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Pure titanium and titanium alloys are well-established standard materials in dental implants because of their favorable combination of mechanical strength, chemical stability, and biocompatibility. Integration of titanium implants with the surrounding bone (osseointegration) is critical for successful bone regeneration and healing of dental implant. The concept of osseointegration was discovered by Brånemark and his co-worker and, has had a dramatic influence on clinical treatment of oral implants. The first generation of successfully used clinical titanium implants, which were machined with a smooth surface texture, now approach 50 years in clinical use. Since then, implant surfaces have long been recognized to play an important role in molecular interactions, cellular response and osseointegration, and scientists all over the world have developed the second generation of implants with surfaces that can accelerate and improve implant osseointegration. This second generation of clinically used implants underwent mechanical abrasion, sometimes coupled with acid etching, bioactive coatings, anodization and, more recently, surface modifications using laser beams[1]. These implants have been extensively documented in vivo, including long-term clinical studies and experimental histological and biomechanical evaluation in animal models.

Currently, the main objective for the development of implant surface modifications is to promote osseointegration, with faster and stronger bone formation. This will likely confer better stability during the healing process, which, preferentially, will improve the clinical performance in the area of poor bone quality and quantity. Furthermore, such promotion may, in turn, accelerate the bone healing and thereby allowing immediate or early loading protocols1.

Recent developments in micro- and nanotechnology is rapidly advancing surface engineering in implant dentistry. Such advances in surface engineering technologies have resulted in more complicated surface properties from micro- and nanometer scales, including morphology, chemistry, crystal structure, physical, and mechanical properties. Such surfaces, intentionally modified with respect to microscale and nanoscale features, may represent a next generation of oral implant systems if it is possible to transfer them to complex three-dimensional geometries1.

Surface roughness has been identified as an important parameter for implants and their capacity for being anchored in bone tissue. There is a variety of different manufacturing methods to increase the surface roughness of the implant, where the most commonly used are: Machining, Sandblasting, Acid etching, Anodic oxidation, Laser modification or a combination of these.

Calcium phosphate coatings on titanium implants are the most common family of bioceramics that are used for the coating of titanium dental implants in order to improve their biocompatibility. Calcium phosphate in the crystallographic form of apatite is an important mineral constituent of bone. Calcium phosphate ceramics are integrated within bone following a well known sequence of events. They are considered to be bioactive and osteoconductive. Different types of methods have been introduced to prepare calcium phosphate coatings on dental implants (see the chart below). These methods can be divided to two groups: physical and chemical methods. Typically physical techniques include plasma spraying deposition, physical vapor deposition, magnetron sputtering deposition, ion beam assisted deposition, pulsed laser deposition, and hot isostatic pressing. Chemical techniques include sol-gel method, biomimetic process, electrochemical deposition, micro-arc oxidation and electrophoretic deposition1.

Figure below shows an overview of surface modification techniques and strategies that target the improvement of biocompatibility and antibacterial property of titanium dental implants.

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Overview of surface modification techniques and strategies that target the improvement of biocompatibility and antibacterial property of titanium dental implants

The same approaches are followed to reach higher biocompatibility and bacterial resistance of titanium dental implants; however the topography improving techniques are much better exploited in biocompatibility promotion than coating techniques, whereas coating techniques are prevailing in the domain of antibacterial implant materials.

The goal of antibacterial coating techniques is to deposit antimicrobial compounds on the surface of dental implants in order to create a local defense system against bacterial contamination. Due to the potential contribution to the spreading of antibiotic resistance the safety of antibiotic loaded coatings is controversial and they fall under strict regulations that make their market entry difficult. As opposed to antibiotic loaded coatings anodic oxidation is known as a topography improving technique that might be suitable for creating bacteria resistant surfaces using nanophase topographies on titanium dental implants. Anodic oxidation is already used for the improvement of the biocompatibility of titanium implants and based upon recent scientific findings it might be exploited in the struggle against dental implant associated infections.

Anodic oxidation is an electrochemical surface modification technique in which the surface of the anode (the material of interest) is oxidized inside an electrolyte. Anodic oxidation requires an inert material (such as platinum or gold) as the cathode and the material to be anodized as the anode. It requires an electrolyte to establish the flow of ions between the electrodes. Upon connection to constant voltage, oxidation occurs at the anode and reduction occurs in the cathode, resulting in the flow of current through the electrolyte. Electrolyte type, electrolyte concentration, pH, temperature, agitation speed, applied voltage, duration and post-heat treatment are all important parameters affecting the nanophase topography of the anodized surfaces.

The photocatalysis mediated antibacterial effect of titanium-dioxide nanoparticles and nanotubes is well known in the art, however, the bacteria killing property of titanium-dioxide nanotubes upon contact has been explored only the last few years. Pucket et al. cultured bacteria on the surface of anodized nanotubular titanium and on conventional titanium samples. They observed, that the numbers of dead S. epidermidis, S. aureus and P. aeruginosa were significantly higher on the anodized nanotubular surface compared to conventional titanium samples[2]. Ivanova et al. observed decreased adhesion and biofilm formation of S. aureus and P. aeruginosa on titanium thin films as nanoroughness increased[3]. The composition and concentration of electrolyte may significantly affect the biological behaviour of anodized surfaces. Omori et al. found that when titanium was anodized by discharge in NaCl electrolyte solution it provided antibacterial activity against oral bacteria, and at the same time improved the attachment of osteogenic cells[4].

Because of the suggested beneficial effects on biocompatibility, TiO2 nanosurfaces created by anodic oxidation has been proposed for the use on implant materials. After anodization, a 33% increase in osteoblast adhesion was observed compared to conventional titanium[5]. Oh et al. found that the density of adherent mouse osteoblast cells on the titanium-dioxide nanotubes significantly increased by 300–400% compared to that of the cells that attached to conventional titanium-dioxide surface.

Elias et al. implanted sandblasted, acid etched and anodized titanium into the tibia of rabbits. Twelve weeks after the surgery, the anodized surface had the highest removal torque[6]. Similar results were also found by Bjursten et al. when anodized titanium and grit-blasted titanium implants were investigated in a pull-out test from rabbit tibia. After four weeks of implantation, pull-out testing indicated that titanium-dioxide nanotubes significantly improved bone bonding strength by as much as nine-folds compared to titanium-dioxide grit-blasted surfaces. Histological analysis confirmed greater bone-implant contact area, new bone formation, and calcium and phosphorus levels on the nanotube surfaces[7].

It was found that the titanium-dioxide nanoparticles could induce the formation of reactive oxygen species (ROS) – without UV light irradiation – that might play a key role in the destruction of bacteria upon contact[8]. With the decrease of the size of nanoparticles the surface free energy increases, which results in the increased number of electron vacancies (electron-holes) on the surface[9]. The formation of electron-holes strongly depends on the surface chemistry of the TiO2 layer, which can be fundamentally influenced by the applied parameters of anodic oxidation (electrolyte and voltage). The effect of ROS on bacteria is supposed to be dose dependent. The quantity of ROS released can be affected by the number of electron-holes on TiO2 nanostructures, which can be fine-tuned via the parameter optimization of the anodic oxidation process. The electron-holes catalyse the formation of ROS by cleaving the proximate water molecules. The ROS are supposed to be responsible for the peroxidation of cell walls and membranes via the free radical chain reaction that leads to the destruction of the DNA of bacteria triggering their death eventually. ROS attack selectively the bacteria on the surface of the nanophase TiO2, without impairing the bone or stem cells.

This presumed selectivity of ROS may derive from the fact that TiO2 nanostructures are immobilized on the surface. They cannot be taken up by stromal cells, like bone, stem or immune cells. Thus the ROS cannot impair the intracellular biochemical pathways of these cells and therefore may significantly reduce the toxicity of immobilized nanoparticles. Therefore, the formation of immobilized TiO2 nanostructures on the surface of titanium implant materials may enable us to exploit nanomaterials for the benefit of medicine in a safe manner.

Dental implant-associated infections pose a significant problem that affects the success rate of implants[10]. There are many reasons for these infections, such as poor aseptic techniques by surgical staff but it might also be stemmed from the patient‘s own oral flora. Once the bacteria adhere to the implant surface, they start to secrete a protective polysaccharide based matrix, creating a biofilm (Figure 6). As the biofilm matures, bacteria are released into the surrounding tissue causing the infection to spread. The development of this biofilm is responsible for many chronic infections and in prevents proper integration of the implant with the juxtaposed tissue[11]. Once the bacteria bind to the biomaterial surface, it becomes harder to target them through antibiotic therapy and also for the host’s immune system to clear them[12]. Biofilm formed on a biomaterial (sessile bacteria) is highly resistant to antibiotic treatment due to the slow transport of antibiotic molecules through the polysaccharide matrix, different environmental cues and changes in gene expression that alters growth rates. Due to this enhanced resistance, host defence mechanisms are also no longer able to remove the bacteria from the implant surfaces. When an infection occurs during implant surgery, the main treatment is the use of antibiotics over prolonged times which aims to reverse symptoms caused by planktonic (free floating) bacteria released from the biofilm. However, this approach does not always provide successful results in eradicating the sessile bacteria from the biofilm.

In fact, the efficacy of the treatments using antibacterial compounds is decreasing due to the increasing presence of strains of antibiotic-resistant bacteria. Often the dental implant must be removed in order to eradicate an infection because surface- bound bacteria cannot be eliminated. Unfortunately, there is an increasing percentage of dental implant failures related to infection, giving solid evidence that current approaches fail to satisfy the need for anti-bacterial dental implants. One innovative approach for reducing infection can be to prevent the adhesion of the bacteria on the implant surfaces in the first place by modifying implant surface properties. Instead of fighting the bacteria after they adhere and form a biofilm, which is hard to target through antibiotic use as discussed in the above paragraph, bacteria adhesion and further bacteria growth would be minimized[13].

Titanium samples

Nowadays pure titanium and α + β type Ti-6Al-4V ELI (extra low level of interstitial content) alloys are widely used as biomaterials for the replacement of hard tissues, such as dental implants, because of its specific strengths and corrosion resistance, and the best biocompatibility characteristic among metallic biomaterials. Under the category of “unalloyed grades” of ASTM specification, there are five materials classified in this group; they include ASTM Grade 1 (99,5% Ti), Grade 2 (99,3% Ti), Grade 3 (99,2% Ti), Grade 4 (99,0% Ti), and Grade 7 (99,4% Ti). Although each material contains slightly different levels of N, Fe, and O, C is specified<0.10 wt% and H is also specified<0.015 wt% ASTM CpTi grades 1‑4 (unalloyed titanium) allow hydrogen content up to 0.015 wt% (i.e., 15 ppm)[14] .

Grade 2 titanium

This commercially pure titanium has been available as mill products since 1950 and is used for applications that require moderate strength combined with good formability and corrosion resistance. The Grade 2 is the most frequently selected titanium grade in industrial service having well-balanced properties of both strength and ductility. The strength levels are very similar to those of common stainless steel and its ductility allows for good cold formability[15]. Grade 2 titanium having a guaranteed minimum yield strength of 275 MPa and good ductility and formability. ASTM Grade 2 titanium has the same nitrogen content limits as ASTM Grade 1 (0.03% max), the same iron content limits as ASTM Grade 3 (0.30% max), and a maximum oxygen concentration of 0.25% that is approximately midway between the 0.18 to 0.40% range in the other three ASTM unalloyed titanium grades. The increase iron and oxygen concentrations of ASTM Grade 2 compared to ASTM Grade 1 impart additional tensile strength (345 vs 240 MPa) and yield strength (275 vs 170 MPa) to Grade 2 but at the expense of ductility (20% elongation for Grade 2 vs 24% elongation for Grade 1). Higher iron and interstitial contents also may degrade corrosion resistance relative to Grade 1. Hydrogen content as low as 30 to 40 ppm can induce hydrogen embrittlement in CP titanium. Grade 2 can be welded, machined, cast and cold worked. Titanium Grade 2 typically has an annealed alpha structure in wrought, cast, and P/M forms[16].

Grade 5 titanium

It is also known as Ti6Al4V, Ti-6Al-4V or Ti 6-4 presently is the most widely used titanium alloy, accounting for more than 50% of all titanium tonnage in the world. To date, no other titanium alloy threatens its dominant position. The aerospace industry accounts for more than 80% of this usage. The next largest application of Ti-6Al-4V is medical prostheses, which accounts for 3% of the market. The chemical composition of Grade 5 titanium is defined by ISO 5823-3: 5.5-6.75% aluminium, 3.5-4.5% vanadium, 0.08% carbon, max. 0.30% iron, max. 0.2% oxygen, max 0.05% nitrogen, max. 0.015% hydrogen (except for billets, for which the maximum hydrogen content shall be 0.010% (m/m). The Grade 5 titanium is significantly stronger than commercially available pure titanium while having the same stiffness and thermal properties (except for thermal conductivity).

Main physical parameters: density: appr. 4420 kg/m3; Young’s modulus: 115 GPa; tensile strength (Rm min.): 860 MPa; proof stress off nonproportional elongation (Rm min.): 780 MPa.

Grade 5 ELI titanium

Ti-6Al-4V is available in ELI (extra-low interstitial) grades with high damage-tolerance properties, especially at cryogenic temperatures. The principal compositional characteristics are low oxygen and iron contents[17].

 Determination of surface energy of samples

The test fluids will be developed by the mean of a drop shape analysis system to measure the contact angle of fluids. Various composition of distilled water, ethanol, formamid, 2-ethoxyethanol, glycerol, and polyethylene glycol, may be used for producing the test fluids. Measurements must be taken 5 sec after placing the droplet on reference surfaces under ambient conditions. Drop shape analysis software should be used to calculate surface free energy.

List of harmonized standards:[MSZ EN ISO 4287:2000]; [MSZ EN ISO 4288:2000] and [ASTM D7334-08 (2013)];

Non-destructive examination of samples

  • Determination of the thickness of the TiO2 layer on the surface of samples (Auger-spectroscopy) and the surface properties (roughness tester, AFM, SEM).
  • Characterization of the crystal orientation by EBSD.

List of harmonized standards:[MSZ EN ISO 4287:2000]; [MSZ EN ISO 4288:2000]

Destructive examination of the samples

  • measurement of the thickness of the oxide layer  by AFM.
  • measurement of the microhardness of the samples
  • investigation of fracture resistance and adhesion of the layers by in vitro scratch tests (INSTRON 5965, a self-developed clamping tool, SEM)

List of harmonized standards:[ISO 1463:2004]; [ISO 9220:2000]; [MSZ ISO 9220:1994]; [ISO 14577-1:]; [ISO 14577-4:2007]; [ISO 6507-1:2005]; [ISO 4516:2002] and [ASTM E2859-11];

Mechanical tests of the samples

Examination of both the compression and bending fatigue by special clamping heads.

List of harmonized standards:  [MSZ EN ISO 14801:2008]; [MSZ EN ISO 6892-1:2010]; [ISO 7438:2005]

Corrosion tests of the samples

Determination of the corrosion resistance of the samples in acidic and lanthanide environments.

List of harmonized standards:[MSZ EN ISO 10271:2011]; [ISO 10993-15:2009 Part 15];[ISO 16701:2008]; [ISO 16429]; and [ASTM F2129-08]

[1]Ahmed M. Ballo, Omar Omar, Wei Xia and Anders Palmquist. Dental Implant Surfaces – Physicochemical Properties, Biological Performance, and Trends. Implant Dentistry

[2]Puckett, S. D., Taylor, E., Raimondo, T., Webster, T. J. (2010). “The relationship between the nanostructure of titanium surfaces and bacterial attachment.” Biomaterials 31(4): 706-713.

[3]Ivanova, E. P., Truong, V. K., Wang, J. Y., Berndt, C. C., Jones, R. T., Yusuf, I. I., Peake, I., Schmidt, H. W., Fluke, C., Barnes, D., Crawford, R. J. (2010). “Impact of nanoscale roughness of titanium thin film surfaces on bacterial retention.” Langmuir 26(3): 1973-1982.

[4] S. Omori, Y. Shibata, T. Arimoto, T. Igarashi, K. Baba, T. Miyazaki: Micro-organism and Cell Viability on Antimicrobially Modified Titanium. Journal of Dental Research. May 11, 2009.

[5]Yao, C., Slamovich, E. B., Webster, T. J. (2008). “Enhanced osteoblast functions on anodized titanium with nanotube-like structures.” Journal of Biomedical Materials Research Part A 85A(1): 157-166.

[6]Elias, C. N., Oshida, Y., Lima, J. H. C., Muller, C. A. (2008). “Relationship between surface properties (roughness, wettability and morphology) of titanium and dental implant removal torque.” Journal of the Mechanical Behavior of Biomedical Materials 1(3): 234-242.

[7]Bjursten, L. M., Rasmusson, L., Oh, S., Smith, G. C., Brammer, K. S., Jin, S. (2010). “Titanium dioxide nanotubes enhance bone bonding in vivo.” Journal of Biomedical Materials Research Part A 92A(3): 1218-1224.

[8]Fenoglio I, Greco G, Livraghi S, Fubini B. Non-UV-induced rad

ical reactions at the surface of TiO2 nanoparticles that may trigger toxic responses. Chemistry. 2009;15(18):4614-21.

[9]Cun Wen, Yi Liu, and Franklin (Feng) Tao. Integration of surface science, nanoscience, and catalysis. Pure Appl. Chem., Vol. 83, No. 1, pp. 243–252, 2011.

[10]Heuer W, Elter C, Demling A, Neumann A, Suerbaum S, Hannig M, Heidenblut T, Bach FW, Stiesch-Scholz M. Analysis of earlybiofilmformation on oralimplantsin man. J Oral Rehabil. 2007 May;34(5):377-82.

[11]Costerton, J. W., Stewart, P. S., Greenberg, E. P. (1999). “Bacterial biofilms: a common cause of persistent infections.” Science 284(5418): 1318-1322.

[12]Donlan, R. M. (2001). “Biofilms and device-associated infections.” Emerging Infectious Diseases 7(2): 277-281.

[13] Batur Ercan. Anodized nanotubular titatnium as an orthopedic implant and the effect of electrical stimulation. PhD thesis 2011, Brown University, USA

[14] Y Oshida: Bioscience and bioengineering of titanium materials. Elsevier (2013) ISBN: 978-0-444-62625-7; pp 15

[15] Y Oshida: Bioscience and bioengineering of titanium materials. Elsevier (2013) ISBN: 978-0-444-62625-7; pp 15

[16] R Boyer, G Welsch, EW Collings: Materials properties handbook: Titanium alloys (1994) pp: 167; ISO 5832-2

[17] R Boyer, G Welsch, EW Collings: Materials properties handbook: Titanium alloys (1994) pp: 483