Qualitative and quantitative evaluation of enamel surface roughness and remineralization after interproximal reduction: An in vivo study

Introduction

Interproximal reduction (IPR) damages the caries protective superficial layer of the enamel, making the enamel surface prone to caries because of the increase in surface roughness. Remineralizing solutions can help in preventing these undesirable side effects. Therefore, this study aimed to compare the effect of nanohydroxyapatite (nHAp) and sodium fluoride (NaF) application on enamel remineralization after IPR and to evaluate changes in surface roughness, composition, and microhardness of the treated enamel.

Methods

A total of 25 patients with Angle’s Class I malocclusion, requiring 4 premolar extractions, were selected and randomly divided into 5 groups (n = 5). Group 1 served as the control. In group 2, the extraction of premolars was done immediately after IPR, and in group 3, the extraction of premolars was done 3 months after IPR. In group 4, the extraction of premolars was performed 3 months after IPR with weekly application of nHAp serum. In group 5, the extraction of premolars was performed 3 months after IPR, along with once-a-month application of NaF varnish. The proximal reduction of premolars in all the groups was done using Strauss IPR burs (Strauss Diamond Instruments, Palm Coast, Fla). The extracted teeth were sectioned, and the enamel surfaces were subjected to energy-dispersive x-ray spectroscopy to evaluate elemental composition. Vicker’s microhardness test was used to evaluate enamel hardness and atomic force microscopy for enamel surface roughness. Descriptive statistics were calculated for the 5 groups using a 1-way analysis of variance, and Tukey’s multiple post-hoc test was used for intergroup comparison.

Results

Calcium-to-phosphorous ratio, enamel microhardness, and surface roughness were found to be closest to untouched enamel in patients treated with nHAp, followed by patients who were treated with NaF. A lower calcium-to-phosphorous ratio and weakened and roughest enamel surface was seen in teeth, which were extracted immediately after IPR.

Conclusions

Among the remineralizing agents tested, nHAp serum can be recommended for better remineralization of enamel surfaces after IPR.

Highlights

•

Interproximal reduction damages surface enamel.
•

Remineralization can reduce surface roughness and increase mineral content.
•

Nanohydroxyapatite serum is an effective remineralizing agent after interproximal reduction.

Interproximal reduction (IPR) is the clinical removal of a portion of the tooth enamel from an interproximal contact region by grinding. It is also referred to as enamel reduction, interdental stripping, air-rotor stripping (ARS), slenderization, or reproximation. In 1944, Ballard advocated for the reduction of the proximal surfaces of mandibular anterior teeth to correct tooth size discrepancies. In 1956, Hudson described a metallic-strip-based enamel reduction method, followed by polishing and fluoride preventive methods. IPR can be accomplished by using either mechanical or manual techniques. Handheld abrasive strips, metallic strip systems, diamond discs, and ARS burs are used for IPR.

ARS can create substantially more space than is usually obtained by conventional IPR procedures and can be done at any time during treatment without causing discomfort to the patient or adversely affecting the function of the dentition, interocclusal relations, or tooth form. The original ARS technique has been modified to enhance the removal of smaller amounts of interproximal enamel in the lower anterior region to a combination technique, in which the enamel is reduced with stripping bur on the lingual and facial surfaces until the contact area is a knife-edge of enamel which is then removed with handheld abrasive strips. Any surface enamel stripping procedure removes the fluoride-rich, caries-resistant layer, which results in undesirable side effects. To avoid these side effects, commercial remineralizing agents have been recommended to smooth out the enamel surface and improve remineralization.

Remineralization is defined as the process in which calcium and phosphate ions are supplied from an external source to a tooth, thereby causing ion deposition into the demineralized enamel. Various remineralizing agents have been introduced to remineralize the enamel. Fluoride varnishes were first introduced in Europe in 1964 and are effective in the prevention of incipient enamel lesions. Gao et al reported that the use of a 5% sodium fluoride (NaF) varnish has the potential to remineralize the enamel. To enhance remineralization, newer agents such as casein phosphopeptide—amorphous calcium phosphate (CPP-ACP) and nanohydroxyapatite (nHAp) have been introduced, which include additional and synergistic effects of fluoride. In addition, nHAp is the most biocompatible and bioactive material, as it has similarities to apatite crystals of natural enamel in morphology and crystal structure. Nozari et al, Maurizio et al, and Grewal et al evaluated the effect of nHAp on enamel and dentin remineralization and reported that nHAp could reduce the enamel surface roughness.

Several studies have focused on the remineralization of initial enamel lesions. ^, However, only a few in vitro studies ^, ^, have evaluated the effects of hydroxyapatite on the enamel surface. Because of the paucity of data on nHAp in clinical studies and to evaluate the effects of hydroxyapatite in clinical situations, this in vivo study was conceptualized. Therefore, this study aimed to evaluate the effect of nHAp and NaF application on enamel surfaces after IPR in patients undergoing orthodontic treatment and to evaluate changes in surface roughness, composition, and microhardness of treated enamel.

Material and methods

A total of 25 patients receiving routine orthodontic treatment with fixed appliances participated in the study. The sample size was calculated using the data obtained from a pilot study conducted earlier (standard deviation [SD] of 5 and margin of error was set at 2%). Patients were informed about the study design, and informed consent was obtained from all adult patients and parents of those aged <18 years. The study protocol was approved by the Institutional Ethics Committee (MDC_KT_D202029). Patients with a Class I skeletal relationship and Angle’s Class I malocclusion aged 13-20 years were included. These patients had crowding, which was categorized as mild to moderate on the basis of Little’s irregularity index, and required extraction of all 4 first premolars as part of their treatment plan. The selected patients were free of active carious lesions and had no history of previous orthodontic treatment. Care was taken to ensure that the first premolars did not have any cracks, hypoplasia, caries, fillings, or exposure to chemical agents.

All patients included in the study were instructed to brush their teeth with a soft toothbrush for 3 minutes using a toothpaste (Colgate-Palmolive, New York, NY) supplied for daily use throughout the study and not to use any other oral agents, including antimicrobial mouth rinses. The patients were randomly allocated to 5 groups of 5 patients each (20 premolars; 40 surfaces). The groups were as follows:

1.

Group 1 (control): no interproximal stripping was performed, and first premolars were extracted immediately.
2.

Group 2: extraction of premolars performed immediately after IPR using Strauss IPR burs (Strauss Diamond Instruments, Palm Coast, Fla).
3.

Group 3: extraction of premolars performed 3 months after IPR using Strauss IPR burs.
4.

Group 4: extraction of premolars performed 3 months after IPR and weekly application of nHAp serum (Repairing Serum PrevDent nHAp ampoules; PrevDent International BV, Ilpendam, Netherlands).
5.

Group 5: extraction of premolars performed 3 months after IPR and monthly once the application of 5% NaF varnish (Embrace, 5% NaF varnish; Pulpdent, Watertown, Mass).

IPR was performed on the mesial and distal surfaces of all the first premolars, which were to be extracted as part of the treatment plan. To prevent the risk of damage to the adjacent teeth during stripping, elastic separators were placed for 3 days before stripping. The teeth were stripped using the Strauss IPR kit following the manufacturer’s recommendations. Strauss bur J316SF was used initially to break the contact point. Subsequently, Z12S/PF IPR bur was used to reduce the proximal surfaces, and 10 strokes were carried out on each proximal surface using a handpiece that had 40,000 rpm under continuous water lubrication. All IPR procedures were performed by the same operator (S.D.).

Immediately after IPR, nHAp serum application on the reduced enamel surfaces was demonstrated to the patients in group 4 in the first appointment. Ampoules of nHAp serum were given to the patients, and they were instructed to apply the serum after brushing their teeth at home for 3-4 minutes once a week for 3 months. After brushing, the patients were instructed to dry the teeth surfaces and squeeze the ampoule containing the serum onto the sponge, which can then be used to apply the serum directly to the tooth. Patients were asked to allow the serum to work for 3-4 minutes and to rinse the mouth with water after 20 minutes. Every week, patients were reminded regarding the application of nHAp serum through a phone call. NaF varnish was applied on the proximal surfaces of the first premolars for patients in group 5 after IPR. The varnish was reapplied during their routine orthodontic visit using an applicator tip once a month for 3 months. The varnish was then air-dried for 10-20 seconds and protected from salivary contamination for 20 seconds ( Table I ).

Table I

Materials and procedures used in this study

Material	Manufacturer	Composition	Application
Repairing serum ampoule containing nHAp	PrevDent International BV, Ilpendam, Netherlands	nHAp (volume of 1 mL)	Serum applied to the tooth surface and allowed to work for 3-4 min
NaF varnish	Embrace, Pulpdent, Watertown, Mass	5% NaF varnish with xylitol, calcium, and phosphate	Apply a very thin coat of varnish on the desired tooth surface with a brush
IPR burs:J316SF and Z12S/PF	Strauss Diamond Instruments, Palm Coast, Fla		Interproximal stripping diamond bur (0.3 mm) and interproximal stripping diamond bur (0.45 mm)

After extraction, the premolars were stored in deionized water, and the root portion of the tooth was removed. The crown of each tooth was then sectioned in the buccolingual direction using a diamond disc under water lubrication. A total of 200 samples were thus obtained. The samples were then ultrasonically cleaned for 10 minutes in deionized water. The samples were then evaluated for surface roughness using atomic force microscopy (SPM-9600; Shimadzu, Kyoto, Japan), and microhardness was tested using a Vickers hardness tester (M-g5037; Shimadzu). The elemental composition of the tooth surface was evaluated using energy-dispersive x-ray spectroscopy (version 4.3; EDAX Microanalysis system with octane plus silicone drift detector, TEAM Enhanced, Kyoto, Japan).

For the evaluation of surface roughness, a total of 15 samples were selected from each group and tested using atomic force microscopy (SPM-9600) and Scanning Probe Image Processor software (version 3.2.5; Image Metrology, Lyngby, Denmark). The enamel surface of each sample was evaluated in 3 randomly selected areas. Images were then acquired at a scan rate of 1 Hz using the direct mode. All topographic evaluations were done with silicone tips, and the enamel surface roughness was quantified in terms of mean roughness (Ra), root mean square roughness (Rq), mean peak to valley height (Rz), and surface skewness (Rsk). These values were recorded in microns, and the results obtained were tabulated and subjected to statistical evaluation.

For testing the chemical composition, representative samples from each group (n = 10) were subjected to energy-dispersive x-ray spectroscopy (EDAX Microanalysis system with octane plus silicone drift detector). Evaluation of the chemical content of calcium, phosphorus, sodium, and fluorine was performed, and the assessment was performed at 3 different areas to reduce variations in the samples being tested. Microhardness of the samples (n = 15) was tested using Vicker’s microhardness tester (M-g5037). A load of 100 g was applied at 3 different locations, and the values were recorded. All the data obtained were then subjected to statistical analysis.

Statistical analysis

All statistical analyses were performed using the SPSS (version 22.0; IBM, Armonk, NY). Descriptive statistics, including the mean, SD, and minimum and maximum values, were calculated for the 5 study groups. Descriptive tests were performed for scale data, 1-way analysis of variance (ANOVA), and Tukey’s multiple post-hoc for intergroup comparison (normality of the data was tested using the relative value of SD with respect to the mean. Because the SD was less than half the mean, the data were considered to be normally distributed). The level of significance was set at a P value of 0.05.

Results

The elemental composition of enamel in all 5 groups is shown in Figure 1 . The mean calcium-to-phosphorous (Ca-to-P) ratio was highest in the control group (1.90 ± 0.04) and lowest in group 2 (IPR + immediate extraction, 1.11 ± 0.03). Calcium mass percentage was highest in the control group (64.69 ± 1.32) and lowest in group 2 (IPR + immediate extraction, 57.48 ± 1.58). However, the mean phosphorus mass percentage was highest in group 2 (IPR + immediate extraction; 51.91 ± 2.15) and lowest in the control group (34.02 ± 1.00). The mean sodium mass percentage was highest in the control group (2.11 ± 0.47) and lowest in group 2 (IPR + immediate extraction, 0.81 ± 0.37). The mean fluoride mass percentage was highest in the control group (2.84 ± 0.40) and lowest in group 2 (IPR + immediate extraction, 0.78 ± 0.29). Comparison of elemental composition using 1-way ANOVA and Tukey’s multiple posthoc procedures ( Table II ) revealed statistically significant differences between and within the groups ( P = 0.0001).

Table II

Comparison of the groups for EDAX parameters using 1-way ANOVA

Sources of variation	Sum of squares	Df	Mean square	F value	P value
Ca-to-P ratio
Between groups	18.27	4	4.57	6435.6670	0.0001 ^∗
Within groups	0.14	195	0.00
Total	18.41	199
Calcium (mass %)
Between groups	1418.78	4	354.69	234.225	0.0001 ^∗
Within groups	295.30	195	1.51
Total	1714.07	199
Phosphorus (mass %)
Between groups	9874.45	4	2468.61	1633.38	0.0001 ^∗
Within groups	294.71	195	1.51
Total	10169.16	199
Sodium (mass %)
Between groups	51.40	4	12.85	54.2430	0.0001 ^∗
Within groups	46.19	195	0.24
Total	97.59	199
Fluoride (mass %)
Between groups	136.28	4	34.07	170.563	0.0001 ^∗
Within groups	38.95	195	0.20
Total	175.23	199