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ORIGINAL RESEARCH  
Year : 2019  |  Volume : 30  |  Issue : 1  |  Page : 37-42
The antibacterial influence of nanotopographic titanium, zirconium, and aluminum nanoparticles against Staphylococcus aureus and porphyromonas gingivalis: An In vitro study


Department of Prosthodontics, SRM Dental College, Chennai, Tamil Nadu, India

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Date of Web Publication20-Mar-2019
 

   Abstract 

Background: Nanoparticle coated implants have revolutionized the field of implantology. Peri-implantitis is one of the main for implant failure. Most implant failures are due to Streptococcus mutans, Staphylococcus aureus, Porphyromonas gingivalis, and Actinomyces concomitant. Aim: To evaluate and compare the antibacterial effect of titanium, zirconium, aluminum nanoparticles against S. aureus and P. gingivalis at 24, 72 hrs and 1 week intervals. Materials and Methods: According to ISO/TR 11175:1993, the samples were prepared in disc shape with 20-mm diameter and 1-mm thickness in Grade IV titanium. The samples were decontaminated by sonicating in acetone and subsequently in water three times for 15 min. A total of eighty samples were made, then coated with nanoparticles, and divided into Group-A (control), Group-B (Ti nanoparticles coating), Group-C (Zi nanoparticles coating), and Group-D (Al nanoparticles coating). The discs were sterilized under ultraviolet radiation and placed in Mueller-Hinton agar medium for S. aureus and blood agar for P. gingivalis. The colonies were counted at 24, 72 hrs and 1 week intervals. Statistical Analysis Used: The values were statistically analyzed using one-way ANOVA and Tukey honestly significant difference test. Results: The P < 0.001 showed that statistically significant difference in colony forming unit had been found in titanium nanoparticles coated samples at 24, 72 hrs and 1 week intervals. Conclusion: TiO2 nanoparticle coated titanium discs showed significant antibacterial effect.

Keywords: Antimicrobial potential, nanoparticles, titanium

How to cite this article:
Karthikeyan M, Ahila S C, Muthu Kumar B. The antibacterial influence of nanotopographic titanium, zirconium, and aluminum nanoparticles against Staphylococcus aureus and porphyromonas gingivalis: An In vitro study. Indian J Dent Res 2019;30:37-42

How to cite this URL:
Karthikeyan M, Ahila S C, Muthu Kumar B. The antibacterial influence of nanotopographic titanium, zirconium, and aluminum nanoparticles against Staphylococcus aureus and porphyromonas gingivalis: An In vitro study. Indian J Dent Res [serial online] 2019 [cited 2019 Oct 17];30:37-42. Available from: http://www.ijdr.in/text.asp?2019/30/1/37/254533

   Introduction Top


Dental implants are surgically fixed substitutes for roots of missing teeth. They are embedded into the bone and behave as anchors for the replacement of a single tooth or teeth.[1] In spite of the high success rate, occurrence of implant failures has also been reported. The etiology of implant failures can be broadly classified into three categories – infection, impaired healing, and occlusal overloading. Peri-implantitis is the most common cause for implant failures.[2],[3]

Numerous microbes are involved in the pathogenesis of peri-implantitis such as Streptococcus mutans, Staphylococcus aureus, Porphyromonas gingivalis, Tannerella forsythia, Actinomyces concomitant, Prevotella intermedia, and Fusobacterium species.[4],[5] Among the microorganisms, the prevalence of S. aureus, P. gingivalis, and A. concomitant are more at the implant site. To avoid late failure of implants due to peri-implantitis, prevention of colonization of microbes around the collars of the implant is mandatory. Thus, to improve the antimicrobial potential of the implants, titanium alloys were surface treated with mechanical blasting, acid etching, bioactive coating, anodized LASER modified surfaces which have their own merits and demerits.[6]

The biomolecules and cells involved in the early healing phase after implant placement interacted at the nanometer level. Reproducing the nano three-dimensional topography in bone may improve early and long-term interaction between host bone and osseointegrated implant.[7] Nanotechnology is defined as “the creation of functional materials, devices, and systems through control of matter on the nanometer length scale (1–100 nm) and exploitation of novel phenomena and properties (physical, chemical, and biological) at that length scale.” In addition to antimicrobial action, nanoparticles applied to the endosseous implant surface resulted in embellishment of the surface with nanometer scale features that led to novel physicochemical behavior such as bone bonding or biochemical events such as altered protein adsorption and cell adhesion with changes in cell behavior.[8] Hence, this study was done to evaluate the antibacterial effect of titanium, zirconium, aluminum nanoparticles coated titanium against S. aureus and P. gingivalis at 24, 72 hrs and 1 week time intervals. The null hypothesis was that titanium surface with titanium, zirconium, and aluminum nanoparticles coating would exhibit excellent antibacterial property against S. aureus and P. gingivalis.


   Materials and Methods Top


Master dye preparation

According to ISO/TR 11175:1993, the samples were prepared with the dimension of 20-mm diameter and 1-mm thickness. Commercially available pure Grade IV titanium (Orotig Verona-Italy) was made into sheets of 1-mm thickness. A Master's Press was used to prepare the individual discs measuring 20 mm in diameter and 1 mm in thickness.

Nanoparticle-coated disc preparation

The nanoparticles were made in the form of discs pallets measuring about 16-mm diameter and 4-mm thickness by compressing the nanoparticles in a palletizer (Hirco tool, Goregaon, Mumbai, India) at 300 N for 5 min. Then, the pallets were retrieved carefully from the palletizer and sintered at a temperature of 1600°C in a furnace (VB Ceramics Pvt. Ltd., Chennai, Tamil Nadu, India) for 8 h and was allowed to cool down to room temperature. The sintered pallets were then retrieved from the furnace after 24 h [Figure 1].
Figure 1: TiO2, ZrO2, and Al2O3 nanoparticle-coated pallots

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The titanium discs were decontaminated by treating them in acetone (Sunil Chemicals, Kolkata, West Bengal, India) and subsequently in water. This procedure was repeated three times for 15 min and then placed in the substrate holder and titanium pallet was kept as target. The pulsed laser deposition (PLD) started with the ablation of the target material by laser irradiation. A plasma plume with high energetic ions, electrons, as well as neutrons developed parallel to the normal vector of the target surface toward the substrate due to Coulomb repulsion. The abraded particles were pushed toward the substrate along the plume resulting in a thin uniform coating over the substrate. This procedure was carried out in the presence of oxygen, and the same procedure was followed for zirconium and aluminum nanoparticles coating using zirconium and aluminum pallets as the target.

Sample distribution

A total of eighty samples were prepared, the samples without any surface coating were considered as Group A (control), samples coated with titanium nanoparticles were Group B, samples coated with zirconium nanoparticles were Group C, and samples coated with aluminum nanoparticles were Group D.

Surface characterization

Scanning electron microscopy using ZEISS SUPRA 55 (Carl Zeiss NTS GmbH, Oberkochen, Germany) was used to observe the surface morphology of Ti, Zi, and Al nanoparticles coating [Figure 2], [Figure 3], [Figure 4].
Figure 2: Scanning electron microscope of TiO2 nanoparticles coating

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Figure 3: Scanning electron microscope of ZrO2 nanoparticles coating

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Figure 4: Scanning electron microscope of Al2O3 nanoparticle coating

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Antibacterial test

The antibacterial activity of titanium, zirconium, and aluminum nanoparticles coated titanium discs was evaluated by bacterial counting method. The coated specimens were sterilized by ultraviolet radiation. Bacterial colonies with a concentration of 106 colony forming unit (CFU)/ml were dripped onto the coated surface of the sample at a density of 0.05 ml/cm2. Then, it was inoculated onto a Mueller-Hinton agar medium for S. aureus and blood agar for P. gingivalis then incubated at 37°C for 24, 72 hrs and 1 week in a vacuum atmosphere [Figure 5] and [Figure 6]. The colonies were counted under a Digital Colony Count Tester (Toshiba, Tokyo, Japan).
Figure 5: Growth of Staphylococcus aureus

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Figure 6: Growth of Porphyromonas gingivalis

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Statistical analysis used

All the datas were analyzed using SPSS for Windows Version 22 (Armonk, NY: IBM Corp. Released 2013, USA). One-way ANOVA was used to compare the number of CFU of S. aureus and P. gingivalis within the groups, and multiple group comparison was done using Tukey honestly significant difference post hoc test. P < 0.001 was considered as statistically significant, and the level of significance was 95% confidence interval.


   Results Top


The results obtained in this study were the CFUs of S. aureus and P. gingivalis. The statistical analysis showed that the CFU values were insignificant while comparing control and aluminum nanoparticles coated titanium disc against S. aureus and P. gingivalis in 24 h of incubation. However, the titanium and zirconium nanoparticles coated titanium disc showed significant effect compared to the control group at 24 h of incubation (P < 0.001) [Table 1], [Table 2], [Table 3], [Table 4]. The control and the aluminum and zirconium nanoparticle-coated titanium discs showed the same antibacterial effect on the CFU of S. aureus and P. gingivalis at 72 h and 1 week of incubation. Hence, zirconium and aluminum nanoparticles had insignificant antibacterial effect on S. aureus and P. gingivalis [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10], [Table 11], [Table 12]. Hence, this study rejected the null's hypothesis. However, the titanium nanoparticle-coated titanium discs showed statistically significant effect (P < 0.001) on CFU of S. aureus and P. gingivalis at 72 h and 1 week intervals [Graph 1] and [Graph 2].
Table 1: One-way ANOVA of colony forming unit for Group A, B, C, and D on Staphylococcus aureus in 24 h

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Table 2: Tukey's honestly significant difference between the groups for Staphylococcus aureus in 24 h

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Table 3: One-way ANOVA of colony forming unit for Groups A, B, C, and D on Porphyromonas gingivalis in 24 h

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Table 4: Tukey's honest significant difference between the groups for Porphyromonas gingivalis in 24 h

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Table 5: One-way ANOVA of colony forming unit for Groups A, B, C, and D on Staphylococcus aureus in 72 h

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Table 6: Tukey's honestly significant difference between the groups for Staphylococcus aureus in 72 h

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Table 7: One-way ANOVA of colony forming unit for Groups A, B, C, and D on Porphyromonas gingivalis in 72 h

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Table 8: Tukey's honestly significant difference between the groups for Porphyromonas gingivalis in 72 h

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Table 9: One-way ANOVA of colony forming unit for Groups A, B, C, and D on Staphylococcus aureus in 1 week

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Table 10: Tukey's honestly significant difference between the groups for Staphylococcus aureus in 1 week

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Table 11: One-way ANOVA of colony forming unit for Group A, B, C, and D on Porphyromonas gingivalis in 1 week

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Table 12: Tukey's honestly significant difference between the groups for Porphyromonas gingivalis in 1 week

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   Discussion Top


Titanium is the most commonly used material for implants in dentistry. The clinical success rates of titanium and its alloys have proved superior than other metals because of their mechanical strength, corrosion resistance, and biocompatibility.[9] The formation of bone directly around the implant surface was due to the formation of a thin layer of oxide on the surface of pure titanium in atmospheric conditions (passivating effect).[10]

The infection initially starts at the implant collars and then spread along the entire length of the bone implant interface resulting in failure of osseointegration. It is important to prevent the colonization of microbes around the collars of the implants to prevent implant failure.[11] In the previous studies, vector ultrasound system and carbon fiber curettes were used to treat peri-implantitis. There were no significant differences between the two techniques, and neither proved sufficient to treat peri-implantitis.[12] Er:YAG laser was used to treat peri-implantitis, but it could only stop bleeding on probing.[13] Furthermore, the treatments with local antibiotics improved clinical parameters such as pocket depth and bleeding.[14]

For the success of the implants, antimicrobial property of the implant surface is mandatory so that the initial attachment and colonization of the microorganisms can be prevented. It had been shown in literature that coating of nanoparticles such as titanium and silver over titanium surfaces is likely to not only change the physical properties but also increase the osteogenic potential and antimicrobial efficacy of the titanium implants.[15],[16] The osteogenic potential of titanium, zirconium, and aluminum nanoparticles has been documented. TiO2, ZrO2, and Al2O3 nanoparticles have a high affinity for bone tissue, and the bone implant interface is similar to that seen around titanium implants. When inserted into subcutaneous tissue, there was development of only a small inflammatory cell infiltrate with the implant completely surrounded by a thin fibrous connective tissue.[17] When the implants were coated with zirconium nanoparticles, significant bone implant contact percentage was found in animal studies.[18] Hence, this study had been done to evaluate the antimicrobial effect of the titanium, zirconium, and aluminum nanoparticles coated titanium discs against the most common microbes involved in peri-implantitis such as S. aureus and P. gingivalis.

In this study, the nanoparticles were coated on the samples by means of PLD. PLD is an advanced and trusted method of coating when compared to sputtering, plasma spraying, chemical vapor deposition, and powder vapor deposition.[19],[20] The results of this study showed the significant value P < 0.001 for titanium nanoparticle-coated titanium discs at all intervals of evaluation; hence, the antibacterial effect of titanium nanoparticles coated titanium discs was significant when compared to zirconium and aluminum nanoparticles titanium discs against S. aureus and P. gingivalis at 24, 72 hrs and 1 week time intervals. The results of this study confirmed the results of previous studies for titanium nanoparticles.[16],[21],[22],[23] However, the zirconium and aluminum nanoparticles did not show significant antimicrobial effect against S. aureus and P. gingivalis 72 h and 1 week time intervals.

According to Allaker, the antimicrobial efficacy of titanium nanoparticles was due to the ion release by the particles. When the Biotrophic Interfacial Complex was invaded by the microbes, the titanium nanoparticles started releasing positively charged titanium ions. These positively charged ions were attracted towards the microbes which carry negative charge. When the ions bind to the cell wall, they slowly started dissolving the cell wall and destroyed the cellular components of the organism, thereby the titanium nanoparticles prevented adhesion and the colonization of the microorganisms.[24],[25]

Clinical significance

Conventional treatment protocols such as antibiotic regimens, ultrasonic scaling, and treatment using LASERs were less effective against peri-implantitis. Coating the implant surface with titanium nanoparticles might be an efficient method to prevent the bacterial adhesion, thereby preventing peri-implantitis.


   Conclusion Top


Within the limitations of the present study, the antibacterial effect of titanium nanoparticles coated titanium was statistically significant against S. aureus and P. gingivalis was statistically significant at 24, 72 hrs and 1 week intervals. Titanium nanoparticles coated titanium showed more antibacterial effect compared to zirconium and aluminum nanoparticles coatings at 24, 72 hrs and 1 week intervals.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

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Correspondence Address:
Dr. S C Ahila
Department of Prosthodontics, SRM Dental College, Ramapuram, Chennai - 600 089, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.IJDR_91_16

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    Figures

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