Influence of Titania Nanotubes Diameter on Its Antibacterial Efficacy against Periodontal Pathogens: An In vitro Analysis.

BACKGROUND: Peri implant infection in dental implantology is a frequently encountered clinical problem. Titania nanotubes (TNTs) are recent improvement in surface characterization, showing promising results. AIM: The nanosurface parameter tweaking has been implicated with profound change in the microbiological and biological response. Hence, it was proposed that alteration in the nanotube diameter could have positive influence in its antibacterial activity against salient periodontal pathogens.
MATERIALS AND METHODS: Commercially, pure titanium discs of 8-mm diameter and 1.5-mm thickness were prepared. Polished titanium discs were used as control (Group A). Vertically oriented, structured TNTs were fabricated by anodization technique and grouped as B and C, having nanotube diameter, 40 and 80 nm subsequently. The surface characterizations of the samples were done by scanning electron microscope analysis. The antibacterial activity was evaluated with the bacterial colony counting method, at 24 h, 72 h, and 1-week intervals. STATISTICAL ANALYSIS: The one-way analysis of variance and Tukey's honest significance post hoc test were employed to assess the statistical significance.
RESULTS: The 80 nm nanotubes showed better antibacterial activity comparatively, at all three-time intervals investigated.
CONCLUSION: The optimal TNT diameter of 80 nm was the most effective from an antimicrobial stand point of view. Copyright:

INTRODUCTION

Dental implants bestow researchers with a unique problem of a trans-mucosal model of implanting system. Its success depends not just on the fulfilment of optimal primary stability and osseointegration but continues in maintaining the same resilient, soft-tissue seal in a hostile environment. Hence, the challenge is even demanding, with dental implants being exposed to constant bacterial attacks.

The formation of oral biofilm on the surface of abiotic dental implant is a multifaceted phenomenon with various factors playing decisive roles. Gristina et al. have appropriately described the above-mentioned concept as “Race for the surface.”[1] A highly diversified oral microorganisms form biofilm with the deposition of an initial protein matrix known as acquired pellicle, which is fundamental for further bacterial attachment and biofilm maturation. Dysbiosis in the microbial environment eventually leads to peri-mucositis and peri-implantitis. Several organisms are involved with the disease progression, including, Staphylococcus aureus, Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actionmycetemcomitans, Prevotella interedia, and Fusobacterium nucleatum.[2]

With the intention of dealing with such clinical challenges, various implant surface bio-functionalization is being attempted. The field of biotechnology has shifted its focus from macro surface level to nanosurface dimensional paradigm. The biomolecular interaction between the host and the implant is at the nano level and hence mimicking the natural condition is an interesting approach.[3] Titania nanotube (TNT) arrays are promising aspirant, with many potential advantages such as improved osseointegration with advantages such as increased surface area available for cell tissue interaction and being a controlled smart drug delivery system. Antibacterial activity of TNT has been well documented against many pathogens.[456]

Hence, the aim of the current work was to evaluate the influence of TNT diameter and the effectiveness of its antibacterial action against major periodontal pathogens.

MATERIALS AND METHODS

Fabrication of titania nanotube arrays

Commercially, pure titanium (CpTi) discs were procured from Hebson Surgicals, Mumbai, with dimensions of 8 mm diameter and 1.5-mm thickness. Five discs were allotted for each group; hence, a total of 15 discs were prepared. The polishing of all the discs was carried out emery silicon carbide sheets of grit up to 1200 (3M, Dry sheet) to achieve an even smooth surface. Ultrasonic cleaner (UT8031/EUK, Shesto) with acetone and double distilled water was used to clean the surface. Etching for 10 s was carried out with Kroll's reagent (1.5 ml of hydrofluoric acid + 2.5 ml of concentrated HNO3 + 6 ml of water). Subsequently, samples were cleaned and dried at room temperature. Group A is the polished CpTi discs.[7]

In a two-electrode anodization cell, the prepared titanium discs were used as anode and platinum as the cathode, with standardized electrode distance of 2 cm. 100 ml of electrolyte solution comprised 50 ml supporting the solution of 1.5 M-H2SO4 and 50 ml of etching solution. The solution was maintained under continuously stirring. With the aim of forming 40 nm and 80 nm nanotube configuration, a direct current voltage of 10 and 20 V was used at room temperature.[8] Later, they were thoroughly rinsed with double distilled water and dried at room temperature. The discs were then subjected to an annealing procedure in a box furnace at 450°C for 3 h to transform the amorphous TiO2 phase to the crystalline anastase phase.[9]

Surface characterization

High resolution scanning electron microscope (HRSEM, F E I Quanta FEG 200) was used to visualize the surface morphological patterns [Figures 1 and 2]. Forty nanometer tubes were categorized as Group B and 80 nm tubes as Group C.

Figure 1

40 nanometer scanning electron microscope image (magnification: ×100)

Figure 2

80 nanometer scanning electron microscope image (magnification: ×100)

Antibacterial analysis

Bacterial counting method was employed to assess the antibacterial activity of different diameters of TNT. All the procedures were done in triplicate. The three specimens groups were sterilized using ultraviolet radiation. The discs were placed on 24 well culture plates with the modified surface facing upward. Selected bacterial strains were inoculated onto the titanium surface at a density of approximately 0.05 ml/cm2. For Staphylococouc aureus (S. aureus), Mueller-Hinton agar medium- aerobically and Brain heart infusion broth agar for Porhyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans)- anaerobically was used for inoculation. Incubation was carried out at 37°C for 1 week. The colony-forming units (CFUs) were assessed with a digital colony counter (Toshiba, Japan) at three-time intervals, namely, 24 h, 48 h, and 1 week.[10]

RESULTS

CFU were the parameters examined in each group. All the variables were statistically analyzed using standard analysis of variance and the statistical significance was set at 0.05. The significance between both the test groups was evaluated statistically with a post hoc test, Tukey's honest Significance post hoc test. All the results are tabulated in Tables 1 and 2 and Graph 1.

Table 1

Analysis of variance analysis of colony forming units for three groups at 24 h, 72 h, and 1 week

Strain	Group	A	B	C	ANOVA	P
Sa	24 h	75.00±3.00	62.33±1.53	54.33±1.53	71.512	0.0001
	72 h	158.67±3.05	124.67±3.06	104.00±3.61	216.547	0.0001
	1 week	284.33±3.06	230.67±3.79	210.00±2.00	478.880	0.0001
Pg	24 h	317.33±2.31	278.67±2.52	245.00±2.65	631.661	0.0001
	72 h	424.67±5.13	314.67±3.06	287.67±4.73	817.190	0.0001
	1 week	687.00±2.65	513.67±4.04	457.67±3.22	3821.624	0.0001
Aa	24 h	377.00±4.36	300.67±4.16	285.33±4.93	357.643	0.0001
	72 h	455.67±3.51	382.67±4.73	308.00±4.00	968.375	0.0001
	1 week	695.33±3.51	599.67±2.08	522.00±3.61	2286.843	0.0001

ANOVA: Analysis of variance

Table 2

Tukey’s honest significance difference test between group B and C

Strain	Time	Mean difference	SE	P
Sa	24 h	8.00	1.74	0.009
	72 h	20.67	2.65	0.001
	1 week	20.67	2.48	0.0001
Pg	24 h	33.67	2.04	0.0001
	72 h	27.00	3.59	0.001
	1 week	56.00	2.74	0.0001
Aa	24 h	15.33	3.67	0.014
	72 h	74.67	3.36	0.0001
	1 week	77.67	2.57	0.0001

SE: Standard error

Graph 1

Comparison of influence of diameter of titania nanotube on antibacterial effect

DISCUSSION

Nanotechnology is a promising alternative for targeting microorganisms, as a possible replacement of antibiotics and its associated antimicrobial resistance concern.[11] Apart from the plethora of advantages in the use of TNT in implantable medical devices over conventional titanium surfaces, dental implants, especially will be benefited from its antibacterial prospectus.[12]

The target mechanisms on which current antibiotics work are: Cell wall maintenance, protein translation, and DNA replication. The bacteria have developed mechanisms to counteract these pathways.[13] The resistance mechanism could be due to new enzyme development such as ß-lactamases, bacterial cell wall modification, bacterial ribosomal alterations, expression of efflux mechanisms.[14] However, the nanosurface related antimicrobial mechanism is unrelated to above-mentioned techniques. Since the mode of action is via direct contact with the bacteria rather than cell penetration, the antimicrobial resistance incidence has been postulated to be less with nanosurfaces.[15]

The above-mentioned action is attributable to its unique physicochemical properties for instance, higher surface area to mass ratio resulting in greater cellular interaction;[16] capacity to alter the metabolic activity of bacteria;[17] ability to disrupt the biofilm.[18] As discussed earlier, the nanosurfaces mandate direct contact with bacteria to elicit its antibacterial action.[19] TNT arrays are one such nanotopographic antibacterial approach with potential clinical results. The current study aimed at delineating the influence of nanotube diameter on the effectiveness of its antibacterial action.

TNT could be prepared on titanium surfaces by various approaches, namely sol-gel method, liquid phase deposition, electrochemical anodization, and hydrothermal technique.[20] Most oriented and structured nanotubes have been found to be produced at a simple, low-cost process with anodization technique.[21] By altering factors such as time, voltage, temperature, cathode anode distance, and electrolyte concentration, morphological control over TNT nanotubes are proven to be possible.[22] Annealing heat treatment was carried out in the current work since it has shown improved antibacterial action compared to that of unheat treated surfaces.[23] Again, as it was carried out in both the test groups, the standardization was maintained, avoiding parameter-induced bias.

Clinical research has investigated the effect of titanium, aluminum, and zinc nanoparticles on titanium surface against S. aureus and P. gingivalis They have ascertained that the titanium nano modification was more efficient compared to that of other groups.[10] Earlier work on carbon nanotubes has postulated that tube diameter may play a role in the antibacterial activity.[24] Similar response to TNTs also has been observed by Ercan et al. on disrupting Staphylococcus epidermis and S. aureus and the authors confirmed that 80 nm tubes exhibited better results.[25] The current study is in accordance with their work, with 80 nm tubes exhibiting the highest antibacterial action against periodontal pathogens.

The possible mechanism involved could be attributed as: Tube diameter may influence the stress responses in the bacterial cell wall and lead to membrane disruption.[26] It was also postulated that smaller TNT tubes could not have had perceptible effect on the cell membranes and thus explaining their reduced antimicrobial action.[25]

TNT with antibiotics (vancomycin, tetracycline, minocycline, and silver) were assessed and had found promising results with establishing TNT as an efficient local drug delivery system.[45627] In the present study, considering the risk of antimicrobial resistance, the inherent antimicrobial potential of TNT arrays and the influence of nanotube diameter were analyzed.

All the pathogens exhibited a statistically significant difference between the three groups in all the three-time frames (P < 0.05). For S. aureus group, the control titanium surface exhibited 284.33 ± 3.06, whereas 40 nm and 80 nm displayed 230.67 ± 3.79 and 210 ± 2 growth, respectively. Reduced colonization of early colonizers such as S. aureus on the 80 nm tube surface may help in reducing the initial biofilm formation. Furthermore, the dental biofilm being a species interdependent model, may also influence and reduce further bacterial adhesion and maturation. The P. gingivalis growth was 687 ± 2.65, 513.67 ± 4.04, and 457.67 ± 3.22 respectively for control, 40 nm and 80 nm groups. A similar pattern was appreciated with the A. actinomycetemcomitans growth pattern: 695.33 ± 3.51, 599.67 ± 2.08, and 522 ± 3.61 respectively for control, 40 nm and 80 nm groups. Hence, apart from early colonizers, late colonizing pathogens were also reduced in 80 nm group.

Later, Tukey honest significance test was carried out to ascertain the significance between the two test groups and established a highly significant difference (P < 0.01) for 80 nm group, compared to that of 40 nm group. The post hoc analysis ascertained that the bacterial inhibition effect was significant for all three pathogens at 24 h itself. However, the mean difference confirmed superior effect at 72 h with all the pathogens examined with 80 nm group.

The current work did not analyze the TNT diameter effect on bacterial biofilm model, which may have an influence on the antibacterial activity of the bacteria involved. Within the limitations of the study, thus we conclude that the diameter of the nanotube system has been proven to regulate its antibacterial action, and 80 nm has been found to be effective, hence will be supportive in future biomedical engineering of dental implants.

CONCLUSION

The risk of peri-implantitis with dental implants is a major clinical problem, requiring pertinent, smart as well as simple solution without major morbidity to the patient. Hence the clinical translation of “Bench to bedside” nanosurface biofunctionalization system requires understanding in-depth about these complex multifunctional arrays and all the influencing variables involved. Fine-tuning the TNT diameter, in particular 80 nm diameter has been proven to positively influence the antibacterial activity of the tube arrays against major periodontal pathogens. The potential of TNT arrays in the dental implantology field is enormous and intriguing. Combining the knowledge of advanced physicochemical characteristics and system biology will enable us a smart and dynamic dental implant surface.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.