Clin Oral Invest (2014) 18:1495–1505 DOI 10.1007/s00784-013-1120-2


Biomechanical and histological evaluation of four different titanium implant surface modifications: an experimental study in the rabbit tibia José Luis Calvo-Guirado & Marta Satorres & Bruno Negri & Piedad Ramirez-Fernandez & Jose Eduardo Maté-Sánchez & Rafael Delgado-Ruiz & Gerardo Gomez-Moreno & Marcus Abboud & Georgios E. Romanos

Received: 7 August 2013 / Accepted: 24 September 2013 / Published online: 18 October 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Objectives This study presents a biomechanical comparison of bone response to commercially pure titanium screws with four different types of surface topographies placed in the tibial metaphysis of 30 rabbits. Materials and methods One hundred twenty implants were tested double-blinded: (a) blasted, acid-etched, and discrete crystal deposition (DCD), (b) blasted, (c) acid-etched, and (d) blasted and acid-etch. Resonance frequency analysis (RFA/ ISQ), reverse torque values (RTV), and bone-to-implant contact (BIC) were measured at the time of implant insertion (day 0), 15, 28, and 56 days of healing. Results All groups tested demonstrated increased RFA/ISQ and RTV results over the time course. At 15 days, the blasted, acid-etched, and DCD group demonstrated a non-significant trend toward higher values when compared to the blasted and etched group (33.0±16 vs. 26.3±12 Ncm, p =.16). At 56 days, the groups utilizing blasting to create additional surface roughness (Sa>1 micron) showed a statistical significant difference in RTQ versus the non-blasted group (38.5±14 vs. 29.5±9 Ncm, p =.03). Conclusions Within the limitations of this study, only the increase in surface roughness (Ra>1) at 56 days demonstrated J. L. Calvo-Guirado : M. Satorres : B. Negri : P. Ramirez-Fernandez : J. E. Maté-Sánchez Faculty of Medicine and Dentistry, University of Murcia, Murcia, Spain R. Delgado-Ruiz : M. Abboud : G. E. Romanos (*) Stony Brook University, Stony Brook, NY, USA e-mail: [email protected] G. Gomez-Moreno Faculty of Dentistry, University of Granada, Granada, Spain

statistically significant effects on RTQ. Other additional surface features, such as sub-micron scale DCD, demonstrated improved healing trends but without significance for clinical applications. Keywords Implant design . Implant surface . Reverse torque . RFA/ISQ . Surface topography

Introduction Macro- and micro-structural implant design has aimed to improve bone formation around dental implants and to reduce the period of implant integration [1–3]. The vast majority of implants are solid parallel-walled or tapered screws with threads and with some kind of surface modification [4]. Different technologies, including sand-blasting, grit-blasting, acid-etching, anodic oxidation, coating, or combinations of techniques, have been used to change the surface topography in an attempt to increase bone-to-implant contacts (BIC), accelerate bone deposition at the implant surface, and improve implant fixation with bone [5–8]. The use of a rabbit bone model has been suggested by several authors, as a valuable screening instrument to select favorable implant surface characteristics/technologies before the conduction of human studies [9–11]. The development of novel mechanical and chemical surface modifications to improve the osseointegration of dental implants is a topic of interest to clinicians worldwide [1]. Surface topography and roughness have been the focus of dental implant research for more than a decade and evaluated in rabbits [12–15]. The range of surface treatments can be divided into two main groups: additive and subtractive [16–19]. One of the


additive methods is the depositing of DCD (which stand for discrete crystal deposition), a nano-technological technique employed for improving surfaces. This surface modification implies changes to both the surface material and surface roughness [20]. Deposition is achieved by submerging the implants in a crystalline calcium phosphate suspension with crystals, or hydroxyapatite crystals of between 20 and 100 microns [21–23]. In addition, other investigators hypothesized that healing of rough surface implants can be improved by the use of bioactive additives such as nano-crystals of calcium phosphate (CaP) [9], which in the presence of a bio-active implant surface enhances the ability to adsorb proteins, such as serum proteins, osteopontin and laminin, and cell surface protein, and in a similar manner, fibronectin increases the osteo-conductivity capacity of the implant itself. However, it is not clear whether it is the micro-texture of a micro-topographically complex implant surface that plays a key role in enhancing the osteoconductivity of the implant itself, or if the nano-crystals of calcium phosphate (CaP) are the main reason for the increased osteoconductive capacity [14, 24]. The subtractive techniques, including sandblasting, consist of blasting the surface with different sized particles of a usually ceramic material (alumina, titanium oxide, or classic phosphate) or silica (sand) at high speed so that the surface is worn away producing surface roughness of approximately 1.6 microns [1, 7]. Sandblasting and acid etching are among the most frequently used and there is a range of variables arising from combinations of the two [18]. There is a demand today for bioactive surfaces that are able to enhance requested properties, such as cell adhesion, cell proliferation, and secretion of paracrine growth factors. Hydrophilic surfaces were developed, which showed encouraging results regarding optimized implant stability and shortened healing times [25–27]. The characteristics of a rough surface like modSLA are a key factor for the bone’s cellular response around the implant, promoting neovascularization by EPCs and subepithelial connective tissue attachment at the transmucosal part of the implant [28, 29]. Ziebart et al. [30] demonstrated that biological behaviors of EPCs may be influenced by different surfaces. The modSLA surface promotes an undifferentiated phenotype of EPCs that has the ability to secrete growth factors in great quantities. Other studies have shown increases in BIC after submerging implants in aqueous solutions of hydrofluoric/nitric acid [8] or phosphoric acid [31]. Implants submerged in ionic solutions have also been tested but failed to produce increases in BIC or reverse torque, such us the use of the bioactive fluoride-modified surface may show no superiority to the bioinert anodized surface in early bone response [2, 32]. One of the ways of evaluating the surface role in the osseointegration process and bone-to-implant contact is with the 2D histological and 3D reverse torque tests [21, 33, 34].

Clin Oral Invest (2014) 18:1495–1505

In fact, the term ‘integration strength’ is understood as the force required, to break the bone formed around implants. The value measured and compared in trials is the peak force immediately before fracture occurs between bone and implant surface measured in Ncm and subjected to statistical analysis [35, 36]. The objective of the present study was to evaluate the influence of four different surface modifications on the formation of newly formed bone and implant adhesion to it, as demonstrated by biomechanical fracture resistance, in tibia rabbits. Resistance was measured using reverse torque testing and osseointegration was measured by resonance frequency analysis system (Osstell ISQ, Gothenburg, Sweden).

Materials and methods Experiment animals Thirty female 3-month-old New Zealand white rabbits, each weighing approximately 3.5–4.0 kg, were used in the study. The animals were kept in purpose-designed room and were fed and watered ad libitum with standard diet. The study was approved by the University of Murcia’s Animal Experiment Ethics Committee in September 2011 (Animal Health Service, Cattle and Fisheries Directorate, Murcia Region) and followed ethical and legal conditions established by Royal Decree 223, March 14th and October 13th in 1988 on the protection of animals used for research purposes. All surgery was performed in an operating room at the University of Murcia Research Support Service. Implants and surface characterization One hundred and twenty titanium grade 3 chemical and grade 4 mechanical implants with four different surface modifications (Table 1) were inserted in 30 New Zealand white rabbits obtained from the Animal Room at the University of Murcia. Table 1 Mean surface roughness (in μm) of the four different surface treatments, characterized by height and spatial parameters Surface treatment

Sa (SD)

Sq (SD)

Sv (SD)

Blast, etch, and DCD (surface A) Blast only (surface B) Osseotite (surface C) Blast and etch (surface D)

1.37±0.10 1.63±0.14 0.47±0.01 1.37±0.07

1.76±0.13 2.15±0.20 0.59±0.02 1.76±0.09

15.63±1.43 20.60±2.55 5.48±0.66 15.98 ±1.65

Sa arithmetic 3D mean of the departures of the roughness profile from the midline, Sq root mean square parameter corresponding to Sa, Sv average height difference between the five highest peaks and the five lowest valleys

Clin Oral Invest (2014) 18:1495–1505

The surfaces were evaluated with field emission scanning electron microscopy at ×30,000 (JEOL JSM-6500F or JSM7500F; Tokyo, Japan), scanning electron microscopy at ×2, 000 (JEOL JSM-6460LV), and a Light interferometer (Micro XAM, ADE Phase Shift ADE Phase Shift, USA), with a ×312 magnification, 52,400 μm2 measured area, three locations per sample were averaged and 50 μm Gaussian Filter, Inverse Fast Fourier Transformed. Surface A: Sandblasted, acid-etched titanium with DCD (nanometric hydroxyapatite crystals); Sa, 1.37 microns (SB+AE+DCD) Surface B: Sandblasted titanium; Sa, 1.63 microns (SB) Surface C: Double acid-etched titanium; Sa, 0.5 microns (AE) Surface D: Sandblasted and acid-etched titanium; Sa, 1.37 microns (SB+AE) The following images show the different surfaces under scanning electron microscopy (SEM) with chemical analysis (Fig. 1). They were threaded conical implants of 3.25 mm diameter and 8.5 mm length (Biomet 3i, Palm Beach Gardens, Florida, USA), adapted to the anatomy of rabbit tibiae. Study groups The animals were divided into three groups (n =10) according to the time between initial surgery and euthanization time (healing period). Surgery was performed on two tibiae of each rabbit, placing two implants per tibia, four implants per rabbit, one in each study group. Premedication and anesthesia The rabbits were anesthetized with an intramuscular injection of tiletamine/zolazepam 15 mg/kg (Zoletil 50, Virbac, Madrid, Spain) and xylazine 5 mg/kg (Rompun, Bayer, Leverkusen,

Fig. 1 SEM images of all dental implant surfaces used in the study


Germany). Before surgery, the shaved skin over the area of the proximal tibia was washed with (Betadine®, Meda Manufacturing, Burdeos, France). Ketamine hydrochloride (Ketolar® Pfizer, Madrid, Spain) was administered as an anesthetic at 50 mg/kg IM. A preoperative antibiotic (Amoxicillin, Pfizer, Barcelona, Spain) was administered intramuscularly and 3 ml lidocaine at 2 % was also administered intramuscularly in the surgical area of each leg, with 0.01 mg/ml epinephrine. Surgical procedure An incision was made with a number 15 scalpel in the hind leg; this was a total thickness incision reaching from the upper part of the tibia for a length of 15 cm. Periosteum was gently lifted to expose the surgical area taking the tibial plateau as a reference point. A site for the first, more distal implant was marked with a graphite pencil. A second implant site was marked in the upper part of the tibia at a distance of 10 mm from the first. The surgical site was prepared using three drill bits (Biomet 3i, Palm Beach Gardens, FL). Four implants were placed in each rabbit (one of each group), two per tibia (Fig. 2). A RFA/ ISQ smart peg was screwed to each implant and once the implants were fully inserted initial stability was tested by a resonance frequency analysis system (Osstell ISQ, Gothenburg, Sweden) at the day of implant insertion (day 0). Two measurements were taken per implant, one corresponding to the outer aspect and the other to the inner, one externally and the other internally, respectively. A 2.9-mm cover screw was placed on each implant (cover screw, titanium alloy, Biomet 3i, Palm Beach Gardens, FL). When the RFA/ISQ evaluations had been taken, the sites were sutured with 3-0 silk (Laboratorios Aragó S.L. Barcelona, Spain) and a plastic spray dressing applied (Nobecután, Laboratorios Inibsa, Madrid, Spain) in order to facilitate


Fig. 2 Clinical and radiographic situation of new surface implants placed in bone tibia rabbits

healing. Then each of the tibiae underwent digital radiography observation using the Kodak system (Kodak RVG 6100 Digital Radiography System, Rochester, NY; Fig. 2). The animals were returned to their cages immediately to prevent unexpected reaction when they regain consciousness following anesthesia, which took place gradually without any need for postoperative medication. Randomization This was a double-blinded, randomized study, whereby the researchers remained unaware of which implant surface was which. The four implants in each rabbit, one of each group, were distributed in the right and the left tibia, allocating the implant surface types to sites by means of the web site http://, study number 24180. Animal euthanization

Clin Oral Invest (2014) 18:1495–1505

that any bias that this might cause in the course of reverse torque testing would be kept to a minimum. Once set in plaster, RFA/ISQ values were obtained following exactly the same procedure as the previous evaluations (Osstell ISQ Gothenburg, Sweden). Reverse torque was measured using a digital reverse torque meter (Mark-10 Corporation, Hicksville, NY, USA). This device provides completely reliable evaluation of the torque required to remove integrated implants (Fig. 3). All data coming from Osstell® device and reverse torque device were evaluated by the statistical analysis Department, University of Murcia, Spain. Histological preparation Two rabbits per study period—euthanized at 14, 28, or 56 days—had been kept apart for histological and histomorphometric analysis. These samples were submerged in 10 % formalin solution for 24 h and then washed in running water for a further 24 h. Then the samples were dehydrated in graded ethanol solutions from 70 % to 100 %. When dehydrated, the samples were set in resin methacrylate blocks (Technovit 7200, Heraeus Kulzer, Wehrheim, Germany) following the manufacturer’s recommendations. After 2 months, the blocks were cut in half, slicing the implants in two, preparing one half for SEM observation (Fig. 4) and optical microscope observation. These were made into laminas with a 0.5 mm thickness and mounted on

The rabbits were euthanized for implant analysis by means of intravenous overdoses of Sodium Thiopental at 2 %, 1 g in 50 ml of physiological saline (NaCl 0.9 %; Pentotal® Braun Medical, Melsungen, Germany), causing instant cardiopulmonary arrest. The 30 rabbits were sacrificed at 3 study times: 10 animals at 15 days, 10 at 28 days, and 10 at 56 days. Sample collection Longitudinal incisions with lateral approach were made into the rabbit legs to obtain the bone samples. When the tibia had been located, this was extracted by removing the complete tibia from the knee to the ankle. After complete periosteum removal and the elimination of all adjacent fibers, a second digital radiograph was taken (Kodak RVG 6100 Digital Radiography System, Carestream Dental Inc. Rochester, NY) and sequenced correctly. The extracted bone samples of one rabbit from each study period were kept back for histological and histomorphometric analysis and SEM observation. In order to stabilize the remaining samples, boxes were fabricated carefully positioning the samples before filling with plaster, with all samples positioned in the same way and held with the same force, so

Fig. 3 Reverse torque device (Mark 10) used in the study

Clin Oral Invest (2014) 18:1495–1505


Fig. 4 Composed images that are showing on the top SEM images of samples of each group, in the middle images of the EDX analysis the implant-bone interfaces and at the bottom the elements distribution. a)

Sandblasted, acid-etched titanium with DCD; b) Sandblasted titanium; c) Double acid-etched titanium; d) Sandblasted and acid-etched titanium, after 56 days

aluminum blocks coated in carbon (using a Polaron sputter coater). They were examined using EDX at a working distance of 19 mm, with 15 kV, acceleration and ×159 magnification using the Oxford Instruments INCA 300 EDX System (Abingdon, Oxfordshire, UK) to obtain SEM images and quantify the elements comprising bone surround the implants. Optical microscope samples were polished and mounted on acrylic plates and dyed with Levai Laczko and then examined under the microscope (Leica microscope Q500Mc, Leica DFC320s, 3,088×2,550 pixels, Leica Microsystems, Barcelona, Spain). The most central areas of the images were analyzed using Microm Image Processing Software 4.5 (Consulting Image Digital, Barcelona, Spain) connected to a Sony DXC-151s 2/3-CCD RGB Color Video Camera (Sony Electronics Inc., San Jose, CA, USA), which provided images used to measure BIC.

also applied to detect significant differences between the four surface modifications tested. Mean values of initial stability quotient (RFA/ISQ), removal torque value (RTV), and boneto-implant-contact (BIC) percentages were calculated and subjected to a two-tailed analysis of variance to test for significant differences between the four investigated surfaces. The first factor was surface topography and the second the block (e.g., subject) factor. Statistical testing was carried out at the 5 % significance level. A p value

Biomechanical and histological evaluation of four different titanium implant surface modifications: an experimental study in the rabbit tibia.

This study presents a biomechanical comparison of bone response to commercially pure titanium screws with four different types of surface topographies...
2MB Sizes 0 Downloads 0 Views