|Year : 2013 | Volume
| Issue : 6 | Page : 694-700
|Evaluation of the antimicrobial and physical properties of an orthodontic photo-activated adhesive modified with an antiplaque agent: An in vitro study
Chanjyot Singh1, Vinay Dua1, Matrishva Vyas2, Sanjeev Verma3
1 Department of Orthodontics, MMCDSR, MM University, Mullana, Haryana, India
2 Department of Orthodontics, Rungta Dental College, Chattisgarh, India
3 Department of Orthodontics, Reader, Swamy Devi Dyal Dental College, Barwala, Haryana, India
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|Date of Submission||09-Mar-2012|
|Date of Decision||12-Apr-2013|
|Date of Acceptance||07-Nov-2013|
|Date of Web Publication||20-Feb-2014|
| Abstract|| |
Introduction: This study was designed to investigate the antimicrobial and physical properties of orthodontic composite resin modified by the addition of an antimicrobial agent.
Materials and Methods: Transbond XT (3M Unitek), a photo-activated, light-cured composite resin, modified by the addition of chlorhexidine, in concentrations 2.5 wt% was formed into a uniform disc and also used for direct bonding of the brackets on the freshly extracted premolars for testing. The tablets of each group and a control group were subjected to the agar plate diffusion test to measure the zone of bacterial inhibition. In addition, they were incubated for 24 h in brain heart infusion medium inoculated with Streptococcus mutans and examined for antimicrobial action. A total of 80 extracted premolars were collected and divided into two sets of 40 teeth each. Stainless steel preadjusted edgewise appliance (PEA) brackets were bonded by using control and experimental composites. A universal testing machine was used to determine the shear bond strength. The first set of teeth was tested after 24 h and the second set after 25 days of storage in distilled water. Time-dependent release of antimicrobial agents from the modified composites was also monitored spectrophotometrically.
Results: The findings indicated that (1) addition of chlorhexidine to the orthodontic composite resin enhanced its antimicrobial properties, (2) there was no significant difference between the bond strengths of the control and the experimental resins tested after 24 h and 25 days and (3) maximum release of chlorhexidine from the modified resin was much higher than the minimum inhibitory concentration level.
Keywords: Antimicrobial, chlorhexidine, white spot lesion
|How to cite this article:|
Singh C, Dua V, Vyas M, Verma S. Evaluation of the antimicrobial and physical properties of an orthodontic photo-activated adhesive modified with an antiplaque agent: An in vitro study. Indian J Dent Res 2013;24:694-700
Etching of enamel prior to bonding and subsequent plaque formation around the orthodontic bracket can create localized demineralization. These areas of localized demineralization are called white spot lesions. These lesions formed after years of orthodontic therapy are several micrometers thick. Ketcham  and co-workers in a long-term follow-up of seven orthodontic patients concluded that the orthodontic lesions are not going to regress on their own and studies that report these regressions might be the result of the surface erosion rather than regression.
|How to cite this URL:|
Singh C, Dua V, Vyas M, Verma S. Evaluation of the antimicrobial and physical properties of an orthodontic photo-activated adhesive modified with an antiplaque agent: An in vitro study. Indian J Dent Res [serial online] 2013 [cited 2019 Oct 21];24:694-700. Available from: http://www.ijdr.in/text.asp?2013/24/6/694/127613
Elisades  advises that the only alternate method is to prevent the occurrence of the lesion itself in the first place and not let them re-occur.
In an attempt to prevent these white spot lesions from occurring, many modifications and alternate materials have been advocated. Bishara  in his study with chlorhexidine in 1998 concluded that when chlorhexidine was mixed with a varnish, it did not affect its shear bond strength.
Chlorhexidine is a base and is stable as a salt. The bactericidal effect of the drug is due to the cationic molecule binding to extramicrobial complexes and negatively charged microbial cell walls, thereby altering the cells' osmotic equilibrium. At low concentrations, low molecular weight substances will leak out, specifically potassium and phosphorous. At higher concentrations, precipitation of cytoplasmic contents occurs, resulting in cell death.  The addition of chlorhexidine would be a significant factor in the reduction of white spot lesion. 
This study was carried out in the Department of Orthodontics SPDC, Wardha, to evaluate the efficacy of chlorhexidine when used in a premixed state with orthodontic bonding adhesives and, also, it was to be determined whether the added chlorhexidine interfered in the internal structure of the resin during polymerization.
| Materials and Methods|| |
This study was carried out using a commercially available bonding material, Transbond XT (3M Unitek) in which chlorhexidine was mixed with the primer in a glass test tube with a plastic stirrer under yellow light in a dark room in a concentration of 2.5 wt% just before application. This acted as an experimental material [Figure 1].
|Figure 1: Transbond XT adhesive and chlosite gel containing chlorhexidine gluconate|
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This experimental material was tested against unadulterated Transbond XT (0.0 wt% chlorhexidine) composite that acted as a control material. Both materials were compared for any difference in their
- Shear bond strength
- Diametral tensile strength
- Antimicrobial action
- Chlorhexidine release over time.
Shear bond strength
The first portion of the study measured the shear bond strength of the brackets bonded with Transbond XT material (control) and chlorhexidine-modified Transbond XT adhesive (MAC). Sixty premolars freshly extracted for the orthodontic therapy were used for the study. All the teeth were mounted in steel moulds of uniform dimensions (45 mm height and 23 mm internal diameter) [Figure 1] and [Figure 2].
|Figure 2: Mounted premolar with brackets and column-type machine used for testing|
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These samples were then divided into two equal groups:
· Control group (Gp XT), consisting of 30 samples in which the teeth were bonded using Transbond XT
· Experimental group (Gp MAC), consisting also of 30 samples in which the teeth were bonded using chlorhexidine-modified adhesive composite.
The labial surfaces were etched with phosphoric acid for 30 s and then rinsed with water and air dried. Then, the brackets were bonded on the marked surface and light cured as per the manufacturer's instructions.
The moulds were then stored in normal saline.
After 24 h, shear bond strength measurement was performed using a column-type motorized constant loading machine delivering a perpendicular force to the bracket by a flat end steel rod from the machine, producing a shear force at the bracket-tooth interface [Figure 2].
All the samples were attached to the lower part of the machine and the shear bond strength of each specimen was tested at a cross-head speed of 1 mm/min.
The results of each test were electronically recorded and tabulated for further statistical analysis.
Diametral tensile strength
The second part of the study involved measurement of the diametral tensile strength of both the experimental and the control groups.
This was investigated by preparing a disc of uniform diameter of 5.5 mm wide and 1.5 mm height. The dimensions of all the discs were kept uniform by preparing a die of this dimension and filling it with excessive composite material, and then the excess was flushed with a probe, Later, the composite was cured twice from both sides for 60 s/side.
A total of 64 discs were prepared, 32 discs each for the control and the experimental groups.
The samples were then divided into two equal groups:
- Control group (Gp XT): Of the 32 discs prepared in this group, 16 discs were stored in normal saline and 16 discs were stored in a dry state at 37°C
- Experimental group (Gp MAS): Similarly, of the 32 discs prepared in this group, 16 discs were stored in normal saline and 16 discs were stored in a dry state at 37°C.
After 24 h, the specimens in both the experimental and the control group were subjected to a diametral tensile strength test. Testing was performed in compression with the disc oriented vertically on the disc diameter.
The column-type loading machine was used for testing with a loading pattern of 1 mm/min [Figure 2].
After 21 days, specimens stored in saline from both the control and the experimental groups. These were then taken out from saline and then tested for diametral tensile strength. Results were recorded and tabulated and subjected to statistical analysis in two states to also determine water aging had any effect on the diametral tensile strength.
A total of 40 discs as previously described were prepared and divided into two equal groups:
- Control group (Gp XT): This group consisted of 20 discs made from the original Transbond XT composite material. These 20 discs were paired in a test tube and sealed. Ten test tubes containing two discs each were made for the control group
- Experimental group (Gp MAC): This group consisted of 20 discs made from the chlorhexidine-modified adhesive composite. Similarly, 10 test tubes containing two discs each were made for this group.
Every 6 th day, one test tube from the control group was removed and tested against one test tube from the experimental group for the antimicrobial properties.
Streptococcus mutans was used as test bacteria; it was grown locally in brain heart infusion broth at 37°C overnight.
At the time of testing, two discs from each of the groups were placed on the preinoculated agar and incubated at 37°C for 48 h. Testing was carried out simultaneously for both the groups.
After 48 h, the diameter of the growth inhibition zone was measured and recorded. Three measurements were made and the mean was taken.
Then, after every 6 th day, the procedure was repeated for one test tube each from the control and the experimental groups. These tests were carried out up to the 60 th day. Ten measurements were made for each group and the recorded results were compiled and tabulated
The fourth part of study was to determine the in vitro release of chlorhexidine from the chlorhexidine-modified adhesive over a period of time. This was performed with an optical density reading of the solution at 254 nm using an ultraviolet spectrophotometer (CAREY-5E-UV-VIS-NIR, varian, Taiwan) [Figure 3].
|Figure 3: Test tubes containing discs and the ultraviolet spectrophotometer (CAREY-5E-UV-VIS-NIR) used for testing chlorhexidine release|
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Testing was performed by fabricating discs of uniform dimension from the prefabricated die (1.5 mm height and 5.5 mm width) as described previously.
A total of six discs of both groups was made and divided into two groups:
· Control group: This group consisted of three discs made from the original Transbond XT composite. Each disc was stored in an air tight test tube containing 3 mL of sterile distilled water. Thus, three test tubes were prepared for the control group
· Experimental group: This group also consisted of three discs made from the chlorhexidine-modified adhesive composite. Similarly, three test tubes were prepared for the experimental group.
The amount of chlorhexidine release was determine from each disc on the 8 th day by taking one test tube from each group. The discs were removed, air dried and transferred to another test tube. The test tube were then sealed and labeled.
Then, chlorhexidine release was measured using a spectrophotometer at 254 nm from the solution, first for the control group and then for the experimental group.
This procedure was carried out on the 8 th day under similar conditions and three measurements were made and mean values were taken at this sampling period.
Then, on the 16 th day again, this procedure was carried out and a mean value was taken. A total of four readings were made and the sampling was performed on the 8 th , 16 th , 32 nd and 60 th days.
The results obtained were tabulated and subjected to statistical analysis.
| Results and Statistics|| |
Shear bond strength
Results obtained for the chlorhexidine-modified adhesive were a bond strength of 9.88 MPa and for the original composite were 10.6 MPa.
Diametral tensile strength
The second variable investigated was difference between the diametral tensile strength.
The result obtained for the chlorhexidine modified adhesive was 39.57 MPa and that for the original adhesive was 40.57 MPa. Both groups showed no statistically significant difference in diametral tensile strength at 0.05 level of significance (P = 0.52).
Effect of water aging
The diametral tensile strength of the chlorhexidine-modified adhesive when soaked in water (39.24 MPa) was comparable to the diametral tensile strength of the original Transbond XT soaked in water for 21 days (43.04 MPa).
Although there was a decrease in the diametral tensile strength in the experimental group, at the 0.05 level of significance (P = 0.10), this was statistically insignificant.
In this in vitro study, an initial surge was reported in the first week, with large inhibition zones at the time of first sampling, and, later, these decreased in size continuously on the second sampling at the 12 th day and at the third sampling on the 18 th day, showing marked reduction of the chlorhexidine activity over a period of 2 weeks. However, after this time, the result obtained at the next sampling periods at the 24 th day until the completion of the last sampling, which was completed on the 60 th day, was more or less constant.
There were initial bursts of chlorhexidine release from the modified adhesive discs at the 8 th day, which was followed by a sharp decrease at Day 16. This was seen on during the 2 nd sampling period, but at the 3 rd sampling on the 32 nd day, and finally at the end of the study, on the 64 th day, the amount of chlorhexidine release was found to be more on less constant.
| Discussion|| |
White spot lesions are reported to result from the high cariogenic challenge prevailing in the plaque around the orthodontic appliance.
pH levels lower than 4.5 have been measured in the plaque around the brackets and the bands. At such a low pH, the remineralization is hampered, and any measures to remineralize the enamel will not be successful. One such method is use of fluoride in combination with antimicrobials.  Glass ionomers were one such promising alternate, but it was not found to be clinically acceptable because of its lower bond strength in a prospective clinical trial by Goworski and Weinstein. 
Othman  in his study clearly stated that the leaking fluoride ion weakens the matrix and the duration of fluoride release is very short.
Rola  states that chlorhexidine inhibits acid production in plaque and thus reduces the pH fall during sucrose challenges; at high pH, the potential for remineralization of the enamel is high.
Results of the physical properties are as below.
Shear bond strength of the modified composite was found to be 9.88 MPa and that for the original composite was 10.88 MPa [Graph 1]. At the 0.05 level of significance, there was no statistically significant difference in the mean shear bond strength (P = 0.21) [Table 1] between the two groups, and the bond strength was within the acceptable limit clinically as given by Reynolds. 
|Table 1: Comparison of the shear bond strength of the original Transbond XT adhesive and the chlorhexidine-modified adhesive |
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Thus, it can be stated that the chlorhexidine-modified adhesive is dependable clinically and is as efficient as the original adhesive.
These results are comparable to the result of the study carried out by Bishara  in 1998, although his bond strength of 11.2 MPa was considerably higher than in this study (9.88 MPa), but this was not a significant difference. Moreover, it was more than the minimum acceptable clinical range, as suggested by Reynolds  ; thus, having a huge benefit over the fluoride-containing glass ionomers, where the fluoride ion is part of the matrix and when leached out it weakens the matrix.
The result obtained for the diametral tensile strength of the chlorhexidine-modified adhesive was 39.86 MPa and that for the original adhesive was 41.02 MPa [Graph 2]. Both groups showed no statistically significant difference between them at the 0.05 level of significance (P = 0.521) [Table 2]. Also, the mean value obtained for the diametral tensile strength of the experimental group was 39.86 MPa, which was within the acceptable range for the all-purpose composites as shown by Elisades. 
|Table 2: Comparison of diametral tensile strength of the original Transbond XT adhesive and the Chlorhexidine-modified adhesive of the unaged sample |
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This is readily comparable with the results obtained by Othman  in 2002 (diametral tensile strength = 42.98 MPa) and Evans  in 2006 (diametral tensile strength = 41.8 MPa), but this minute difference is not significant and can be attributed to the different settings of the study and apparatus and also of the operator, but the range for the acceptance for the all-purpose composite allows both to be used clinically.
This study show a slight decrease in the diametral tensile strength of 1 MPa as compared with the original adhesive, but this minute decrease is statistically insignificant.
The physical properties of the modified adhesive remained comparable to the original adhesive and all three studies by Othman  in 2002 (42.98 MPa), Evans  in 2006 (41.8 MPa) and the present study (39.86 MPa).
The difference in value may be due to the difference of the set up and design of the study and use of a different machine for the measurement of the diametral tensile strength, but this is insignificant and can be ignored statistically.
Effect of water aging
In measuring the effect of water aging, which starts from 15 min after polymerization and requires a minimum of 7 days to reach equilibrium and about 4 days to show the majority of expansion, the result of this study clearly showed a statistically insignificant change in the diametral tensile strength of the water-aged chlorhexidine-modified adhesive soaked in water for 21 days (39.24 MPa) when compared with the diametral tensile strength of the unaged original Transbond XT adhesive (43.04 MPa) [Table 3].
|Table 3: Comparison of diametral tensile strength of the original Transbond XT adhesive and the Chlorhexidine-modified adhesive of the water-aged sample |
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Also, when the same was compared with the diametral tensile strength of the unaged chlorhexidine-modified adhesive (39.57 MPa), the result was not similar [Table 4] and Graph 3].
|Table 4: Comparison of diametral tensile strength of the original Transbond XT adhesive and the Chlorhexidine-modified adhesive of the water-aged and unaged samples |
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Although there was a decrease in the diametral tensile strength in the experimental group, but at the 0.05 level of significance (P = 0.80), this was statistically insignificant.
Also, the mean value obtained for the diametral tensile strength (39.57 MPa) was within the acceptable range for the all-purpose composites, as shown by Elisades. 
The Streptococcus mutans activity was reported to be inhibited by chlorhexidine, but the rate at which this activity decreased or continued was important for clinical benefits.
In this in vitro study, an initial surge was reported in the first week, with large inhibition zones at the time of first sampling, and, later, these decreased in size continuously on the second sampling on the 12 th day and at the third sampling on the 18 th day, showing marked reduction of the chlorhexidine activity over a period of 2 weeks. But, after this time, the result obtained at the next sampling period on the 24 th day until the completion of the last sampling, which was completed on the 60 th day, was more or less constant [Graph 4].
The value achieved on the 18 th day was consistent till the 60 th day. Therefore, it was clear that the potency of the antimicrobial activity is maximum in the first week, which then decreases till the second week, and, at the end of the third week, it achieves a balance that is consistent, exhibiting a potent stable antimicrobial activity till the 60 th day, while on other hand the control group showed zero antimicrobial activity.
This is in contrast to the finding in Ogaard's  prospective clinical trial for the efficiency of fluoride release from glass ionomers, where no difference from the control in plaque formation was seen.
This consistency is comparable to the material used by Othman  in 2002 in his study with Benzalkonium chloride (BAC), which showed similar antimicrobial properties as chlorhexidine, but with a disadvantage of being a relatively new material and not readily available). Chlorhexidine has a wide research in his background as compared with BAC.
Chlorhexidine, when incorporated in composite resins, has to be released freely from the cured material to be effective. This study clearly showed that there were initial bursts of chlorhexidine released from the modified adhesive discs at the 8 th day, followed by a sharp decrease on Day 16, which was in the 2 nd sampling period, but, at the 3 rd sampling on the 32 nd day and finally at the end of the study on the 64 th day, the amount of chlorhexidine release was found to be more on less constant [Graph 5]; however, the reduced level of chlorhexidine release was equally effective in inhibiting the growth of Streptococcus mutans activity, as evidenced by the zone of inhibition around the disc.
The minimum clinically significant amount of chlorhexidine was not determined in this study. However, the pattern of chlorhexidine release might offer several advantages in clinical orthodontics, such as slow and continuous release of chlorhexidine over a prolonged period and, secondly, chlorhexidine release from the modified adhesive site specific to the area most susceptible to plaque accumulation and enamel decalcification adjacent to the bonded orthodontic brackets and independent of patient compliance.
This could sharply decrease the demineralization zone present at these sites at the time of debonding.
This study has clearly shown that chlorhexidine when mixed with the primer of the bonding adhesive provides antimicrobial activity, which is derived at the site of plaque accumulation and is sustained over a period of time. This modified adhesive has a similar internal structure and physical properties as the original adhesive.
Othman and co-workers  have shown that BAC is effective in reducing the bacterial count significantly and also that it has bactericidal properties.
But, in this study, chlorhexidine was used because it is readily available in a developing economy like ours.
Sandham et al.  in 1992 showed that chlorhexidine is not toxic was not causing any staining when used in orthodontic therapy. Therefore, this study preferred chlorhexidine, which already has a wide acceptance and availability.
| Conclusion|| |
· There was no statistically significant difference in the physical properties of the experimental material when compared with the original material
· There was a significant increase in the antimicrobial activity of the modified adhesive, which was absent in the original material
· Chlorhexidine release was continuous from the experimental adhesive and is sustained over a long period of time.
This study has validated the use of chlorhexidine premixed with the orthodontic bonding adhesive as an effective material for the prevention of white spot lesion.
However, this study was performed for a limited period of time. Chlorhexidine release was evaluated for a 60-day period, whereas in a clinical situation, the orthodontic treatment will last for about 18-24 months. Also, the safe level of chlorhexidine intake could not be determined as it was an in vitro study.
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|10.||Reynolds I. A review of direct orthodontic bonding. Br J Orthod 1975;2:171-8. |
|11.||Al-Musallam TA, Evans CA, Drummond JL, Matasa C, Wu CD. Antimicrobial properties of an orthodontic adhesive combined with cetylpyridinium chloride. Am J Orthod Dentofacial Orthop 2006;129:245-51. |
|12.||Øgaard B, Larsson E, Henriksson T, Birkhed D, Bishara SE. Effects of combined application of antimicrobial and fluoride varnishes in orthodontic patients. Am J Orthod Dentofacial Orthop 2001;120:28-35. |
|13.||Sandham HJ, Nadeau L, Phillips HI. The effect of chlorhexidine varnish treatment on salivary mutans streptococcal levels in child orthodontic patients. J Dent Res 1992;71:32-5. |
Department of Orthodontics, MMCDSR, MM University, Mullana, Haryana
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]
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