Study Design
The study was evaluated and approved by the XXX Clinical Studies and Publication Ethics Committee (decision no: XXX) and received support from the XXX University Scientific Research Projects Coordination Unit (Project no: XXX). Nisin was synthetically produced under in-vivo conditions at the XXX University Biochemistry laboratory, and its antimicrobial efficacy was evaluated. The sample size required to assess the effect of Nisin-added adhesive resin on shear bond strength (SBS) and adhesive remnant index (ARI) scores of orthodontic brackets was determined using G*Power software. It was estimated that 72 teeth would provide the trial with a 0.6 effect size and 95% power (control group: 36 teeth, experimental group: 36 teeth). To increase the reliability of the study results, a total of 80 maxillary premolar teeth extracted for orthodontic purposes were gathered as the study material and divided into 40 teeth for each group.
Antimicrobial Peptide Synthesis and Evaluation of Antimicrobial Efficacy
Nisin production
Nisin was produced by Lactobacillus lactis strains obtained from generated culture collections. MRS Broth medium was used to produce nisin from Lactobacillus lactis culture. Once the desired incubation period was completed, the bacterial culture was harvested through centrifugation and filtration to separate the bacterial cells from the culture medium.24-25
Synthesis of AMP-added adhesive resin
The AMP-added adhesive resin was produced by synthesising amino acids that bound to the acid end in an environment in which the amino acid N-terminal was preserved. The minimum inhibitory concentration against S. mutans (ATCC 35688) and Lactobacillus cariogenic microorganism strains was determined to detect the optimal adhesive-antimicrobial peptide concentration. The synthesis followed the Fmoc solid-phase peptide synthesis principle.26
Extraction and purification of nisin
The nisin was extracted from the harvested culture, followed by purification of the compound from other cellular components. Precise extraction and purification methods included solvent extraction, filtration, chromatography, or other specialised methods. The concentration and purity of the extracted nisin were measured using analytical techniques involving enzyme-linked immunosorbent assay (ELISA).
Purification of AMP
The AMP produced from L. lactis (ATCC 7962) (Cas No. 1414-45-5) bacteria was purified to synthesise nisin. Disc diffusion test was performed to determine the effectiveness of the purified antimicrobial peptide against the microorganism. In this test, the zone diameter determines the sensitivity of the microorganism to the antimicrobial agent used. The zone diameter obtained because of an antibacterial test applied to a sample taken from the AMP synthesis environment was 16 mm. After ammonium sulfate precipitation, the zone diameter increased to 20 mm. With Sephadex G75 column loading (gel filtration chromatography), the zone diameter reached 25 mm. Finally, with DEAE Sepharose Sephadex G75 column loading (ion exchange chromatography), the zone diameter increased to 27 mm.27
Optimum conditions for synthesised nisin antimicrobial peptide:
The conditions in which synthesised nisin antimicrobial peptide exhibited optimum efficacy were determined by variable manipulation, and it was found to be at pH 6.7. The antimicrobial efficacy was then tested at different concentrations (1-1/80) using scotch bond. The antimicrobial peptide alone formed a zone with a diameter of 2.7 cm, while the mixture of AMP-bond at 1/40 concentration formed a zone with a diameter of 1.2 cm (Figure 1). The zone diameter values indicated minimal risk of tooth decay development.
Preservation of nisin in different solutions
A pilot study was conducted to determine the solution that exhibited the highest bond strength for nisin preservation. The buffer solution that provided the most suitable and highest SBS for AMP mixed with 1/40 bond and preserved in four different buffer solutions (sodium citrate pH=3, sodium citrate pH=4, sodium phosphate pH=4, and medium sample) was found to be sodium citrate at pH=3.
Antimicrobial effect test
Initially, culture media preparation involved sterilising nutrient agar. Streptococcus mutans (S. mutans) (ATCC 35688) and Lactobacillus strains (cariogenic microorganisms) were inoculated onto the agar plates by streaking a standardised suspension of the microorganisms. The nisin was dissolved to achieve the desired concentration, and discs containing the nisin samples were placed on the surface of the agar plates. After incubation, the agar plates were examined for zones of inhibition, which appeared as clear areas around the nisin discs where the growth of microorganisms was inhibited. The diameters of the zones of inhibition were measured using a calliper.28
Appropriate positive and negative controls were included in the assay. Positive controls consisted of discs impregnated with a known antimicrobial agent, while negative controls were discs or wells containing the solvent or diluent without nisin. The zone sizes of the nisin discs were compared with the positive and negative controls. The average diameter of the zones of inhibition for each concentration of nisin was measured, and the data analysed to determine the antimicrobial activity of nisin against Streptococcus mutans (S. mutans) (ATCC 35688) and Lactobacillus microorganisms.29
Tooth preparation
A total of 140 upper premolar teeth extracted for orthodontic reasons were collected over a period of 4 months and preserved in distilled water containing 0.1% (weight/volume) thymol at room temperature. The inclusion criteria for the study teeth were an absence of any carious lesion, the absence of buccal enamel cracks or fractures, the absence of hypoplastic enamel tissue, the absence of restorations and developmental enamel disorders. Eighty teeth that met the inclusion criteria were accepted as the study material. The tooth surfaces were cleaned with pumice without fluoride (Ortho Teeth Cleaner, Shofu) and a rubber prophylaxis brush (Merssage Cup No.15, Shofu) for 15 seconds, rinsed with air-water spray, and dried before bonding the brackets to the tooth surface. The 80 teeth were randomly assigned to two equal groups (n = 40) identified as:
Group 1 (G1): A Control Group (teeth bonded with standard adhesive resin resin without AMP)
Group 2 (G2): An Experimental Group (teeth bonded with AMP-added adhesive resin)
The following materials were used: 0.022 inch slots mean bracket base area of 9.94 mm2, Roth system, Mini Master premolar metal brackets (Mini Master Series, American Orthodontics, California, USA), Universal bond (3M Espe, Scotchbond Universal Plus Bonding, Germany), 37% orthophosphoric acid (3M™ ESPE™ Scotchbond™ Etchant Gel, 3007), Transbond XT bracket adhesive composite (3M Unitek), and VALO™ Cordless light device. All materials were used according to the manufacturers instructions, and brackets were appropriately bonded to the teeth. The materials and contents used in the study are presented in Table 1.
Table I. Contents, origins and serial numbers of materials used in the study.
Materials
|
Lot number
|
Manufacturer
|
Country
|
Ingredients
|
Acid etch
|
220686
|
BLUE HIGH VISCOSITY
|
USA
|
Contains Phosphoric Acid and Benzalkonium Chloride
|
Adhesive-1
|
10450A
|
3M ESPE-Single Universal Bond
|
Germany
|
Ethyl Alcohol %25- 35, Bisphenol A Diglycidyl Ether Dimethacrylate %10 – 20, Silane Treated Silica (Nanofiller) %10 – 20, 2-Hydroxyethyl Methacrylate %5-15, Glycerol 1,3-Dimethacrylate %5-10, Copolymer Of Acrylıc And Itaconic Acids %5- 10, Diurethane Dimethacrylate %1- 5, Water %<5
|
Adhesive-2
|
-------------
|
3M ESPE-Single Universal Bond added to Nisin AMP (%13)
|
Türkiye
|
Ethyl Alcohol %25- 35, Bisphenol A Diglycidyl Ether Dimethacrylate %10 – 20, Silane Treated Sılıca (Nanofiller) %10 – 20, 2-Hydroxyethyl Methacrylate %5-15, Glycerol 1,3-Dimethacrylate %5-10, Copolymer Of Acrylıc And Itaconic Acids %5- 10, Diurethane Dimethacrylate %1- 5, Water %<5,Nisin AMP %25
|
Composite
|
NE23926
|
Transbond XT,3M Unitek
|
USA
|
—Bis-GMA, bis-FMA, acrylate, monomers, filler
|
Bracket
|
L-000373…
|
American Orthodontics Mini Master Metal Bracket
|
USA
|
Contains Nickel and /or Chromium
|
Curing Light
|
SNO05354
|
VALO ORTHO-CORDLESS
|
USA
|
Utilizable wavelength range:385-515
Demetron L.E.D. Radiometer 1600nW/cm2
|
Shear bond strength (SBS) test
After embedding the tooth samples in acrylic blocks, SBS measurements were performed using an INSTRON universal test machine (Z020; Zwick/Roell, Ulm, Germany). The SBS test was set up such that a 2.5 kg load was applied on the bracket base by a compression shear force in an occlusogingival direction (force applied perpendicular to the bracket base) (Figure 2). The force required to separate the bracket from the tooth surface was recorded in Newtons at a crosshead velocity of 1.0 mm/min, and the SBS value was calculated using the following formula:30 Shear Bond Strength (SBS) Value = Force (N) / Bracket Base Area (mm2)
Adhesive remnant index
After removing the brackets, photos of the bracket bases and buccal enamel tissues were taken using a stereomicroscope with 10x magnification (Stemi 305, ZEISS, Tokyo, Japan). The photos were recorded on a computer and numbered by an assistant who was not involved in the study to avoid analytical bias. The adhesive remnant index (ARI) was determined on the numbered photos by an experienced researcher using the method recommended by Bergland and Artun.31 The ARI index ranged from 0 to 4, using the following interpretations:
- Index 0: No adhesive on the tooth surface,
- Index 1: Less than 50% adhesive on the tooth surface,
- Index 2: More than 50% adhesive on the tooth surface,
- Index 3: All adhesive on the enamel surface with bracket traces,
- Index 4: Fracture on the enamel surface (Figure 3).
Statistical analysis
Statistical analysis was performed using SPSS software (Windows version 20.0 for SPSS; SPSS Inc, Chicago, Illinois). After applying the normal distribution test, non-parametric tests (Mann-Whitney U test) were used for data that did not follow a normal distribution. Fisher's exact test was used for the ARI index. A significance level of P<0.05 was considered statistically significant.