In vivo evaluation of micro-rough and bioactive titanium dental implants using histometry and pull-out tests

J O U R N A L O F T H E M E C H A N I C A L B E H AV I O R O F B I O M E D I C A L M AT E R I A L S

4 (2011) 1672–1682

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/jmbbm

Research paper

In vivo evaluation of micro-rough and bioactive titanium dental implants using histometry and pull-out tests Conrado Aparicio a,1 , Alejandro Padrós b , Francisco-Javier Gil a,∗ a Cátedra UPC-Klockner, Department of Materials Science and Metallurgical Engineering, Technical University of Catalonia, Barcelona, Spain b Instituto Padrós, Barcelona, Spain

A R T I C L E

I N F O

A B S T R A C T

Article history:

We report on the in vivo histological and mechanical performance of titanium dental

Received 21 May 2010

implants with a new surface treatment (2Step) consisting of an initial grit-blasting process

Received in revised form

to produce a micro-rough surface, followed by a combined chemical and thermal treatment

7 April 2011

that produces a potentially bioactive surface, i.e., that can form an apatitic layer when

Accepted 3 May 2011

exposed to biomimetic conditions in vitro. Our aim was to assess the short- and mid-

Published online 27 May 2011

term bone regenerative potential and mechanical retention of 2Step implants in mandible and maxilla of minipigs and compare them with micro-rough grit-blasted, micro-rough

Keywords:

acid-etched, and smooth as-machined titanium implants. The percent of bone-to-implant

Titanium dental implants

contact after 2, 4, 6, and 10 weeks of implantation as well as the mechanical retention

Osseointegration

after 4, and 6 weeks of implantation were evaluated with histometric and pull-out tests,

Bioactivity

respectively, as a measure of the osseointegration of the implants. We also aimed to

Mechanical retention

assess the bioactive nature of 2Step surfaces in vivo. Our results demonstrated that the 2Step treatment produced micro-rough and bioactive implants that accelerated bone tissue regeneration and increased mechanical retention in the bone bed at short periods of implantation in comparison with all other implants tested. This was mostly attributed to the ability of 2Step implants to form in vivo a layer of apatitic mineral that coated the implant and could rapidly stimulate (a) bone nucleation directly on the implant surface, and (b) bone growing from the implant surface. We also proved that roughness values of Ra ≈ 4.5 µm favoured osseointegration of dental implants at short- and mid-term healing periods, as grit-blasted implants and 2Step implants had higher retention values than as machined and acid-etched implants. The surface quality resulting from the 2Step treatment applied on cpTi provided dental implants with a unique combination of rapid bone regeneration and high mechanical retention. c 2011 Elsevier Ltd. All rights reserved. ⃝

∗ Corresponding address: Department of Materials Science and Metallurgical Engineering, ETSEIB, Universitat Politècnica de Catalunya, Av.Diagonal 647, 08028 Barcelona, Spain. Tel.: +34 934016708; fax: +34 934016706. E-mail address: [email protected] (F.-J. Gil). 1 Present address: MDRCBB-Minnesota Dental Research Center for Biomaterials and Biomechanics, Department of Restorative Sciences, School of Dentistry, University of Minnesota, Minneapolis, MN, USA. c 2011 Elsevier Ltd. All rights reserved. 1751-6161/$ - see front matter ⃝ doi:10.1016/j.jmbbm.2011.05.005

J O U R N A L O F T H E M E C H A N I C A L B E H AV I O R O F B I O M E D I C A L M AT E R I A L S

1.

Introduction

A requisite for the clinical success of titanium dental implants is the achievement of a strong and long-lasting connection between the implant and bone (Buser, 2001). Even though under healthy conditions reliable results with implant success rates of more than 90% after 15 years have been reported (Schwartz-Arad et al., 2005), the improvement of the implant–bone interface for compromised clinical scenarios (Ronold et al., 2003; van Steenberghe et al., 2002), including procedures following immediate loading of the dental implant, is still an open problem (Schliephake and Scharnweber, 2008). The modification of the surface of dental implants can overcome this limitation (Le Guehennec et al., 2007). Implant surfaces with improved topographical, physical, and chemical properties will provide a suitable environment where the natural biological potential for bone functional regeneration can be stimulated and maximized. These therapeutic strategies should ultimately enhance the osseointegration process of dental implants for their immediate loading and long-term success. Surface roughness of dental implants at the micro-level is known to be a very important factor for establishing their clinically-reliable bone fixation (Albrektsson et al., 1981; Bagno and Di Bello, 2004; Buser et al., 1991; Giavaresi et al., 2003; Ronold et al., 2003; Thomsen et al., 1997). Surface topography can be modified by plasma-sprayed coatings, by grit blasting with various types and sizes of abrasives, by acid etching and electrochemical processes with different solutions, or by a combination of some of them (Le Guehennec et al., 2007; Schliephake and Scharnweber, 2008). Results from in vitro studies suggested a positive correlation between surface roughness and cellular attachment and osteoblast-like cell activity (Han et al., 1998). Changes in roughness furthermore correlate with selective protein adsorption, collagen synthesis as well as with the maturation of chondrocytes, which all of them have a significant influence on implant osseointegration (Boyan et al., 1996; Martin et al., 1995; Pegueroles et al., 2010). Implants with microstructured surfaces have been reported to have a more intensive bone implant contact than implants with smooth machined surfaces resulting in higher mechanical retention when implanted in humans (Cochran et al., 1998; Ivanoff et al., 2001). Those are the reasons why most of the dental systems nowadays have a micro-rough surface. In a majority of those implants, the micro-rough surfaces is obtained by grit blasting and/or acid-etching the implant (Chang et al., 1999; Rocci et al., 2003; Wennerberg and Albrektsson, 2000). Although some studies indicated that micro-rough implants can also be successful in compromised clinical scenarios (Iezzi et al., 2005) the bioinert nature of titanium surfaces cannot stimulate a more rapid bone regeneration and mechanical fixation of dental implants and so, are not appropriate for compromised immediate-loading applications. However, the deposition of osseoinductive calcium phosphate minerals, such as apatite, can enhance implant performance at an early stage after implantation (Ronold et al., 2003). The biological nature of apatites, which are the mineral phase in bone, have the potential to actively signal the cells that interrogate the surface after implantation for them to

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rapidly regenerate bone in contact with the mineral coating. A first generation of thick coatings applied on titanium dental implants by a plasma-sprayed or electrodeposition processes showed that response (Geesink et al., 1987; Shrikhanzadeh, 1991). But later, it was also demonstrated that the chemical and structural heterogeneity of the layers obtained resulted in heterogeneous degradation of the layer and eventually mechanical failure and fragmentation (Hulshoff et al., 1997). Then, in vivo reactivity of those fragments resulted in adverse tissue reactions and failure of the implants at mid- and long-term after implantation. New treatments have been developed to obtain coatings of calcium phosphate minerals that overcome the aforementioned problems (Schliephake and Scharnweber, 2008). Among them, the chemical modification of titanium by alkaline etching and further thermal treatment (Kokubo et al., 1996) with potential for growing an apatitic layer under biomimetic conditions at 37 ◦ C still constitutes one of the most promising. The treated titanium is potentially bioactive and so, it might develop the layer of mineral in vivo after implantation. We have developed a new surface treatment, 2Step, for titanium dental implants that combines micro-roughness and potential bioactivity by first, grit blasting, and second, alkaline etching and thermally treating the implant surface. We optimized the properties of the grit blasting treatment (Aparicio et al., 2003; Pegueroles et al., 2008). We also demonstrated the potential bioactivity of the 2Step surfaces (Gil et al., 2002; Aparicio et al., 2007) by growing in vitro hydroxyapatite layers in simulated body fluids. Those implant surfaces have been also tested to prove that they induce preferential differentiation of MG63 cells into the osteoblastic lineage (Aparicio et al., 2002). As a further step, our aim in this work was to assess the short- and mid-term bone regenerative potential and mechanical retention of 2Step implants in mandible and maxilla of minipigs and compare them to micro-rough gritblasted, micro-rough acid-etched, and smooth as-machined titanium implants. We also aimed to assess the bioactive nature of 2Step surfaces in vivo.

2.

Materials and methods

2.1.

Implants

Bar of commercially-pure grade III titanium (cpTi, ASTMB348) was used to machine both screw-shaped implants for histometric analysis and cylinders for pull-out tests. The screw-shaped dental implants were 3.8 mm in diameter and 12.0 mm in length with 1.0 mm pitch and 1.5 mm long collar (Fig. 1(a)). The cylinders were 3.8 mm in diameter and 10.0 mm in length, and had a 1.4 mm diameter transversal threaded hole to adjust the pull-out fixture while performing the mechanical tests.

2.2.

Surface treatments

cpTi implants and cylinders were prepared with four different surface treatments and divided into four groups with 16 implants and 8 cylinders per group:

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a

b

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c

Fig. 1 – (a) SEM picture of an AEtch implant before being implanted; (b) picture showing the location of the implant after surgery in the mandible of one of the animals used; and (c) histology image (×80) showing identification of the tissues for calculating the BIC values.

1. as-machined (Ctr); 2. acid-etched (AEtch) in 0.35 M hydrofluoric acid for 15 s at room temperature; 3. grit-blasted (GBlast) with alumina particles (600 µm size) with 0.25 MPa blasting- pressure until achieving roughness saturation; and 4. grit-blasted and alkaline-etched + thermo-chemical treatment (2Step) obtained as previously described (Aparicio et al., 2002). First, grit-blasting was performed as for GBlast-surfaces. Second, alkaline-etching + thermochemical treatment was carried out as described in Kokubo et al. (1996). Briefly, the cpTi device is immersed in 10 ml 5M-NaOH at 60 ◦ C for 24 h. The device is then thoroughly rinsed with distilled water and dried at 40 ◦ C for 24 h. Following, thermal treatment is performed in a tubular furnace at 600 ◦ C for 1 h. Ctr and GBlast implants and cylinders were provided by SOADCO, S.L., Escaldes Engordany, Andorra. AEtch and 2Step implants and cylinders were obtained at the Technological University of Catalonia labs from Ctr and GBlast implants, respectively. After surface treatments were performed, all implants were ultrasonically cleaned in soap and distilled water for 10 min, dried with nitrogen gas, and sterilized in ethylene oxide at 37 ◦ C and 760 mbar for 5 h. Then, implants were aerated for 42 h before being packed for surgery. Sterilization and packing of all implants was carried out at SOADCO, S.L.

2.3.

Surface roughness and topography

Roughness was evaluated in the framework of the recommendations by Wennerberg and Albrektsson (2000) on topographic evaluation for dental implants. A white light interferometer microscopy (Wyko NT1100, Veeco) was used. The surface analysis area was 189.2 × 248.7 µm2 for the smooth Ctr surfaces and 459.9 × 604.4 µm2 for all the microrough surfaces. Data analysis was performed with Wyko Vision 232TM software (Veeco, USA). A Gaussian filter was used to separate waviness and form from the roughness of the surface. Cut-off values, λc = 0.8 mm, for micro-rough AEtch, GBlast, and 2s surfaces and λc = 0.25 mm for Ctr surfaces were applied, according to previous tests (Aparicio et al., 2003). The measurements were made in three different surfaces of each type of surface treatment to characterize the

amplitude and spacing roughness parameters Ra and Pc , respectively. Ra and Pc were calculated by averaging the values of all individual profiles that were evenly distributed along the surface analysed. Ra (the average roughness) is the arithmetic average of the absolute values of the distance of all points of the profile to the mean line. Pc is the number of peaks in the profile per length of analysis. A scanning electron microscope (SEM) (JSM 6400, Jeol, Japan) was used to qualitatively analyse the surface topography of the implants before being implanted.

2.4.

Animals

Six 6-year-old female minipigs were used following a protocol approved for this study by the Faculty of Veterinary Sciences of the University of Santiago de Compostela. The animals were fed a powder and liquid food diet throughout the study. Oral prophylaxis was performed using aseptic technique within 3 weeks before the initial experimental surgeries.

2.5.

Experimental surgeries

All experimental surgeries were performed at the Hospital Clinico Veterinario Rof Codina of the University of Santiago de Compostela, Lugo, Spain. Mono- and multiradicular extractions of all teeth of each animal were performed four months before the surgical implantation. After exodontias, haemorrhages were spontaneously resolved. Radiographic pictures of the mandible and maxilla of each animal were taken one day before the surgeries were performed to assess appropriate bone regeneration, to discard the presence of radicular pieces, and to plan the location of the dental implants during surgery. Food was withheld the night before the surgery. The animals were preanesthesized with xylazine and ketamine and maintained on gas anaesthesia (5% isofluorane/O2 ). The animals were kept hydrated during the procedure by infusion of lactated Ringer solution. Depth of anaesthesia was continuously monitored measuring heart rate, depth of respiration, and respiratory rate. The animals were frequently toe-pinched and tested for corneal reflex. Implants were located in the mandible and maxilla of the minipigs following a semi-submerged technique; i.e. after

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surgery collars of the implants and above-the-hole areas of the cylinders were exposed and maintained at the gingival level (Fig. 1(b)). Additional lateral incisions were performed to avoid tension in the area of implantation. After locating the implants in the bone bed, flap margins were adapted and tension-free sutured using bioresorbable polyglactin 910c 3-0, Ethicon, USA. Vycril⃝ After surgery, buprenorphine HCl was administered for pain control and amoxicillin for infection control. The animals were routinely monitored for swelling, dehiscences, and infection. Each animal received 4 implants or cylinders with each of the surface finishings. One implant of the same type was placed in each quadrant. Four animals received dental implants and were sacrificed after 2, 4, 6, and 10 weeks after surgery. Two animals received cylinders and were sacrificed after 4, and 6 weeks after surgery.

2.6.

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Fig. 2 – Scheme of the experimental set up used to perform the pull-out mechanical tests.

Histometry

After euthanasia, 16 mm thick block sections including implants, alveolar bone, and surrounding mucosa were collected by cutting with an irrigated diamond-saw (Accutom 50, Struers, Germany) and radiographed. The specimens were thoroughly rinsed in sterile saline solution and immersed in buffered 10% formol solution. The tissue blocks were fixed for 5 days in the 10% formol solution, dehydrated in ethanol solutions (70%, 80%, 96%, and 100% alcohol for 3 days each), and embedded into moulds (Exact 41440–4150, Exact Apparatebau GmbH, Germany) in a photopolymerizable resin (Technovit 7200 VLC, Sulzer, Germany) using a polymerization unit (Exact 520–530, Exact Apparatebau GmbH, Germany). Activation of the polymerization of the resin was achieved by irradiation with yellow and blue light for 12 h. The embedded implants were cut (Accutom 50, Struers, Germany) midaxially in a buccal–lingual plane into sections of approximately 200 µm thick; and further treated using the cutting-grinding technique (Donath and Breuner, 1982) to obtain a final polished 50 µm thick section. Sections were then stained with toluidine blue (Toluidine Blue O, Fisher Scientific, USA) for 20 min. The histopathologic and histometric analysis were performed with a digital camera system (DP12, Olympus, Japan) attached to a light microscope (BX51, Olympus, Japan) and an image analyser software (MicroImage 4.0, Olympus, Japan). ×80 images were taken from end to end of the bottom part of the implant collar. One blinded and calibrated examiner (CA) performed the histometric measurements by taking the pictures as well as discerning and marking straight lines of contact between either soft tissue (blue lines) or bone tissue (red lines) and the surface of the implant (Fig. 1(c)). Osseointegration of the implants was assessed by calculating the percent of bone-implant contact (BIC) along the total length covered by the pictures.

2.7.

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Scanning electron microscopy

Blocks of the different implants from the animal that was sacrificed after 10 weeks of the surgery were also prepared for

inspection of the tissue-implant interface at the ultrastructural level. To that purpose, one block of each type of implant surface was coated with gold and visualized in a scanning electron microscope (SEM) (JSM 6400, Jeol, Japan) with and attached energy-dispersive X-ray Spectrometer (EDAX) (LZ-5, Lynk Analytical, UK) for semi-quantitative elemental analysis.

2.8.

Pull-out tests

The retrieved bones were mechanically-stabilized during the pull-out tests in a customized device. The set-up was adjusted using a level tube to place the test area aligned with the load-cell. A threaded pin with a head was then fit in the prethreaded hole of the implant. To minimize the effect of shear forces in the outcome of the mechanical test, a 350 mm long wire with high rigidity (piano wire) was connected to the loadcell and the pinhead (Fig. 2). An Adamel (MTS, USA) mechanical testing machine fitted with a calibrated load-cell of 1000 N was used to perform the pull-out tests. Cross-head speed range was set to 1.0 mm/min. Force measuring accuracy was ±1%. The load was recorded until loosening of the implant and plotted as load vs. time. The maximum load during the test; i.e. the retention value is reported in this work to compare mechanical stability of the implants with different surface finishing.

2.9.

Statistical analysis

Statistically significant differences among test groups for both histometry and mechanical evaluation were assessed using statistical software (MinitabTM 13.1, Minitab Inc., USA). ANOVA tables with multiple comparison Fisher test were calculated. The level of significance was established at pvalue <0.05. Standard deviations of the test groups and concordance correlation coefficient, ρc , for assessing the reliability of the examiner were also calculated. ρc ranges between 0 and 1 with values close to 1 indicating high reliability. In this study the calculated ρc was 0.98.

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b

300 µm

c

300 µm

d

10 µm

150 µm

300 µm

Fig. 3 – SEM pictures of (a) CTr, (b) AEtch, (c) GBlast, and (d) 2Step surfaces before being implanted. Notable differences in the topography of the implants depending on the surface treatment can be assessed. Insert on the picture for 2Step implants shows nanotopographical features superimposed on the micro-rough topography.

3.

Results and discussion

3.1.

Surface roughness and in vitro bioactivity

The aim of this study was to evaluate the influence of a new treatment, 2Step, on the osseointegration of dental implants at short- and long-term after surgical implantation in minipigs. Thorough surface and in vitro characterizations of the new 2Step surface as well as the controls for this experiment, i.e. grit-blasted, GBlast; acid-etched, AEtch; and as-machined, Ctr cpTi surfaces have been previously reported (Aparicio et al., 2002, 2003, 2007, 2011; Gil et al., 2002; Pegueroles et al., 2008, 2010, 2011). Those characterizations included roughness, wettability, surface free energy, X-ray diffractometry, protein adsorption, osteoblast adhesion and differentiation, corrosion resistance, and bioactive potential by in vitro growing in simulated body fluid and structural integrity of hydroxyapatite coatings. Roughness and topographical features are the most relevant of the surface properties for a dental implant for its clinical success (Buser, 2001). For that reason, we studied here the surface topography (Fig. 3) and surface roughness (Table 1) properties of the implants. GBlast surfaces were significantly rougher than AEtch and Ctr surfaces, and AEtch surfaces were significantly rougher than Ctr in accordance with others (Ohtsuki et al., 1998; Yan et al., 1997; Kokubo et al., 1990; Kokubo, 1997). GBlast and 2Step surfaces did not have significantly-different values of roughness. However, a skeletal nano-porous structure resulting from the alkaline-etching treatment is superimposed to the micro-rough structure resulting from

Table 1 – Mean ± standard deviation of surface roughness parameters Ra and Pc for the different types of cpTi implant surfaces. Implant surface Ctr AEtch GBlast 2Step

Ra (µm) 0.33 ± 0.1 1.69 ± 0.1 4.74 ± 0.2 4.23 ± 0.2

Pc (cm−1 ) 150.9 ± 69 198.3 ± 34 82.1 ± 10 92.1 ± 13

the initial blasting treatment in 2Step samples. The sizes of those nano-topographic features are smaller than the lateral resolution of the technique used to measure roughness. The acid-etched surfaces were characterized by a myriad of small craters and groves. The walls of the craters had a micro-patterned structure and pitting at the bottom of the craters was observed. The blasted surfaces had a heterogeneous surface structure with peaks and valleys of varied geometry showing several flat facets. The facets also had small irregularities appearing as pits and stripes. Also of significance for the discussion of the results reported here is the fact that we previously demonstrated (Gil et al., 2002; Aparicio et al., 2007) the in vitro bioactivity of 2Step surfaces by obtaining homogeneous hydroxyapatite layers using a biomimetic route. The apatite layers on 2Step surfaces were obtained when the 2Step implants were immersed in simulated body fluid at 37 ◦ C for a period of time as short as 3 days. That result suggested that the 2Step surfaces could grow the same type of hydroxyapatite layers when implanted in vivo. Thus, the 2Step implants used in this work were not in vitro coated with a hydroxyapatite layer previously to their

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2 weeks

4 weeks

6 weeks

10 weeks

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Fig. 4 – Representative histological images (×20) of Ctr implants after different times of implantation.

Table 2 – Number of implants failed/total number of implants per type of implant (rows) and time after implantation (columns). Total percent of implants failed are reported in between brackets. Type of implant 2 weeks Ctr AEtch GBlast 2Step Total

0/4 0/4 0/4 0/4 0/16 (0%)

4 weeks 2/4 2/4 1/4 0/4 5/16 (31%)

in vivo implantation. We also decided to do so because 2Step surfaces, without hydroxyapatite layer, induced preferential differentiation of osteoblast-like cells in comparison to Ctr and GBlast surfaces (Aparicio et al., 2002).

3.2.

Failed implants

Table 2 shows the total number and percent of implants that failed per type of surface and period of implantation. Failure in all cases was determined by assessing mechanical instability of the implant in the bone bed, which resulted in the impossibility of treating the sample for the histometric analysis. None of the 2Step implants failed. At least a total of two implants failed for the other type of implants tested. This was an initial evidence of an improved in vivo performance of the micro-rough and bioactive surfaces in comparison to the other types of implants tested.

3.3.

Histology and histometry

Figs. 4–7 show representative histologies at each period of implantation for Ctr, AEtch, GBlast, and 2Step implants,

Implants failed 6 weeks 1/4 1/4 1/4 0/4 3/16 (19%)

10 weeks 0/4 0/4 0/4 0/4 0/16 (0%)

Total 3/16 (19%) 3/16 (19%) 2/16 (12%) 0/16 (0%) 8/64 (12%)

respectively. Only 2Step surfaces showed new immature bone formation around the dental implants after 2 weeks of implantation (Fig. 7). For the other types of implants, after 2 weeks of implantation most of the bone in contact with the surface was originally-machined bone during the surgery procedure in contact with the threads of the implants (Figs. 4–6). This provided good primary stabilization to all implants, even before the hard tissues started to significantly regenerate. That primary stabilization might explain the fact that no implant, irrespective of their surface treatment, failed after 2 weeks of implantation (Table 2). At longer periods after implantation the accumulated micromovements on the implants due to biting could have induced a higher number of failed implants with those surfaces that showed slower bone regeneration rates. The newly-formed and mature bone almost completely colonized large surface areas of both GBlast and 2Step implants after 6 weeks of implantation (Figs. 6 and 7). However, (a) at the same period after implantation GBlast implants still showed some areas of immature bone (Fig. 6); and (b) the same degree of bone colonization was observed on Ctr and

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2 weeks

4 weeks

6 weeks

10 weeks

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Fig. 5 – Representative histological images (×20) of AEtch implants after different times of implantation.

2 weeks

4 weeks

6 weeks

10 weeks

Fig. 6 – Representative histological images (×20) of GBlast implants after different times of implantation.

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2 weeks

4 weeks

6 weeks

10 weeks

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Fig. 7 – Representative histological images (×20) of 2Step implants after different times of implantation.

Fig. 8 – BIC values for the different types of implants after different times of implantation. Straight lines bellow the columns join groups with statistically non-significant differences.

AEtch implants only after 10 weeks of implantation (Figs. 4 and 5).

regeneration. This was our initial hypothesis based in the potential in vivo bioactivity of that type of surfaces.

Thus, the evolution of the regeneration of bone around the different types of implants obtained in this study qualitatively showed that the blasted surfaces with roughness Ra ≈ 4 µm, GBlast and 2Step, had faster tissue colonization than Ctr and AEtch surfaces. The alkaline and thermo-chemically treated surfaces, 2Step, showed the fastest induction of bone

Fig. 8 shows the results of the histometric analyses. The highest BIC were near 60 % after 6 weeks of implantation in GBlast and 2Step surfaces, confirming our previously discussed qualitative evaluation of the regenerated bone. GBlast and 2Step implants had a significantly higher BIC than Ctr and AEtch implants for almost all periods of implantation,

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including 10 weeks, which can be attributed to the effect of the optimized roughness for those types of implants (Anselme, 2000; Boyan and Schwartz, 1999). Statistically significant differences in BIC after 10, 6 and 4 weeks of implantation in comparison with the initial values after 2 weeks of implantation were assessed for AEtch, GBlast, and 2Step implants, respectively. Ctr implants did not significantly increased BIC values during the whole period of implantation tested. Again, 2Step surfaces showed an accelerated regeneration of the hard tissues in comparison to all the other implants tested. Additionally, 2Step implants had a significantly higher BIC than all the other types of implant after 4 weeks of implantation. This further demonstrated that the differences in BIC between week 2 and week 4 for 2Step implants were a consequence of a stimulative effect on bone growth due to their surface treatment. The surface treatment of 2Step surfaces combines appropriate microtopographical features –provided by the blasting treatment– with changes in their chemical oxides –provided by the alkaline etching– with potential induction of beneficial polar and ionic interactions, such as increase in hydrophilicity (Pegueroles et al., 2008) and nucleation of apatite crystals (Aparicio et al., 2007).

3.4.

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a

b

Pull-out tests

Table 3 summarizes the retention values for the differentlytreated cylinders after 4 and 6 weeks of implantation. The results of mechanical retention confirmed most of the results obtained for BIC (Fig. 8). Both, GBlast and 2Step cylinders had significantly higher retention values than Ctr and AEtch cylinders in both periods of implantation. The mechanical retention of AEtch cylinders was significantly higher than the Ctr surfaces. This set of results suggested that the surface roughness at the micro-level is the main variable affecting mechanical retention at the periods of implantation studied. The effect of roughness on mechanical retention was assessed even at short implantation times, when the tissues were far from being fully regenerated around the implants— as in the case of AEtch cylinders after both 4 and 6 weeks of implantation (Fig. 5). It was also notable that during the pullout tests parts of bone remained attached on the surfaces of some of GBlast and 2Step cylinders. This same occurrence did not happen when Ctr or AEtch cylinders were tested (Fig. 9). From our results one could conclude that the higher the roughness, the higher the retention of the implants. However, Ronold et al. (2003) showed that implant surface roughness higher than Ra ≈ 5.0 µm resulted in a decrease in their functional attachment. Thus, both GBlast and 2Step have optimized roughness values, as confirmed by our results. Although small, the differences between GBlast and 2Step cylinders after 4 weeks of implantation were statistically significant different. The fastest growth of bone resulting in higher BIC at the 2Step/bone than at the GBlast/bone interface, as discussed in the previous section, can be the main cause for the improvement of mechanical retention at the short-term after implantation for the 2Step implants. The results and analysis of the in vivo bioactivity of 2Step implants reported in the next section can further confirm

Fig. 9 – Pictures of representative cylinder-shaped implants after pull-out tests. (A) AEtch implant; and (b) GBlast implant with an attached piece of the surrounding bone tissues.

Table 3 – Mean ± standard deviation of maximum load to pull cylinders out of the bone bed (mechanical retention) for the different types of surface treatments after 4 and 6 weeks of implantation. Type of surface treatment Ctr AEtch GBlast 2Step

Retention (N) 4 weeks 60 ± 15 118 ± 23 352 ± 21 385 ± 24

Retention (N) 6 weeks 66 ± 12 140 ± 25 390 ± 35 396 ± 44

this hypothesis. Clinically, this might be relevant because our results suggest that 2Step implants can be functionallyloaded at shorter periods after surgery than the other types of implants. The significant differences in retention values disappeared when the GBlast and 2Step cylinders were tested after 6 weeks of implantation, following the same trends observed for the BIC results (Table 2).

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b

Fig. 10 – (a) SEM picture of the interface between an 2Step implant and bone after 10 weeks of implantation. The image shows the presence of an a apatitic layer on top of the 2Step cpTi implant. Points 1 and 2 are the locations used for EDAX chemical analysis on the apatitic layer and bone, respectively; (b) representative histology (×80) of a 2Step dental implant after 2 weeks of implantation showing the growth of immature bone from the surface of the implant and that nucleated on top of the surface.

3.5.

In vivo bioactivity

Hench, 1991 defined a bioactive material for bone replacing applications as that material forming a layer of hydroxyapatite on its surface in vivo. That is because hydroxyapatite is the mineral phase in our bones and the cells interrogating the surface in vivo will respond accordingly; i.e., leading to the regeneration of bone around the area where the bioactive material is implanted. As aforementioned, the 2Step surfaces demonstrated their potential bioactivity by being able to form a layer of hydroxyapatite in vitro under biomimetic conditions. Here, an analogous hydroxyapatite layer was formed in vivo, as shown in Fig. 10(a). The representative SEM picture showed a layer of crystalline material between the implant and bone on 2Step implants after 10 weeks of implantation. The mineral layer showed a range of thicknesses between 3 and 10 µm. The EDAX chemical microanalysis on the mineral layer (point 1 on Fig. 10(a)) and on bone (point 2 on Fig. 10(a)) showed the presence of Ca and P in both materials with the Ca/P molar ratio being 1.70 and 1.66, respectively. This demonstrated the apatitic nature of the mineral layer formed in vivo on 2Step implants. Moreover, no presence of C was detected in the mineral layer, which suggested that no organic material is associated to the mineral in the interfacial layer. Those results confirmed the in vivo bioactive nature of the titanium surface after alkaline etching and thermochemical treatment. All the rest of implants did not show any presence of interfacial growing of a mineral layer and thus, they were not bioactive. On the one hand, 2Step surfaces and GBlast surfaces did not show significant differences in retention values (Table 3) after 6 weeks of implantation, once the two types of implants have reached their highest BIC values (Table 2). This means that the presence of the apatitic layer did not significantly influence on the mechanical retention of the implants, which reinforces our previous conclusion; i.e., optimal roughness is the main parameter for increasing mechanical retention of dental implants. On the other hand, the acceleration of the regeneration of bone tissue in contact with the 2Step implants can be

attributed to the presence of the apatitic layer. We did not investigated here the time needed to form the apaptitic layers on 2Step surfaces in vivo. However, the apatitic layers were nucleated and homogeneously grown on 2Step surfaces after 3 days of being immersed in simulated body fluid (Aparicio et al., 2007), which suggests that the same layers could be formed in vivo during the first week after implantation. In fact, new immature bone was formed with direct contact on 2Step implants and grown from the surface after short periods of implantation (Fig. 10(b)). That proves a unique osseostimulative effect on the 2Step surfaces. For all the other types of implant the bone reached contact with their surfaces growing from the old bone. In that case, the Ctr, AEtch, and GBlast implants proved the very-well known osseoconductive nature of cpTi.

4.

Conclusions

The new blasted and alkaline-etched+thermally-treated surfaces, 2Step, produced micro-rough and bioactive implants that accelerated bone tissue regeneration and increased mechanical retention in the bone bed at short periods of implantation in comparison with all other implants tested. This is mostly attributed to the ability of 2Step implants to form in vivo a layer of apatitic mineral that coated the implant and could rapidly stimulate (a) bone nucleation directly on the implant surface; and (b) bone growing from the implant surface. Roughness values of Ra ≈ 4.5 µm favoured osseointegration of dental implants at short- and mid-term healing periods, as grit-blasted implants, GBlast, and 2Step implants, had higher retention values than as machined, Ctr, and acidetched, AEtch, implants at 4 and 6 weeks after implantation. The surface quality resulting from the 2Step treatment applied on cpTi provided dental implants with a unique combination of rapid bone regeneration and high mechanical retention during osseointegration. Thus, 2Step implants are good candidates to be used in immediate-loading clinical scenarios.

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Acknowledgements The authors would like to acknowledge the Ministry of Science of Spain for funding this project as well as Klockner, S.L. for donating the implants used. REFERENCES

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