INTRODUCTION

Dental plaque, a biofilm form, is considered the main causative factor of dental caries and periodontal diseases. High levels of acidogenic bacteria are present in the plaque, notably Streptococcus mutans (S mutans).¹ The insertion of an orthodontic appliance tends to create new surfaces available for plaque formation, thereby increasing the level of microorganisms in the oral cavity.² Lundstrom and Krasse³ reported an increase in the level of S mutans after the insertion of orthodontic appliance. Components of fixed orthodontic appliances such as brackets, bands, and archwires serve as plaque-retentive niches impeding effective oral hygiene and causing a high cariogenic challenge. The orthodontic wires remain a good habitat for oral microorganisms sufficient to cause dental caries and periodontal diseases.⁴

Nickel titanium (NiTi) alloys are the most common aligning archwires. Copper NiTi (Cu-NiTi), a quaternary alloy of copper, nickel, titanium, and chromium, increases the effectiveness of tooth movement.⁵ Literature has reported on the antimicrobial property of a metallic copper surface and that bacteria, yeasts, and viruses are rapidly killed on metallic copper surfaces.⁶ Although the mechanical properties of NiTi and Cu-NiTi orthodontic archwires have been extensively studied, very little in vivo research has compared the bacterial adhesion to these wires.

Bacterial adhesion is influenced by the surface characteristics of biomaterials, particularly surface roughness and surface free energy (SFE).⁷ In accordance with thermodynamic rules, a material with high SFE attracts more bacteria to its surface than one with a low SFE.⁷ Surface roughness promotes bacterial adhesion by increasing the adhesion areas and preventing the dislodgement of bacterial colonies.⁴

Thus, this clinical study was designed to compare the adhesion of S mutans to NiTi and Cu-NiTi archwires and correlate the surface roughness and surface free energy of these wires to bacterial adhesion.

MATERIALS AND METHODS

Approval for this clinical study was obtained from the Institutional Ethical Committee, Sri Ramachandra University. Based on preliminary data and power analysis (power of the study as 0.80, α- error as 0.05), the sample size was calculated to be 16 patients.⁸ Hence, 18 patients (expecting an attrition rate of 10%) who were to undergo orthodontic treatment at the Department of Orthodontics were included after being screened as per the inclusion and exclusion criteria. The inclusion criteria were patients with a Little irregularity index of 1 to 3 (minimal crowding),⁹ good or fair oral hygiene with a Simplified Debris Index ¹⁰ of 0 to 1.5, and an extraction treatment plan involving the extraction of four premolars. The exclusion criteria were patients who underwent unilateral or single arch extractions, patients with tooth restorations and fixed or removable prosthesis, and patients with poor oral hygiene with a Simplified Debris Index of more than 1.5.

Patients were bonded with 0.022” × 0.028” preadjusted edgewise brackets (Mini Master Series, American Orthodontics, Sheboygan, Wis.). 0.016” NiTi and Cu-NiTi orthodontic archwires (Ormco Corp., Glendora, Calif.) were placed as initial aligning wires in the upper and lower arches on random allocation. NiTi archwires were placed in the upper and Cu-NiTi archwires in the lower arches for nine patients and vice versa for the other nine patients. After 28 days of an initial aligning phase, the patients were recalled and the archwires were stepped up to 0.016” × 0.022” NiTi and Cu-NiTi archwires.

Archwire Insertion and Retrieval Protocol

The archwires, before insertion, were sterilized in an autoclave. Oral prophylaxis was performed just prior to archwire insertion. Patients were instructed on oral hygiene measures¹¹ and the usage of identical dentifrices. The patients were then recalled 28 days postinsertion of the 0.016” archwires, and the archwires were removed cautiously to avoid any contact with the oral mucosa. These retrieved archwires were stored in a phosphate buffer solution (PBS) in a 50 ml sterile falcon tube.

After the initial aligning, the archwires from the same set of patients were stepped up to 0.016” × 0.022”, reversing the pattern that was followed in the first phase. Oral prophylaxis was again performed just prior to archwire insertion. Patients were recalled after 28 days of insertion of the 0.016” × 0.022” archwires, and a similar retrieval protocol was followed for these archwires.

The estimation of microbial adhesion was done following the procedure by Yang et al.¹² Briefly, the retrieved archwires were washed with PBS. The adherent bacteria were then detached by vortexing and then by a sonication method using a sonicator in 15 ml PBS with four 30-second pulses and three 30-second intermittent cooling periods. After the detachment of the microbes, S mutans adhesion was evaluated using real-time polymerase chain reaction (PCR). This was performed using the Fast 7900HT Real-time PCR equipment (Applied Biosystems, Foster City, Calif.) to study the bacterial quantification by SYBR green chemistry for relative quantification. The sequence for the primer Sm F5 was AGC CAT GCG CAA TCA ACA GGT T with 22 bases, and the sequence for the primer Sm R4 was CGC AAC GCG AAC ATC TTG ATC AG with 23 bases. The results were analyzed using relative quantification CFQ Software, (Applied Biosystems, Foster City, Calif).

The three-dimensional (3D) surface profilometry analysis was performed to assess the surface roughness (R_a) of the archwires. The mean roughness of each specimen was measured using the 3D surface profilometer Contour GT-K (Bruker, Mass.) at a magnification of 60× and vertical scanning interferometry mode.

Contact angle measurements were performed to calculate the SFE of both archwire materials. Advancing and receding contact angles were measured for the 0.016” and 0.016” × 0.022” sizes of the NiTi and Cu-NiTi archwires in as-received condition and after 4 weeks of intraoral exposure. The dynamic contact angle measurements were performed using the Dynamic Force Tensiometer (9000 series; NIMA Technology Ltd, Coventry, UK, England). Solid total SFE (γ_s) was calculated from the surface tension of the probe liquid and the contact angle hysteresis of the liquid using the following formula, where Θ_a and Θ_r are the advancing and receding contact angles, respectively, and γ_l is the surface tension of the probe liquid water, which is equal to 72.8 mJ/m²:

RESULTS

S mutans Adhesion

The real-time PCR showed the relative quantification of S mutans present in the samples by evaluating the cycle threshold (Ct) values. Ct values are the threshold values at which there is an expression of the bacterial genome. The value is inversely proportional to the amount of bacterial genome present, which means that a higher Ct value indicates a lower S mutans count and vice versa.

Round and rectangular Cu-NiTi archwires showed higher S mutans adhesion than that of corresponding NiTi archwires, and this difference was statistically significant (P = .00). Rectangular archwires (NiTi and Cu-NiTi) showed higher S mutans adhesion than that of round archwires, and this difference was statistically significant (P = .00; Table 1).

Table 1. Cycle Threshold (Ct) Values of Adherent Streptococcus mutans on Nickel Titanium (NiTi) and Copper-NiTi (Cu-NiTi) Archwires^a

View Fullscreen

Surface Roughness

Surface roughness of the different groups at T₀ (as-received condition) and T₁ (after 4 weeks of intraoral exposure) is shown in Table 2.

Table 2. Average Surface Roughness of Nickel Titanium (NiTi) and Copper-NiTi (Cu-NiTi) Archwires (N = 10)

View Fullscreen

The images generated by 3D profilometry for the archwires in as-received condition and after 4 weeks of intraoral exposure are shown in Figures 1 and 2.

View Figure

• Download as Powerpoint
• Download Figure

View Figure

• Download as Powerpoint
• Download Figure

Round and rectangular Cu-NiTi wires showed higher surface roughness than THE corresponding NiTi wires in as-received condition as well as after 4 weeks of intraoral exposure, but these changes were statistically significant only in the rectangular wires (P = .0000). All of the archwires studied increased in surface roughness after 4 weeks of intraoral exposure, and this change was statistically significant only for 0.016” Cu-NiTi and 0.016” × 0.022” NiTi wires (P = .035 and .008, respectively).

Dynamic Contact Angle and SFE

The advancing and receding contact angles, which are components of dynamic contact angles, were measured with the help of a contact angle tensiometer. From these, the SFE of the archwires at T₀ (as-received) and T₁ (after 4 weeks of use) were calculated (Table 3).

Table 3. Dynamic Contact Angles (Advancing and Receding) and the Calculated Surface Free Energy Value of 0.016” and 0.016” × 0.022” Nickel Titanium (NiTi) and Copper-NiTi (Cu-NiTi) Wires at T₀ and T₁

View Fullscreen

The 0.016” and 0.016” × 0.022” Cu-NiTi archwires showed greater SFE than the corresponding NiTi archwires at T₀ and T₁, and this change was statistically significant (P = .000). All of the archwires studied demonstrated greater SFE after 4 weeks of intraoral exposure, and these changes were statistically significant (P = .015 for NiTi and P = .008 for Cu-NiTi).

DISCUSSION

The aim of the present study was to evaluate the adherence of S mutans to the NiTi and Cu-NiTi wires. Furthermore, the physical properties, such as surface roughness, dynamic contact angle, and SFE, were assessed to correlate these surface characteristics to the microbial adhesion. The real-time PCR showed the relative quantification of S mutans present in the sample by evaluating the Ct values. Ct values are the threshold values at which there is an expression of the bacterial genome. The value is inversely proportional to the amount of bacterial genome present, which means that a higher Ct value indicates a lower S mutans count and vice versa.

The results showed that there was greater adhesion of S mutans to Cu-NiTi wires than NiTi wires (Table 1). This was evident in both the round and rectangular wires, with the difference being statistically significant.

Grass et al.⁶ have explained the antimicrobial property of metallic copper surface and the toxicity mechanisms of ionic copper. Bacteria, yeasts, and viruses are rapidly killed on metallic copper surfaces by the process termed contact killing,⁶ but the present study demonstrated no reduction in the microbial adhesion to Cu-NiTi archwires. Rather, there was an increase in the adhesion of microbes to these archwires. This increased adhesion could be attributed to an increase in both the surface roughness and the SFE of the Cu-NiTi archwires. The antimicrobial effect of copper was not elicited probably because of the small amount (5.09% in 35⁰ Cu-NITI⁵) of copper that is incorporated in the NiTi matrix. Furthermore, the copper in the Cu-NiTi alloy is present only in its oxide form in the surface of the archwire.⁵ Also, real-time PCR quantifies the amount of deoxyribonucleic acid present in the given sample, evaluating all live and dead bacteria alike. Hence, the assessment of the antibacterial effect of copper in Cu-NiTi requires further studies.

While comparing the round and rectangular wires, it was observed that the rectangular NiTi and Cu-NiTi wires had greater microbial adherence than their corresponding round wires. These findings were statistically significant. This could be a result of the increased surface area of the rectangular wires and their octagonal cross section because of the edge bevels, but this finding should also be considered with the bacterial count differing at the various stages of treatment.

Surface roughness of the archwires has been studied by various methods, such as contact profilometry, scanning electron microscopy, and atomic force microscopic analysis. The 3D profilometer analysis, used in the present study, is a newer method of noncontact profilometry used to measure the surface roughness. Although bacterial adherence to archwires has been extensively studied, their correlation to surface characteristics in an in vivo condition has not been addressed to the best of our knowledge.

At their respective dimensions, Cu-NiTi wires exhibited greater roughness than the NiTi wires (Table 2). This was evident for both the as-received and retrieved arch wires (T₀ and T₁). However, this increased roughness demonstrated was statistically significant only for the rectangular wires. The round wires exhibited greater surface roughness than the rectangular wires in both the NiTi and Cu-NiTi. The round cross section exhibited a curvature in the surface to be measured, whereas the 3D profilometry required a flat surface to measure the surface roughness. Hence, the surface roughness evaluation of the round wire should be viewed with caution.

This observed increased roughness associated with Cu-NiTi wires has been explained by Gravina et al.,⁵ who demonstrated that Cu-NiTi 35°C wires showed irregular morphological characteristics when compared with other wires, presenting with drawing marks, slots, and micro-cavities formed as a result of a pullout of particles, possibly NiTi. The findings of the present study also affirm this by measuring the surface roughness of the archwires by 3D profilometry. The increased roughness of the Cu-NiTi wires could have been the reason for the greater microbial adherence demonstrated by these wires.

Both the 0.016” and 0.016” × 0.022” NiTi and Cu-NiTi wires demonstrated an increase in surface roughness after 4 weeks of intraoral exposure (Table 2). However, this change was statistically significant only for the 0.016” Cu-NiTi and 0.016” × 0.022” NiTi wires.

Many studies¹³ have documented the importance of the increase in surface roughness. The increase in surface roughness of wires after intraoral usage may profoundly modify the reactivity of the archwire surface with undetermined effects on the corrosion resistance, nickel dissolution, and frictional resistance of the archwires. Eliades et al.¹³ and Zegan et al.¹⁴ observed that intraoral exposure altered the micro-structural and chemical composition of archwires. Taha et al.¹⁵ and Ghazal et al.¹⁶ showed a significant increase in surface roughness and biofilm adhesion after intraoral exposure. Eliades et al.¹³ attributed the surface changes to the development of microcrystalline NaCl, sodium chloride; KCl, potassium chloride; and CA-P, calcium phosphate deposits that substantially altered the surface composition and topography of the archwire alloy, causing pitting and crevice corrosion defects as well as a reduction in the alloy grain size, which they identified in retrieved NiTi archwires. Bahije et al.¹⁷ demonstrated the impact of acidogenic bacteria on the corrosion behavior of the NiTi wires, hence affecting the surface properties of the arch wires.

The SFE is the energy associated with the intermolecular forces at the interface between two media. The apparent SFE value was derived from the dynamic contact angles (advancing and receding contact angles) measured with the help of a dynamic surface tensiometer. Because the flat surfaces of the archwires were too small to allow goniometric methods for measuring contact angle, the dynamic contact measurements were performed.

The Cu-NiTi wires showed greater SFE than the NiTi wires in both the round and rectangular dimensions studied. These changes were also statistically significant (Table 3).

The 0.016” and 0.016” × 0.022” NiTi and Cu-NiTi wires demonstrated an increase in SFE after 4 weeks of intraoral exposure (Table 3). This change was statistically significant, but the difference in mean SFE between T₀ and T₁ was greater for the 0.016” NiTi wires than that of the Cu-NiTi wires. Therefore, the inference is that the NiTi wires went through higher surface changes than the Cu-NiTi wires in terms of SFE.

Materials with greater surface roughness were found to have greater SFE. This was evident from the results of the current study. These two parameters have been shown to be closely associated with the adherence of S mutans to the archwires.⁸ Hence, these were subjected to correlation analysis.

The Pearson correlation suggested that there was a predominantly negative correlation between SFE, surface roughness, and Ct values. A statistically significant, moderate negative correlation was evident between the SFE and microbial adhesion for the 0.016” Cu-NiTi wires. However, the Ct values are inversely proportional to microbial adhesion, thus leading to the inference that SFE and surface roughness are directly proportional to the S mutans microbial adhesion. Similar results between surface roughness and biofilm adhesion have been reported in NiTi archwires by Taha et al.¹⁵

Based on this in vivo study, S mutans adhesion could be attributed to the SFE and surface roughness of NiTi and Cu-NiTi archwire materials. Further research would be required to conclusively prove the same for other archwire materials and microbes.

Some of the limitations of the present study are that it has evaluated only 35°C Cu-NiTi and NiTi for surface properties and microbial adhesion. Future studies may evaluate the rheology of different types of coatings, which are added for esthetic purpose and for reducing the friction in archwires as well as archwires made of other materials. Other oral pathogenic bacteria such as Lactobacilli, Staphylococci, Veillonella, and yeast, which have been proven to be increased after the bonding of fixed orthodontic appliances, may also have to be evaluated.

NiTi alloy wires are the most commonly used initial aligning archwires. Because the present study showed an increase in surface roughness and SFE following intraoral exposure, which is a critical factor to greater microbial adhesion, the clinician should be prudent and aware of this situation, especially when using the same archwire for an extended period of time.

CONCLUSIONS

• S mutans adhesion was significantly greater in Cu-NiTi than NiTi archwires for both cross sections studied. S mutans adhesion was significantly greater in rectangular archwires when compared with round archwires for both NiTi and Cu-NiTi.

• The surface roughness (R_a) of the Cu-NiTi wires was greater than that of the NiTi wires for both cross sections studied. Surface roughness increased after 4 weeks of intraoral exposure for all the archwires, but the mean difference was more for the Cu-NiTi archwires.

• The surface free energy of Cu-NiTi wires was greater than that of the NiTi wires for both cross sections studied. Surface free energy increased after 4 weeks of intraoral exposure for all of the archwires, but the mean difference was more for round NiTi archwires.

• A predominantly negative correlation was seen between the Ct value of adherent bacteria and surface characteristics. This indicates that an increase in surface roughness and surface free energy results in an increase in bacterial adhesion.