; 25/5: 619-624.

[17] Song WH et al. Biomaterials 2004 ; 25 : 3341-3349.

[18] Liu X et al. Materials Science and Engineering R 2004; 47: 49-121.

[19] Gao Y, Luo E, Hu J, Xue J, Zhu S, Li J. Effect of combined local tratment with zoledronic acid and basic fibroblast growth factor on implant fixation in ovariectomized rats

[20] Tengvall P, Skoglund B, Askendal A, Aspenberg P. Surface immo-bilized bisphosphonate improves stainless-steel screw fixation in rats. Biomaterials 2004; 25: 2133 – 2138