TiNOx coatings on roughened titanium and CoCr alloy accelerate early osseointegration of dental implants in minipigs

Bone 52 (2013) 230–237

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Original Full Length Article

TiNOx coatings on roughened titanium and CoCr alloy accelerate early osseointegration of dental implants in minipigs☆ Stéphane Durual ⁎, Philippe Rieder, Giovanni Garavaglia, Anna Filieri, Maria Cattani-Lorente, Susanne S. Scherrer, H.W. Anselm Wiskott Laboratory of Biomaterials, University of Geneva, Geneva, Switzerland

a r t i c l e

i n f o

Article history: Received 4 June 2012 Revised 17 August 2012 Accepted 12 September 2012 Available online 19 September 2012 Edited by: David Burr Keywords: SLA titanium TiNOx coatings In vivo study Göttingen minipigs CoCr alloys

a b s t r a c t Introduction: Titanium nitride oxide (TiNOx) coatings are known for their biocompatibility, hardness and high resistance to corrosion and wear. Further, they can be applied by plasma vapor deposition onto a wide variety of metallic, mineral, or organic substrates. In cell cultures, TiNOx coatings applied onto SLA (sandblasted, large grit, acid etched)-roughened titanium surfaces increased human primary osteoblast proliferation by 1.5 times in the first 2 weeks after seeding, while maintaining a high degree of cell differentiation. Therefore, the objectives of the present study were (i) to determine whether these findings would translate into the enhanced osseointegration of TiNOx-coated implants in vivo and (ii) to compare the osseointegration of Ti-SLA (titanium–SLA) and CoCr-SLA (cobalt–chromium–SLA) implants coated with TiNOx. Methods: Forty‐eight cylinders made of Ti-SLA, Ti-SLA-TiNOx (TiNOx‐coated Ti-SLA) and CoCr-SLA-TiNOx (TiNOx‐coated CoCr-SLA) were implanted into the lower jawbone of 8 minipigs. The bone-to-implant contact was determined after 1 week, 2 weeks, 1 month and 3 months. Results: Osseointegration proceeded normally on all 3 surfaces, with equal activity after the first week of healing. After 2 weeks, bone-to-implant contact was 1.8 times higher on TiNOx coatings, either deposited on Ti or on CoCr. These differences fell off after 1 and 3 months of healing. Conclusions: When compared to standard SLA titanium, TiNOx coatings enhance implant osseointegration during the first month of healing. Furthermore, this stimulating effect is independent of the substrate, leading to similar results whether the coating is applied onto SLA-Ti or onto SLA-CoCr. © 2012 Elsevier Inc. All rights reserved.

Introduction Since the early days of dental implantology [1], endosseous applications have been almost exclusively limited to titanium (Ti), to the extent that data on the osseointegration of other metals are scarce to non-existent [2–5]. While some commercial systems still use Ti6Al4V (essentially due to its superior mechanical strength), the vast majority of contemporary implants is made of Ti grade 4 [6,7]. While the alloy's surface chemistry is of importance, it will not per se ensure osseointegration as microtexturing is the other dominant factor. In this regard, roughening a surface was shown to markedly increase osseointegration, cellular differentiation and mechanical fixation [8,9]. To create the appropriate type of roughness, several methods were developed, that is, primarily plasma spraying [10],

☆ No benefit of any kind has been or will be received either directly or indirectly by the authors. ⁎ Corresponding author at: Laboratory of Biomaterials, University of Geneva, School of Dentistry, 19, rue Barthélemy-Menn, 1205 Geneva, Switzerland. Fax: +41 22 379 41 14. E-mail address: [email protected] (S. Durual). 8756-3282/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bone.2012.09.014

sand blasting, acid etching, anodization [11] as well as various combinations of these [9], most specifically the SLA treatment (sand blasting, large grit, acid etching). More recently, a procedure geared at increasing hydrophilicity was shown to favorably influence the kinetics of osseointegration [12–15]. Another strategy to improve osseointegration is to coat the surface with “bioactive” components. The addition of bone matrix derivatives (chiefly hydroxyapatite) or organic biomolecules (i.e. growth factors, enzymes, and collagen) [16] as well as the chemical modifications of the titanium surface by metal coatings such as gold/palladium [17] or titanium nitride (TiN) [18,19] were reported. As to the latter, the underlying hypothesis of TiN (i.e. a ceramic) coatings was to increase the surfaces' resistance to abrasion and corrosion [20,21] as well as to reduce the risks of bacterial colonization [22,23]. TiN coatings, though, had no “bioactive” effect as they increased neither osseointegration nor osteoblastic differentiation and/or proliferation [18,19]. In this context, our group investigates the anabolic effects of plasma vapor deposited (PVD) titanium nitride oxide (TiNOx) coatings. Such coatings can be deposited in submicron thicknesses on titanium, other alloys or polymer surfaces. In vitro, human primary osteoblasts grown on Ti-SLA-TiNOx (TiNOx‐coated Ti-SLA) start their proliferation early

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after seeding. Relative to (bare) Ti-SLA surfaces, the kinetics of cell proliferation is ahead by ca. 50% during the first 2 weeks after seeding without detrimentally affecting the normal differentiation process [24]. TiNOx coatings also open perspectives regarding metal substrates other than Ti as their effects on bone cell proliferation and differentiation is independent of the metal substrate [25]. TiNOx coatings, therefore, might allow the endosseous application of metals such as stainless steels or cobalt–chromium high-performance alloys [26]. In light of encouraging results in vitro, the objective of the present study was to verify the hypothesis that TiNOx coatings would accelerate the initial stages of in vivo osseointegration as well. Materials and methods Experimental design The experiment was conducted on minipigs. The overall time sequence is shown in Fig. 1A. After initial acclimation, six lower premolars (left and right P2,3,4) were extracted under general anesthesia. After a period of 3 months, 6 experimental implants per animal were inserted in the edentulous zones: 2 Ti-SLA (bare SLA-textured titanium), 2 Ti-SLA-TiNOx (TiNOx-coated SLA titanium), and 2 CoCr-SLA-TiNOx (TiNOx-coated SLA cobalt–chromium alloy). Two animals were sacrificed after 1 week, 2 weeks, 1 month and 3 months. The mandibles were block-sectioned and subjected to histomorphometric analysis. Outcomes were analyzed in terms of new bone apposition and bone remodeling. Geometry, fabrication and coating of the experimental implants The experimental implants were designed as metal cylinders, 4.2 mm in diameter and 12 mm in length (Fig. 1B). Forty Ti grade 4 and 20 CoCr (Co60Cr28Mo7) implants were machined to specifications (Divisa Precision, Geneva, CH). The titanium implants were brought to an SLA surface by sand blasting and hot acid etching according to a protocol described previously [24]. The CoCr implants were also prepared to an SLA texture but the protocol was adapted to closely duplicate the texture of Ti-SLA. The surface treatment consisted in grit-blasting using large grit corundum (250 μm) at 7.5 bars, 2× cleaning for 2 min in isopropanol under multifrequency ultrasonics, acid etching (HCl 7.4%–H2SO4 76%, 100 °C, 6 min) followed by nitric acid (30%, 50 °C, 6 min). Finally, the implants were rinsed for

231

5 min under tap followed by distilled water and sonicated again in isopropanol. 20 Ti-SLA and 20 CoCr-SLA implants were coated with a 125 nm TiNOx layer using PVD [24]. The implants' surface roughness was assessed using an electro-mechanical profilometer (Pertometer M1, Mahr, Göttingen, D). The resulting surfaces presented micropits 0.5–3 μm in depth (Supplemental Fig. 1), a texture which was preserved after coating. All Ra values were within the same range, that is, Ti-SLA: 2.49 ±0.34, Ti-SLA-TiNOx: 2.26± 0.66 and CoCr-SLA-TiNOx: 2.75± 0.39 μm. Typical implant cylinders are shown in Fig. 1Ca and b. Animals Eight adult Göttingen minipigs (18–23 months, approx. 40 kg) were used in the experiment (Göttingen minipigs, Ellegaard A/S, Dalmose, DK). The animals underwent an acclimation period of 1 week prior to tooth extraction. In line with Swiss legal requirements, all animal experiments were approved by an academic committee and supervised by the local veterinary agency. Surgical procedure The first surgery (i.e. extraction of the lower premolars) was performed under general anesthesia. To prevent vomiting, the animals were deprived of food 12 h prior to the procedure. A prophylactic antibiotic coverage (amoxicillin, 1 g/day, per os, Sandoz, CH) was dispensed 1 day before and 3 days after the surgery. The animals were first anesthetized by an intramuscular injection of midazolam (Dormicum, 0.5 mg/kg, Roche, CH), azaperone (Stresnil, 0.5 mg/kg, Janssen-Cilag, CH) and atropine (25 μg/kg, Lavoisier, France). A transdermal patch of fentanyl (Durogesic, 50 μg/h, Janssen-Cilag, CH) was then applied at the groin for long term analgesia. Deep anesthesia was induced by intravenous injection of fentanyl (Sinthenyl, 2.5 μg/kg, Sintetica, CH) and atracrium besilate (Tracrium, 0.6 μg/kg, Glaxo-Smith-Kline, UK) before intubation. Deep anesthesia was maintained under a gaseous mixture of 2% isoflurane (Baxter, USA) in pure oxygen. Porcine premolars are slender and friable. Therefore, for tooth extraction, a flap was elevated before the left and right P2, P3, P4 were removed from the lower jaw. In case of breakage, root fragments were removed after conservative osteotomy. Wound closure was achieved with single resorbable loop sutures. One month after surgery, the wounds were examined for healing. The animals were sedated by intramuscular injections of midazolam (dormicum, 0.5 mg/kg), azaperone

A implant placement

tooth extraction

sacrifice of 2 animals block sections

healing day 0

t

3 mos

phase 1 day 0

1 wk

2 wks

1 mo

3 mos

phase 2

B

4.2 mm 2 mm

0.5 mm

12 mm

C

a

b

6 mm

M2 Thread

Fig. 1. (A) Time frame of study. (B) Specifications of implant cylinders. (C) Typical implant cylinders: a: Ti, b: implant with TiNOx coating.

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(Stresnil, 0.5 mg/kg) and atropine (25 μg/kg). The edentulous zones were palpated for smoothness. A few hard tissue remnants were detected which were removed with a forceps. After another 2 months, the experimental implants were placed under general anesthesia (same protocol as above). First a crestal incision was made and a mucoperiosteal flap was reflected. Then the edentulous zones were flattened using a round bur under abundant irrigation with saline. The implant bores were first prepared with 2.2 mm diameter pilot drills and then sequentially enlarged to 4.1 mm. For implant placement, ancillary “holding screws” were tightened into the cylinders' thread and functioned as handles during insertion. Primary stability was achieved after press-fitting the implants into their respective bores. Then the holding screws were removed and replaced with 'closure screws' (Fig. 1B) which remained in situ for the rest of the experiment. Each animal was fitted with 6 implants, that is, two of each experimental surface (Ti-SLA, Ti-SLA-TiNOx, CoCr-SLA-TiNOx). The distribution of the implant types within the edentulous zones was determined from a table of random allocations (SPSS software). Wound closure was again carried out with single resorbable loop sutures. Supplemental Fig. 2 illustrates a typical surgical procedure. The animals were euthanized 1, 2, 4 and 12 weeks after implant placement. Immediately after sacrifice, the jaws were dissected and immersed in a fixative solution of phosphate-buffered saline and 4% formaldehyde.

Histological preparation and histomorphometric analysis First the pertinent zones of the jaws were block-sectioned to isolate the implants. Then the blocks were rinsed for 3 h in tap water to remove the formalin from the tissues. They were dehydrated by increasing the ethanol concentrations in a dedicated agitation system and embedded in polymethyl methacrylate resin (Technovit 7200 VLC, Kulzer). Using a precision band saw (312, Exakt, Norderstedt, D), undecalcified specimens were obtained by placing longitudinal cuts parallel to the long axes of the implants. The specimens were brought to shape by microgrinding to a thickness of 50 μm and polished (400CS, Exakt, Norderstedt, D). They were stained with Sanderson's Rapid Bone Stain (Dorn and Hart, Villa Park, USA) which contains methylene blue and potassium permanganate. The sections were examined under standard microscopy (6000DRB, Leica Microsystems, Wetzlar, D) and stereomicroscopy (MZ16 A, Leica Microsystems). The length of the implants' interface in contact with bone tissue was determined using a software for image analysis (ImageJ, NIH, USA). The amount of new bone-to-implant contact (N-BIC) was expressed as the percentage of the total length of tissue (hard or soft) in contact with the implant surface. The closure screw and artifacts and/or broken tissues were excluded from total length computations. Four specimens of each experimental surface were analyzed at each time point (total: 48 specimens). Among these, four specimens were excluded from the analysis due to unmanageable artifacts (Table 1).

Table 1 Specimens analyzed. Healing period

Implant surface

Number of specimen analyzed (n)

1 week

Ti-SLA Ti-SLA-TiNOx CoCr-SLA-TiNOx Ti-SLA Ti-SLA-TiNOx CoCr-SLA-TiNOx Ti-SLA Ti-SLA-TiNOx CoCr-SLA-TiNOx Ti-SLA Ti-SLA-TiNOx CoCr-SLA-TiNOx

3 4 4 4 4 3 3 3 4 4 4 4 Total analyzed (44)/Total placed (48)

2 weeks

4 weeks

12 weeks

Statistical analysis Data obtained from the different implant surfaces were compared using Wilcoxon's test for paired observations (SPSS software). The null hypothesis was rejected at p b 0.05.

Results None of the 48 implants presented signs of inflammation or did not integrate. One week after implantation, primary stability was essentially due to old bone. There were no notable differences between the 3 surfaces. All implants were surrounded by a highly vascularized soft tissue, with local intratissular foci of new bone formation. The latter originated from old bone or bone debris whose surface was lined with osteoid and osteoblasts (Figs. 2B, K and 3) with incipient attempts at trabecular organization - a process that was more advanced on TiNOx coatings (Figs. 2A, B, J, K and 3). Sparse foci of new bone were also developing at the implants' interface (Fig. 3B), hence the N-BIC of ca. 10% on each surface (Fig. 4). Two weeks after implantation, the N-BIC increased markedly on Ti-SLA-TiNOx and CoCr-SLA-TiNOx, reaching 23% as compared to Ti where an N-BIC of 13% was determined (Ti-SLA vs. Ti-SLA-TiNOx: p = 0.028; Ti-SLA vs. CoCr-SLA-TiNOx; p = 0.04) (Fig. 4). The deposits of osteoid had doubled in thickness (Figs. 2C, D, L, M and 5). Mineralization was under way and new bone was growing to link to the old bony bed (Fig. 5B). Heavy bone remodeling was noted within the old bone (Fig. 5B). The soft tissue gradually gave way to a well-organized osteoid trabecular network (Figs. 2C, L and 5A). The network was more dense on TiNOx coatings with respect to uncoated Ti-SLA (Fig. 2C, L). Moreover, the extensions from the old bone bed were longer and thicker on TiNOx (Fig. 2D, M). After a healing period of 1 month, no differences were evidenced between the three surfaces in terms of N-BIC which now reached a mean value of about 30% (Fig. 4). The new bone deposited on the implants’ surfaces was now largely connected to the old bone and continued to mineralize (Figs. 2E, F, N, O and 6). Continuous remodeling of old bone was observed throughout (Figs. 2E, F, N, O

Fig. 2. New bone apposition kinetics on Ti-SLA and Ti-SLA-TiNOx. The process initiates from bone debris within soft tissue (A, C, E, H, J, L, N, and P) or from larger portions of old bone (B, D, F, I, K, M, O, and Q). At 1 week, the implants are surrounded by a highly vascularized soft tissue, containing bone debris of various sizes (A, B, J, and K). Note that a reduced distance between the implant and old bone promotes the concentration of debris and the organization of new trabeculae as well as early bone apposition (B and K). At 2 weeks, the peri-implant tissue is organized into a network of new trabeculae; the process is more advanced on Ti-SLA-TiNOx (L) as on Ti-SLA (C). Trabeculae originating from the old bony bed have started to mineralize (D and M). Thin layers of new bone are deposited on the implant surface (C, D, L, and M); the coverage of Ti-SLA-TiNOx surfaces (L and M) is significantly larger than that of Ti-SLA (C and D). At 1 month (E, F, N, and O), the mineralized layer and the trabeculae's thickness (i.e. the peri-implant bone density) are increased. After 3 months of healing (H, I, P, and Q), a compact zone of bone has filled the peri-implant space. Note that the new bone originating from large fragments of old bone is much more mineralized (I and Q) when compared to the new bone issued from bone debris (H and P).

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Ti-SLA

233

Ti-SLA-TiNOx

A

B

J

K

C

D

L

M

E

F

N

O

H

I

P

Q

1

2

4

12

Wks

500µm

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% of new bone on the implant surface 70

A

60

BD

BD

50

O O

%

40

**

30 20

*

*

* 200 µm

B

10 0

1 wk

2 wks

1 mo

3 mo

C OB BD

30 25

** Ti-SLA

20

Ti-SLA-TiNOx

% 15

CoCr-SLA-TiNOx

10 5 200 µm Fig. 3. Higher magnifications of Ti-SLA-TiNOx peri-implant zones after 1 week of healing. (A) Bone debris (BD) that are concentrated in close vicinity of the implant serve as initiators for a primary network of trabecular osteoid. New osteons (O) are clearly visible, delimited by rows of osteoblasts. Some of the newly formed trabeculae connect to the implants’ surface (*). (B) A deposit of osteoid at the implant's surface is visible; note the osteoblasts lining (arrows). The soft tissue adjacent to this thin line is rich in capillaries (C), osteoblasts and bone debris. The old bone (OB) as well as the bone debris are lined by osteoid and osteoblasts. This newly formed, still unmineralized bone connects to the implant, either by coating the bare surface or by contacting a pre-existing deposit.

and 6B). The trabecular network of osteoid had grown in thickness, density and mineralization (Fig. 6A). After 3 months, the implants were intimately integrated on about 80% of their surface in a tissue surrounding that was made of approximately 75% new mineralized bone and 25% primary old bone. The N-BIC had doubled on the 3 surfaces, reaching a mean value of about 60%. No significant differences were observed between the 3 surfaces (Fig. 4). Bone density and mineralization had notably increased (Figs. 2H, I, P, Q and 7) and the borders between old primary bone and new mineralized bone tended to fade (Figs. 2I, Q and 7B). New bone deposition was still observed locally in close contact to the implant (Fig. 7A). Discussion The present work aimed at investigating the kinetics of osseointegration of implants coated by a thin layer of titanium nitride oxide (TiNOx) in a minipig model. Titanium– and cobalt–chromium TiNOx-coated implants integrated at the same rate. Further, relative to standard SLA titanium, both demonstrated a net increase of bone apposition after 15 days of healing. TiN and TiNOx coatings are widely used in biomedical and surgical applications. They share the same properties regarding hardness, biocompatibility and their repelling effect for bacterial adhesion. Because of their high hardness, these coatings are suitable for the watch, cutlery and in the tools industries to reinforce surfaces exposed to wear. Their resistance to abrasion also spurred their use in orthopedic joint prostheses as they resist sliding against CoCr and lessen the wear rate of UHMW

0

2 wks

Fig. 4. New bone–implant contact after 1 week, 2 weeks, 1 month and 3 months of healing. Data are expressed as mean ± SE. *: significantly different (p b 0.05) from Ti-SLA implants at the same time point.

polyethylene in vitro [18,27–30]. In addition, they limit the corrosion and ion release of the substrate metal [31,32]. TiNOx surfaces have also found acceptance in cardiovascular applications as they largely prevent restenosis – an insidious form of tissue overgrowth and relapse after a stent has been placed to dilate a coronary artery. Presently, about 80% of angioplasties are treated by stenting, although restenoses are observed in 20–30% of patients [33,34]. On the upside, though, 10 clinical studies (2005–2010) reported highly encouraging results when the stents were coated with TiNOx as compared to bare stents or drug-eluting stents of identical geometry. Histologically, an overgrowth of the smooth muscle from the vessel wall, in addition to a net decrease of the inflammatory process was described. To date, the TiNOx coating's mechanism of action has not been elucidated but could be linked to the presence of nitric-oxide (NO) species on the surface [35]. In cell cultures, the growth rate of human primary osteoblasts is increased by 50% when the titanium substrate is coated with TiNOx. This increase takes place without detrimentally affecting the cells' differentiation [24]. In consideration of the enhanced wear resistance of TiNOx coatings and their accelerating effect on the growth of bone cells, the working hypothesis of the present experiment was that TiNOx coatings could accelerate the osseointegration of titanium implants. Further, they would “isolate” the substrate metal from the bone bed and thus permit endosseous applications of alloys of (possibly) inferior biocompatibility but of vastly superior mechanical strength. SLA titanium was used as the “gold standard” for osseointegration. On Ti-SLA, we produced a roughness of Ra = 2.5 ± 0.34 μm and micropits 0.5–3 μm in depth. This texture was preserved after PVD coating with TiNOx [24]. The surfaces of the CoCr test implants were also brought to an SLA texture and coated with TiNOx. Their Ra was 2.75 ± 0.4 μm (before and after coating) which was not significantly different from Ti-SLA. The microtexture presented pits of about 3 μm and was similar to that found on the Ti-SLA (Supplemental Fig. 1). The similarity

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235

A

A

NB

NB

200 µm

B

OB

OB

200 µm

B

BR

OB

BR

OB

*

*

*

*

200 µm 200 µm

Fig. 5. Higher magnifications of Ti-SLA-TiNOx peri-implant zones after 2 weeks of healing. (A) The trabeculae's thickness is increased and the trabecular network has augmented in density. There is close contact with the implant surface and the size of the deposits has increased; osteoid extensions lined by osteoblasts are visible (white arrows). Note that all the osteoid tissue is lined by rows of osteoblasts (black arrows) and that the surrounding soft tissue has become well organized. (B) The bone deposits at the implant surface are thicker; they increase in size and begin to mineralize. The same observation is made at the surface of the old bone (OB). Note that the link between implant surface and old bone is now firmly established (*).

Fig. 6. Higher magnifications of Ti-SLA-TiNOx peri-implant zones after 4 weeks of healing. (A) The trabecular network is now compact and mineralization is in an advanced stage. The soft tissue compartment has considerably decreased. The implant surface is intimately coated by new mineralized bone (NB). (B) The old bone and the implant surface are firmly linked by new mineralized bone (*). The peri-implant zone continuously fills with bone, as shown by the rows of osteoblasts lining the bone surfaces (arrows). Note the signs of remodeling (BR) in the old bone (OB).

between both surfaces (in terms of roughness and texture) was such that Ti-SLA-TiNOx and CoCr-SLA-TiNox surfaces were considered “micromorphologically equivalent.” Consequently, any change in bone repair in direct contact or near an implant should be attributed to the coating and not to a difference of roughness or morphology of the substrate. CpTi is considered as the material of choice for endosseous dental implants [6]. The typical contemporary dental implant is 10–12 mm in length and 3.8–4.2 mm in diameter. These specifications are large enough to compensate for the mechanical properties of titanium, which are inferior to those of other surgical metals, such 316L stainless steel and CoCr alloys. Yet in some applications, either in the presence of a bone crest of reduced width or small interdental spaces, the clinic calls for implants of lesser diameter. In these instances, the inferior mechanical properties of CpTi may become critical [36]. CoCr alloys are good substitute candidates as they are hard, highly resistant to wear and corrosion and of superior tensile strength and toughness [37]. CoCr alloys are largely used in orthopedic applications whenever strong materials are required (e.g. joint arthroplasties). On the downside, their biocompatibility is inferior to that of titanium. In vitro, for instance, cells were less prone to grow and differentiate on CoCr alloys and under in vivo conditions, a thin fibrous layer was always observed around CoCr endosseous implants [38]. Coating CoCr implants with a thin layer of TiNOx thus appears as an elegant and expedient method to optimize both biocompatibility and mechanical strength. Irrespective of the surface, osseointegration proceeded at a fast pace leading to an N-BIC of ca. 60% and a total BIC of ca. 80% after 3 months of healing. While these rates are congruent with human data [14,15], some animal studies using similar experimental protocols yield higher osseointegration ratios. In a minipig model, Ti-SLA implants integrated to BICs of 20% and 50% at 2 and 4 weeks [12],

while 12%, respectively 30% were found herein. Higher rates were also recorded in dogs [39]. These two experiments, though, were conducted using wound chambers and not plain cylinders as in the present study. Also, the roughness of the present implants was Ra = 2.5 μm while the typical industrial implant is brought to a roughness in the 3.5 μm range (Straumann, Basel, CH) [40,41]. Still, at 3 months, the implants were almost fully osseointegrated as 80% of the surface was in contact with bone. Compared to the bare Ti-SLA surface, TiNOx coatings, either on Ti-SLA or CoCr-SLA, yield significantly larger N-BICs at 2 weeks. This difference, however, was no longer perceivable after 3 months of healing. Except for the accelerated bone growth on TiNOx coatings during the first 2 weeks, we did not observe any difference between the 3 surfaces regarding the general mechanism of osseointegration. In the initially soft and highly vascularized tissue, gradually a new trabecular network developed from structurally intact old bone and isolated bone remnants. The sequence of events made TiNOx appear as a catalyst for bone repair. This observation is in line with data previously published by our group, in which osteoblasts were shown to increase their proliferation on TiNOx as compared to bare Ti-SLA in the first week after seeding [24]. At 2 weeks in vitro, the number of osteoblasts had increased by 1.5 times on TiNOx while in vivo the N-BIC on Ti-SLA-TiNOx and CoCr-SLA-TiNOx was 1.8 times higher than on uncoated Ti-SLA. In effect, these results parallel those obtained with an industrial process in which the surface is rendered more hydrophilic by a special packaging in a controlled atmosphere and marketed under the name “SLA-active.” In pig and dog models, these “SLA-active” implants yield 1.7 times larger BICs after 2 weeks of healing as compared to standard Ti-SLA surfaces [12,15,39].

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A

also is gratefully acknowledged for his histological processing. Our gratitude also goes to Dr. M. Mekki (Hexacath, Paris, France) for coating depositions and hours of constructive discussions. Last but not the least, this study was supported by grant #659‐2009 from the ITI Foundation (ITI, Basel, CH).

References

200 µm

B

OB

NB 200 µm Fig. 7. Higher magnifications of Ti-SLA-TiNOx peri-implant zones after 12 weeks of healing. (A) There is an ongoing increase of the thickness of the bone deposits at the implant surface as evidenced by the rows of osteoblasts lining the bone surface (arrows). Note the gradient of bone mineralization (Δ) decreasing from the implant surface, where a dense bone is observed, to the soft tissue where a delicate layer of osteoid tissue has appeared. (B) An area where all the soft tissue has been replaced by highly mineralized bone structure is shown. Note that the border between old (OB) and new bone (NB) now becomes barely perceptible (white arrows), thus illustrating the degree of the new bone's maturation and mineralization.

Finally and importantly, whatever the alloy (Ti or CoCr), the effects of TiNOx coatings on implant osseointegration were identical, both in terms of BIC and histological healing processes. It thus appears that TiNOx coatings act as a biological barrier that isolates the substrate material from the biological environment. In a recent study on minipigs, rough CoCr implants were allowed to heal for 6 months and yielded BICs of 60% vs. 80% on standard Ti-SLA [42]. Yet in the present study TiNOx‐coated CoCr implants not only provided similar integration after 3 months but also demonstrated accelerated integration after 2 weeks. Conclusion TiNOx coatings on roughened titanium accelerate osseointegration during the first 2 weeks of healing after implantation. Clinically, these findings might translate into an improved short term stability of implants. Further, these observations also apply to TiNOx coatings on roughened CoCr. TiNOx coatings could thus lead to the use of CoCr alloys for endosseous dental implants of reduced diameter implants but of mechanical strength comparable to standard titanium cylinders. These observations, however, require confirmation by long term data on implants under functional conditions. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.bone.2012.09.014. Acknowledgments The authors are indebted to Pr. D. Morel (Department of Experimental Surgery, University of Geneva) and his friendly and efficient team. A particular thanks goes to Mr. J.P. Giliberto for his excellent technical assistance. Dr P. Schüpbach (Schüpbach LTD, Horgen, CH)

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