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available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jmbbm
Research paper
Relationship between surface properties (roughness, wettability and morphology) of titanium and dental implant removal torque Carlos Nelson Elias a,∗ , Yoshiki Oshida b,c , José Henrique Cavalcanti Lima d , Carlos Alberto Muller e a Biomaterials Laboratory, Instituto Militar de Engenharia, Pr Gen Tibúrcio 80, 22290-270 Rio de Janeiro, RJ, Brazil b Indiana University, United States c Department of Mechanical Engineering, Syracuse University, Syracuse NY, 13244, United States d INCO 25, Av Rio Branco 124, Rio de Janeiro, RJ, Brazil e Instituto Oswaldo Cruz, Av Brasil 4366, Rio de Janeiro, RJ, Brazil
A R T I C L E
I N F O
A B S T R A C T
Article history:
The biological properties of titanium depend on its surface oxide film. Several mechanical
Received 15 April 2007
and chemical treatments have been used to modify the surface morphology and
Received in revised form
properties of titanium dental implants. One possible method of improving dental implant
19 December 2007
biocompatibility is to increase surface roughness and decrease the contact angle. In the
Accepted 20 December 2007
present work, the biological properties of dental implants were investigated through in vivo
Published online 31 December 2007
and in vitro tests. The effects of surface roughness, contact angle and surface morphology on titanium dental implant removal torque were investigated. Machined dental implants
Keywords:
and discs made with commercially pure titanium ASTM grade 4 were submitted to
Dental implant
sandblasting treatments, acid etching and anodizing. The sample surface morphologies
Removal torque
were characterized by SEM, the surface roughness parameters were quantified using a laser
Contact angle
non-contact profilometer, and a contact angle measurement was taken. Dental implants
Surface implant treatment
were placed in the tibia of rabbits and removed 12 weeks after the surgery. It was found
Implant roughness
that: (i) acid etching homogenized the surface roughness parameters; (ii) the anodized
Wettability
surface presented the smallest contact angle; (iii) the in vivo test suggested that, in similar conditions, the surface treatment had a beneficial effect on the implant biocompatibility measured through removal torque; and (iv) the anodized dental implant presented the highest removal torque. c 2007 Elsevier Ltd. All rights reserved.
∗ Corresponding author. Tel.: +55 21 2546 7244; fax: +55 21 2546 7244. E-mail address:
[email protected] (C.N. Elias). c 2007 Elsevier Ltd. All rights reserved. 1751-6161/$ - see front matter doi:10.1016/j.jmbbm.2007.12.002
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1.
Introduction
Initially, the clinical dentist used dental implants only to improve a patient’s masticator function. Nowadays, the typical patient wants more than an improvement in masticator function; other desires include aesthetics, short treatment time and procedural reliability. In order to meet patients’ needs, dentists have been seeking dental implants whose manufacture allows for easy surgical procedures, various prosthetic options, good aesthetic solutions, prosthetic components with dimensional accuracy, and superstructures with mechanical stability. Prosthetic stability minimizes rotation and reduces the possibility of screw loosening in unitary prostheses. Dentists also prefer implants with surface treatments that accelerate osseointegration mechanisms, and with geometries that provide good primary stability, good loading distribution and maintenance of osseointegration (Buser et al., 2004). To meet the demands of both the professionals and their patients, manufacturers invest in research and development of new products. Each year, new dental implant systems with different shapes, sizes, surface treatments and prosthetic components are sold. The conventional dental implant protocol proposed by Professor Branemark requires that the procedure be done in two stages. In the first stage (surgical stage), the preparation of the alveolus and the implant installation are performed. In the second stage (the prosthetic stage), the prosthesis is molded, made and installed. Only after these stages is the implant ready to function. According to this procedure, there is a wait time of 4–6 months between the surgical stage and prosthetic stage for healing. During the healing period, there is significant reduction in the patient’s comfort, especially when installation of the temporary prosthesis is difficult. The first week is uncomfortable for patients, and their function during the healing time is limited. The success and loading resistance of dental implants is determined by their physical–chemical surface properties. To support cell attachment, spread and growth, and to improve cell function, a number of reports have been published concerning roughened (Martin et al., 1995; Schwartz et al., 1996) implant surface treatment (Martin et al., 1995; Schwartz et al., 1996; Chehroudi et al., 1997; Brunette, 1988) as well as controlled microtopography (Martin et al., 1995; Schwartz et al., 1996; Chehroudi et al., 1997; Brunette, 1988). Over the years, the dental implant protocol proposed by Branemark has been altered (Cochran and Morton, 2004). Currently, there are some dental implant protocols that describe conditions for immediate loading (installation and prosthetic activation within 48 hours). The treatment options using immediate prostheses loading have been developed to minimize the patient’s masticator function disability during the healing wait time. The overall success rate for immediate prosthesis healing of the edentulous patient is similar to that for the traditional 2-stage method. The change in the protocol can be attributed to better knowledge of surgical care, an understanding of implant biomechanics, implant shape modification and the development of new surface treatments to obtain better biological response from titanium.
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According to Sul (2002), healing around the machined titanium implant occurs through a gradual mineralization process from the bone towards the implant. The cells in contact with the turned surface allow the bone mineralization, but titanium does not act as an inductor. Besides this, the healing process occurs over several days, and the re-modeling process takes weeks or years. The healing time for dental implants without surface treatment is higher than that for implants with treated surfaces. With smooth surfaces, the biological processes at the bone–implant interface are slower, and the properties of the native titanium oxidized layer take a longer time to be affected. To minimize the mineralization time, titanium surface treatment is carried out. This procedure accelerates the adhesion micromechanisms between the implant and the bone. With surface treatment, it is possible to change the surface features of the titanium dental implant, such as chemical composition, energy level, morphology, topography and roughness (Upp et al., 2006). Surface chemical changes include hydroxyapatite deposition and the incorporation of calcium ions, phosphorous and fluorite (Ellingsen et al., 2004). Sandblasting and acid etching treatment can change the surface topography and energy. The morphology and roughness can be controlled by treatment with acid solution or oxidation. The data in the literature show that these modifications have their advantages and disadvantages (Martin et al., 1995; Schwartz et al., 1996; Chehroudi et al., 1997; Brunette, 1988). In most cases, they improve the success rate of the surgery procedure, but in others, they cause inflammatory complications in the tissue close to the implant (Rosenberg et al., 2004). Normally, the dental implant manufacturer develops the implant’s shape, topography, morphology and roughness. After several years of use, some commercial dental implant systems with positive clinical results and extensive documented scientific studies have been abandoned and replaced by new systems. On the other hand, some new systems that do not have clinical approval have been sold and described by the manufacturers as capable of providing better benefits (Albrektsson and Wennerberg, 2004a,b). Currently, dental implant manufacturers use commercially pure titanium and titanium alloy (Ti6Al4V) with a treated surface in order to optimize the osseointegration process. To improve the healing process, some cells are added to the surgical hole as growth factors; these include PGDF (platelet derived growth factor), BMP (bone morphogenetic proteins), IGF (insulin growth factor) and TGF-b (transforming growth factor) (Sycaras et al., 2004). However, the results obtained up to this point have not been as expected. The objective of the present work is to compare some features of commercial dental implants through the following: (i) to treat the dental implant surfaces of available commercial implants; (ii) to characterize the surface morphologies of machined, acid etched, sandblasted and electrochemical anodized dental implants; (iii) to measure the roughness and the contact angle of the surfaces before and after treatment; and (iv) to indirectly quantify the implant’s osseointegration process through the removal torque from the tibia of rabbits. The goal of these treatments
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is to improve the surface roughness and consequently the osseointegration, fixation, and stability of the dental implant. Some authors, such as Lim and Oshida (2001), consider other parameters more important than surface roughness for ensuring biocompatibility. They cite the angle of contact, which can be used to determine the surface free energy. Based on this citation, the surface wettability was also measured in this work.
2.
Materials and methods
A total of 140 dental implants were machined from a 5 mm bar of grade 4 unalloyed Ti (ASTM F67; “Unalloyed titanium for surgical implant applications”). The implants were screw shaped with a nominal outer diameter of 3.3 mm, pitch height 0.6 mm and length 6.0 mm. The implants were supplied by Conexão Sistemas e Protese (São Paulo, Brazil), machined, and subjected to surface treatment with the objective of replicating commercial dental implant morphologies. The titanium dental implants were treated by acid immersion, sandblasting and anodizing as the proprietary embodiment of the Conexão System dental implant surface technology. The samples were divided into four groups: — Machined Group: machined implant without surface treatment; — Acid Group: acid etching implant surface (hydrochloric acid and sulfuric acid solution); — Sandblasted Group: sandblasting implant with TiO2 particles (70 µm) and acid etching surface (hydrofluoric acid/nitric acid); — Anodized Group: anodized implant surface (anodic oxides were prepared by galvanostatic mode up to the high forming voltage of dielectric breakdown and spark formation) (Black, 2006). With the objective of quantifying implant roughness and hydrophobicity, 120 disks of commercially pure titanium (ASTM grade 4) with a 5 mm diameter and a 3 mm height were prepared. Commercially pure titanium discs were machined from the same bar used to manufacture the dental implants. The disks were gradually wet ground with 120-grit alumina paper. The ground and cleaned discs then received the same surface treatment as the dental implants and were sterilized by cobalt gamma irradiation (25 kGy). The ground, cleaned and sterilized discs were then divided into four groups: nontreated (machined), acid etched, sandblasted and anodized. The surface morphology (of two implants and two discs from each Group) was observed on a scanning electron microscope (SEM, JEOL 5800LV, Japan) with energy dispersive spectroscopy (EDS) for chemical analysis. Surface roughness parameters (of three discs from each Group) were measured two-dimensionally in noncontact mode by a laser profilometer (Perthometer Concept, Mahr GmbH, Brauweg 38 Gottingen, Germany). The mean roughness (Ra), peak-to-valley roughness (Rz), quadratic average roughness (Rq) and maximum roughness height (Rmax) were calculated as typical height parameters. Space descriptive parameters were calculated, including highest peak (Rpkx), highest valley (Rvkx), highest difference between
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the maximum and the average surface heights (Rp), peak area (A1) and valley area (A2). This range of parameters was selected to include the relevant parameters able to describe and explain the investigated wetting behavior and removal torque. The contact angle measurement (of 5 discs from each Group) was performed with a Contact Angle Goniometer (Ramé-Hart Inc. USA). Distilled water was used as a reference, and the results were compared with the contact angle of liquids with different hydrophobicity: (i) highly hydrophilic liquid (0.145M NaCl); (ii) lightly hydrophobic (dimethylsulfoxide — (DMSO)); and (iii) donor’s human blood (O+), which had not been submitted to any medication. Five samples of each surface treatment were used, and three readings were made with the application of 5 ml drops. The contact angle considered for each surface–liquid combination was the arithmetic average of 15 readings. To confirm the results, a second experimental contact angle measurement was taken. These surface contact angle measurements were conducted at room temperature, distilled water was used, and one microliter droplet was used. During the wettability test of the anodized samples, as soon as one droplet was placed on the surface, water started to be absorbed to some extent, and the reading was initiated after 5 seconds. The same procedure was applied to all other surfaces. The droplet height “h” and width “d” were measured, and the contact angle “θ” was calculated as follows: θ = 2 tan−1 (2h/d) [degrees]. The first biomaterial biological behavior is determined using in vitro techniques (cell, culture or simulated body fluids). After the in vitro test, it is common practice to test new implant materials, or old materials with different surface treatments, in whole-animal tests, usually employing rabbits, pigs or dogs as animal models. Although the use of nonhuman species involves many limitations and compromises, the evaluation of the dental implant osseointegration through in vivo tests is widely used by dentistry and implant producers. This decision is based on the assumption that such tests, involving the exposure of materials to systemic physiological processes, are necessary, practical and ethical precedents to human clinical testing (Black, 2006). A rabbit tibia model of osseointegration was used. Eight sterilized dental implants with each finished surface were inserted in the tibia of New Zealand rabbits. Rabbits weighing between 5.0 and 6.0 kg were selected, all of which were treated according to the FIOCRUZ (Rio de Janeiro, Brazil) research protocols and Norms of work with animals. The implants were removed 12 weeks after the surgery, and the peak removal torque was recorded using a digital torquimeter. All surgery procedures were performed as described by Morais et al. (2007). The measurements of the removal torque were performed according to the methodology of Morais et al. (2007) and Elias et al. (2006). Removal torque has been used as a biomechanical measure of anchorage, or endosseous integration; greater torque required to remove implants may be interpreted as an increase in the strength of bony integration.
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Fig. 1 – SEM micrographs of the dental implants surfaces. (A) machined, (B) acid etched, (C) sandblasted and (D) anodized.
Table 1 – Mean value ± SD of titanium cylinder surface roughness parameters Group Machined Acid etched Sandblasted Anodized
Ra (µm)
Rq (µm)
Rz (µm)
0.65 ± 0.11 0.51 ± 0.10 0.75 ± 0.05 0.87 ± 0.14
0.81 ± 0.17 0.71 ± 0.07 0.98 ± 0.04 1.12 ± 0.18
6.09 ± 0.37 5.09 ± 0.46 5.55 ± 0.21 5.14 ± 0.69
Rmax (µm) 7.76 ± 1.37 6.78 ± 1.33 12.44 ± 9.7 19.84 ± 2.13
To determine any statistical difference between the Groups, the data were subjected to a one-way analysis of variance (ANOVA) and Tukey’s HSD test. To determine any statistical significance among the values, data obtained were subjected to an independent-sample t-test. Significance was determined at 95% confidence level.
3.
Results
Fig. 1 shows the dental implant surface morphologies after treatments. No morphological difference was observed among the disks and dental implants finished with the same surface treatment. However, it was possible to observe that the prepared surface morphologies were similar to some commercial dental implants available from various companies. The dental implant with acid etching presented imR plant surface morphology similar to Master Plus (Conexão R
Sistemas e Protese) and Frialit Plus (Maillefer, Swaziland). The sandblasted Group presented implant surface morpholR (Institut Straumann AG, Waldenburg, CH), and ogy like SLA the implant surface morphology of the anodized Group was like TiUnite surface (Nobel Biocare, Goteborg, Sweden) and
Rpkx (µm) 21.6 ± 0.41 1.77 ± 0.37 6.75 ± 0.76 16.71 ± 2.47
Rvkx (µm) 3.14 ± 0.52 2.74 ± 0.42 9.91 ± 1.71 6.25 ± 1.23
A1 (µm2 ) 24.71 ± 5.42 34.76 ± 7.35 99.75 ± 6.76 97.67 ± 11.43
A2 (µm2 ) 70.66 ± 16.20 103.86 ± 14.80 190.13 ± 4.90 215.37 ± 1.67
R Vulcano Actives (Conexão Sistemas e Protese, São Paulo, SP – Brazil). The mean value ± SD of the titanium cylinder surface roughness parameters are displayed in Table 1. The dental implant surface treatment significantly changes the roughness parameter values, which influence the cell surface interaction as measured by implant removal torque. Multiple comparison Tukey’s HSD tests demonstrated a statisticallysignificant difference in Ra between the acid treatment and the other surface treatments, except for machined. The machined surface has grooves, which increased the roughness. The surface area (A1 and A2 in Table 1) after sandblasting and anodizing increased; statisticallysignificant differences were found between these groups and the others. Table 2 shows the dental implant removal torque and contact angles. The surface treatment changes the surface wettability. For all contact angle tests, a statisticallysignificant difference (P < 0.05) was noted between the values for the anodized surface and the other surface treatments. A statistically-significant difference (P < 0.05) in the contact angle of water with titanium occurred for the anodized group only. The contact angle of NaCl (highest hydrophilic liquid tested) with the anodized surface showed the smallest
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Table 2 – Mean value ± SD contact angle of treated titanium discs surfaces and dental implant removal torque Implant treatment Water Machined Acid etched Sandblasted Anodized
85.20 ± 3.55 96.24 ± 9.20 79.86 ± 4.85 47.25 ± 2.94
Mean value ± SD contact angle (degree) DMSO NaCl 72.4 ± 2.3 68.7 ± 2.5 52.5 ± 2.6 38.3 ± 2.1
value. No statistically-significant difference (P < 0.05) in the contact angle between blood and any titanium surface was observed, except between blood and the anodized surface. Comparing the contact angle of the same liquid with different treatment surfaces, the anodizing treatment produced the most hydrophilic surface. Table 2 shows that the removal torques of dental implants with treated surfaces are higher than those with machined surfaces. This behavior has been ascribed to the greater union between the de novo bone and the microroughness surface on the implant. A statistically-significant difference (P < 0.05) in removal torque occurred for the anodized group only.
4.
Analysis and discussion of the results
Machined titanium dental implants were modified in order to improve implant wettability, increase the surface available for bone ingrowth and fixation, and/or increase blood clot retention on the material’s surface. The data in Tables 1 and 2 show that the topography features and physical and chemical properties of the titanium dental implant surface can be modified with treatment and can improve osseointegration mechanisms. In the present work, the secondary dental implant stability measured through removal torque has been considered as indicative of osseointegration. While primary stability is a function of bone quality and dental implant design, secondary stability is more dependent on the union between cells and the implant surface. Ogawa et al. (2002) have observed that the topography of the implant surface could induce phenotypic cell alteration during healing. The results in Tables 1 and 2 show this tendency. Success warranty occurs when there is an attachment of the cells on the implant surface, and the activation mechanisms of certain genes (bmp-2, for instance) occur. These phenomena involving transcription factors (osterix, for instance) characterize the differentiation of bone cell formation. Consequently, it is important to analyze the biological signs of the cells that identify titanium as the appropriate biomaterial for use in dental implants, and the signals of the necessary conditions for adhesion and bone formation to occur. Ideal osseointegration is an adhesion of the de novo bone to the implant surface. However, it is important to consider that between the bone tissue and the implant exists a thin amorphous structure layer of glycoprotein rich in proteoglycans, glicosaminoglycans, collagen and fibronectin. It is currently accepted that osseointegration occurs through the formation of a fine layer composed of an extracellular matrix, mainly rich in proteoglycans, at the bone–implant interface (Sycaras et al., 2004; Ogawa et al.,
51.6 ± 1.8 44.4 ± 1.6 43.0 ± 0.9 25.3 ± 1.3
Removal Torque (N cm) Blood 66.4 ± 0.5 61.6 ± 0.7 61.8 ± 0.1 55.5 ± 0.3
57.0 ± 18.6 75.45 ± 10.5 72.15 ± 14.9 83.15 ± 12.7
2002). An activated osseointegration mechanism is a function of previous implant surface treatment and the presence of some components of an extracellular matrix or some of their related peptides (RGD, for instance), which serve as a signal and transmit instructions to cells to adhere to the surface, spread, differentiate and induce bone tissue formation. The osseointegration process is a function of cell contact with the implant surface and cell surface adhesion. Consequently, the implant’s surface wettability influences the cells’ behavior. In the present work, the anodizing surface presented the lowest contact angle. However, cell adhesion to the surface of metallic implants is not enough to guarantee implant osseointegration. The cells must receive some signs or information to induce them to differentiate and proliferate. In this system, the material surface characteristics (morphology, roughness, macroporosity and microporosity) are very important. The implant topography, chemistry and surface energy play an essential part in cell adhesion on dental implants, especially in terms of osteoblast adhesion. Thus, attachment, adhesion and spreading are the first phases of interaction between the cell and the biomaterial, and the quality of this first phase will influence the proliferating cells’ capacity to differentiate themselves on contact with the implant. For instance, coating the titanium surface with a protein BMP-2 induces the division of the osteoblasts after 92 hours of adhesion (Sycaras et al., 2004). The removal torque shows that anodized implants have the best surface feature. The fibronectin present in the environment or the protein associated with the surface of the implant reduces the time of cellular division of human osteoblasts to 6 hours. This phenomenon is ascribed to the fact that the fibronectin has a sequence of amino acids (RGD) that signals the activation of cell cycles to the human osteoblasts and, consequently, initiates division. Therefore, the absorption, adhesion and behavior of the cells on the surface of the implants should be analyzed in order to look for a means of accelerating cell division. At the same time, it is also important to avoid apoptosis or cellular death when these cells are in contact with the implant surface. The removal torque results of the present work show that the osteogenic cells recognize important material surface properties, like surface texture, and accordingly adjust their attachments. The composition of the protein films on the surface of the biomaterial influences the whole tissue adjacent to the host, and this could lead to alterations in the coagulation time, absorption of the cells and tissue repair in general. The thickness of the adsorbed protein layer on the surface of the titanium implants is 1 to 10 nm. Osseointegration is faster, for instance, once superficial
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treatment with Ca++ ions is undertaken and compatibility with the host increases (Sul et al., 2002). Controversies related to the influence of roughness in bone formation exist. Suzuki et al. (1997) observed larger bone formation in implants with roughness. Wong and Eulenberger (1995) verified that treatment of the surface with acids does not increase bone formation on the surface of the implant. To obtain much needed answers concerning the phenomena involved, it is necessary to analyze the influence of the physical parameters (topography and texture at micrometer and nanoroughness scales), physiochemical aspects (electrostatic interaction, superficial tension, hydrophobicity and connection forces) and biochemical aspects involved in the process. This analysis is complex due to the difficulty in isolating the influence of each of the parameters mentioned. The peak removal torque needed to loosen the dental implant screws recorded in the present work can be explained by Takeuchi et al. (2005) and Brunette’s (2001) results. Similar to these studies, it was observed that the implant surface morphology affects titanium biocompatibility and cell function. The surface roughness changes the cells’ adhesion force to the surface and modifies the connection between them. The force of the union of the cells to the surface can be modified by the presence of different cells, which are more numerous when fibronectin exists on the surface. The shape of the cells regulates their growth, genetic expression and secretion of proteins. It also modifies differentiation and cellular death. The roughness levels can influence osteoblast and osteoclast formation. Cell differentiation in osteoblasts depends on the communication between caderins and integrins. Cellular expression depends on caderin and conexine, as these are responsible for bone formation. Through photoluminescence analysis, it is possible to observe that cell attachment on the titanium surface varies with the cell sizes; some areas can have more proteins than others.
4.1.
Machined dental implant surface
Fig. 1A shows the surface morphology of received machined implants without any surface treatment. The tool marks created by the turning process made the surface anisotropic with clear directional surface irregularities. No correlation was observed between Ra (Table 1) and removal torque (Table 2), but a correlation among surface peak parameters (Rq, Rz, Rmax and Rpkx) and removal torque was detected. The heterogeneous topography affects bone cells and reduces the speed of the osseointegration mechanism. The larger the depth of the grooves, the greater the tendency of the cells’ growth to be unidirectional. During cell growth, the cells show a high tendency to follow the orientation of these grooves. This behavior demands a longer healing time. The results of the present work show that the removal torque of machined implants is lower than that of the treated surfaces, which means that osseointegration mechanisms are also lower. The biomechanical properties of machined implants for initial healing time are smaller than those for treated implant surfaces. Table 2 shows that the smallest removal torque of the machined implants is associated with the smallest resistance of the bone–implant interface, which
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depends strongly on the interlacement of the new bone to the tool mark and the dental implant roughness. The growth of the bone occurs preferentially in the superficial irregularities on machined dental implants (Albrektsson and Wennerberg, 2004b). On the surface of turned implants, cell retraction occurs, as well as contact guidance, in which cells grow along the grooves of the substrate (Brunette, 1988). In the machined surfaces, the fibroblasts take the plane shape, and there is a growth tendency along the machining tool marks. Inside the groove marks with 0.5 mm depth and 4.9 to 220 µm width, there is guided growth of the cells along these grooves. This behavior is different from that on surfaces with homogeneous roughness, in which the epithelial cells and fibroblast cells are round and grow in all directions. The cells present haptotaxis, rugopholy and rugophobia properties, making it important to determine the area of contact of the cells, the number and density of the cell surface interaction, the diffusion tax and the necessary force to break the connection. In any situation, there is no direct contact between bone and implant surface. Due to the surface morphology characteristics shown in Fig. 1A and the smallest resistance to removal torque (Table 1), the implants without surface treatment, denoted as smooth implants, machined implants or turned surfaces, are being removed from the market. Nowadays, commercial dental implants are available with surfaces treated with acids, sandblasted, acid etched, anodized, coated with hydroxyapatite and coated with a fine layer of bioactive material.
4.2.
Acid-etched implant surface
The acid-etched dental implant surface presents a superficial morphology that varies with the treatment conditions (acid, chemical composition percentage, etching time and temperature treatment). Through acid etching, it is possible to control the roughness, number, size and porous distribution on micrometer and nanometer scales. The surface morphology shown in Fig. 1B is more isotropic than the turned surface and presents microvoids with defined borders. This surface type facilitates osteogenic cell retention and allows for cell migration at the implant surface. Some dental implant manufacturers point out that the dental implant surface morphology, similar to that shown in Fig. 1B, induces fibrin retention and facilitates the osseointegration process (Brunette, 2001; Geis-Gerstorfer et al., 2004). As Table 2 shows, the removal torque of acid-etched implants is higher than that of machined implants, which means that the osseointegration mechanisms are faster in acid-treated implants than machined implants.
4.3.
Sandblasted dental implant surface
After the surface treatment by sandblasting with silicon oxide (silica), aluminum oxide (alumina), titanium oxide (rutile), hydroxyapatite and phosphate of calcium, there is a surface work hardening process, and the surface energy increases. The sandblasting process induces the formation of a fine superficial layer with residual stress. The compressive stress level is a function of the hardness and size of the particles, as
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well as the pressure and time of the sandblasting process. The superficial layer with residual compressive stress increases the material’s fatigue resistance; this parameter has not been analyzed in the field of dental implants. It can be observed in Fig. 1C that the sandblasting process changes the implant surface morphology. The surface roughness after sandblasting is larger than after acid treatment and smaller than that for oxidized surfaces. The removal torque showed the same tendency. However, there is a roughness size that induces the best osseointegration process and increases the implant removal torque. Sandblasted or acid-etched implants have more homogeneous surface roughness and porosities with a porous size that allows for better cell adhesion than in the implants without surface treatment. It can be observed in Table 2 that all dental implants with treated surfaces have equivalent removal torque.
4.4.
Oxidized dental implant surface
Depending on the anodization conditions (mainly potential, electrolyte, temperature), the solid oxide layer can be either compact or nanotubular (nanoporous). Macak et al. (in press) give an overview and review on self-organized TiO2 nanotube layers and other transition metals. The tubular oxide structures are grown by controlled anodic oxidation of a metal substrate. They describe the mechanistic aspects of the tube growth and discuss the electrochemical conditions that must be fulfilled in order to synthesize these layers. To achieve an ideal self-ordering of the nanotubes, use of the optimized anodization parameters is crucial. Depending on the anodizing conditions, the crystal structure has been reported to be anatase, a mixture of anatase and rutile, or rutile. In general, the morphology and structure of porous layers are affected strongly by the electrochemical conditions (electrolyte, voltage, temperature). Fig. 1D shows the experimental oxidized dental implant surface morphology. It can be observed that the surface presents volcano-shaped saliencies. The commercial dental implant TiUnite (Nobel Biocare) has a titanium oxidized layer thickness, which increases towards the apex; in the present work, the thickness layer was homogeneous. Some authors mention these implants as “osteoattractive”, avoiding the designation “bioactive” (Albrektsson and Wennerberg, 2004a,b). This study presents the results of measurements of removal torque changes during healing of dental implants with different surfaces, using the machined surface as a reference. Table 2 shows that the removal torque of an anodized implant is 45.6% higher than that of a machined implant. These findings demonstrate that the anodized surface increases bone formation in the early stages of healing. This difference can be ascribed to surface morphology, roughness, chemical composition and a thicker oxide layer. As Strnad et al. (2007) speculate, differences in the rates of osseointegration in the initial stages of healing for the anodized surface could be related to different surface reactivities stemming from different surface material properties, e.g. surface area or surface wettability. In general, the surface reactivity, which is a common characteristic
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of bioactive materials, increases with increasing surface area. In addition, Strnad and coworkers observed that an easily wettable hydrophilic surface allows the establishment of contact between the body environment (blood) and the complicated rough and porous structure of the implant, and thus contributes to cell and biomolecule migration and adhesion.
4.5.
Dental implant surface roughness
Measurement of the roughness parameter of the dental implant surface morphology is important because its value influences the adhesion, adsorption and differentiation of the cells. The most commonly used dental implant roughness parameter is Ra, the arithmetic medium value of the deviations of the roughness profile in relation to a medium line. Other important roughness parameters are the quadratic medium value of the roughness (Rq or Sq), the medium value of five unitary roughness measurements measured by the difference between the lowest and highest point of the profile (Rz), the maximum roughness (Rmax), the medium distance among grooves (Sm), the value of the medium roughness of the peaks that are above a profile plane (Rpk), and the value of the medium roughness of the valleys that are below the profile plane (Rvk). Data from the literature (Albrektsson and Wennerberg, 2004b; Suzuki et al., 1997; Geis-Gerstorfer et al., 2004) show that good machined dental implants present Ra between 0.5 and 1.0 µm. The Ra roughness parameter of the implant surface treated with acid presents a medium roughness between 0.54 and 1.97 µm. In the present work, the Ra values were between these values. The Ra of the sandblasted implants is from 0.84 to 2.12 µm, and the Ra of the oxidized implants is above 2.0 µm. Treatment of the implant surface with acid, besides modifying its roughness, makes the surface more isotropic. On the other hand, the sandblasting process changes the roughness and the surface energy. The surface energy expresses the chemical composition, the residual stress, the local spatial arrangement of atoms and the implant–bone contact as well. In the present work, it was observed that the biomechanical resistance of the implants after osseointegration, measured by the removal torque, is a function of roughness, surface energy and chemical composition. Up to a certain Ra value, as the roughness increases, the removal torque increases. Albrektsson and Wennerberg (2004b) observed that when the value of the roughness is higher than a critical value, the removal torque is reduced. Among the roughness parameters measured in the present work, peak area (A1) and valley area (A2) presented the best correlation with removal torque. As A1 and A2 increase, the removal torque increases.
4.6.
Wettability
The physical surface properties and the surface energy can be quantified by the wettability and by the contact angle of liquids with the surface (Lim and Oshida, 2001; Lim et al., 2002). The values of the contact angle indicate whether the surface is hydrophilic or hydrophobic. Table 2 shows that the oxidized surface is the most hydrophilic surface. The behavior
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of the surfaces analyzed shows that wettability is reduced when the contact angle is measured with human blood. Comparing the wettability data with the removal torque data (Table 1), it can be observed that, 12 weeks after surgery, wettability influenced the mechanical properties of the bone–implant interface. The behavior of the proteins on the surface of the titanium implant is dependent on the surface properties, especially adsorption and adhesion. The implant surface wettability (hydrophobic or hydrophilic) influences cell behavior in the initial osseointegration process. The osseointegration mechanism starts when the implant gets in contact with the blood. In the hydrophobic surfaces, the signs of the antibodies reduce cell adsorption, while in the hydrophilic surfaces, the signs of the trombines and prontotrombines are predominant and adsorption is stimulated (Macak et al., in press). The results of “in vitro” and “in vivo” tests in the present work show that, to increase the implant surface area for human osteoblast adhesion, it is necessary to increase the surface wettability. Some researchers also observed that proliferation of the cells increases with surface wettability; the fibroblasts have greater adhesion on hydrophilic surfaces than on hydrophobic surfaces (Boyna et al., 2001). Comparison of “in vitro” results with “in vivo” results demonstrates that surface features influence cell behavior. The mechanisms of cell adhesion on titanium have great influence on the differentiation, proliferation and formation of an extracellular matrix. The topographical surface properties like roughness, energy and wettability modify cell behavior and alter the function of the cells in the initial mechanisms of osseointegration. In the present work, the removal torque indirectly quantified the osseointegration mechanism. Feng et al. (2003) observed that, besides the surface characteristics, the number of hydroxyl groups on the titanium surface influences the behavior of the osteoblasts in contact with the titanium surface. They also observed that the greater the roughness, the larger the surface energy and the higher the number of hydroxyl groups, the greater the number of adhered osteoblasts and cell activity. The acid-etched implants had higher removal torque than the machined implants (Table 1). This result can explain by cell behavior, especially proliferation and replication, which depend on implant surface morphology. The production of the extracellular matrix is sensitive to the surface roughness. Cell proliferation varies with the surface roughness, once the cells can identify the surface roughness. Cells cultivated in rough surfaces increase osteocalcin production and alkaline phosphatase. These parameters are indicative of differentiation and increased osteoblastic production (Webb et al., 1998). The production of growth factors of the TGFb1 type increases in rough surfaces. This growth factor is a known stimulator for the formation of collagen and prostaglandin production. There is a dependence between the increase in roughness and the increase in the production of PGE 1 and PGE 2 with osteogenic differentiation (Boyna et al., 2001). The inhibition of prostaglandin production depends on the reduction of the proliferation and the phenotic expression, as well as of the production of PGF-b1, suggesting that prostaglandin production involved in the mechanism of the surface roughness depends on the stimulation of osteogenic differentiation (Boyna et al., 2001).
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Conclusions
The behavior of titanium dental implant samples submitted to surface treatments was investigated through contact angle analysis, roughness measurements and in vivo tests. The scanning electron microscopy analysis showed that surface treatment changed the surface morphology. The results have shown that the surface roughness and wettability of implants may influence biological responses such as the removal torque of dental implants. The highest contact angle and the lowest removal torque were presented by machined implants. The lowest contact angle and the highest removal torque were presented by the anodized dental implants.
Acknowledgments The work described in this paper was supported by two grants from CNPq (Process 472449/2004-4, 400603/2004-7 e 500126/2003-6) and from FAPERJ (Process E-26/151.970/2004).
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