I-Materials:
Table 1
Brand names, composition, and manufacturers of materials used in the study.
Brand name | composition | manufactures |
Charisma composite | 61% filler by volume, with a particle size of 0.005–10 µm. Barium, Aluminium Fluoride glass, and Pre-polymerized fillers. BIS-GMA matrix. | Kulzer, Germany. |
HV Etch | 37% phosphoric acid gel | Bisco, USA. |
Gluma Bond Universal | Acetone/water-based solution of light-activated methacrylate monomers and Silane. | Heraeus Kulzer, Germany |
II- Methods:
Ethical approval
This in vitro study was approved by the Institutional Review Board at Misr University for Science and Technology with approval number: 2022/0066.
Sample size calculation
Based on Bin‑Shuwaish et al., 2021 21 using the G power statistical power. Analysis program (version 3.1.9.4) for sample size determination 22, the total sample size was 40 (8 in each group) which was sufficient to detect a large effect size (f) = 0.61, with an actual power (1-β error) of 0.8 (80%) and a significance level (α error) 0.05 (5%).
Grouping of samples
Group 1: No disinfection was applied (negative control).6
Group 2: 2% Chlorhexidine nanogel (positive control): The cavity was dried with an air syringe and 1 ml of the prepared gel was applied for 60 seconds then it was washed with saline for 30 seconds.2
Group 3: Propolis nanogel 10% was applied for 120 seconds then it was washed with saline for 30 seconds 2,11
Group 4: Liquorice nanogel 10% was applied for 120 seconds then it gel was washed with saline for 30 seconds.11
Group 5: Diode laser 980 nm (Doctor Smile, Wiser, LAMBDA SpA, Italy) with power 1 W was applied for 60 seconds divided into 2 cycles, 30 seconds each.2
Preparation of samples
A total of 40 intact human premolar teeth recently extracted for orthodontic reasons were collected. All teeth were cleaned and examined under a stereomicroscope to exclude fractured tooth then they were stored until the beginning of the experiment. Standardized class V cavities (3 mm mesiodistally ×2 mm occlusogingivally ×1.5 mm buccolingually) were prepared on the facial surface of each tooth with a diamond bur (HoricoDiament, Germany), 1mm occlusal to the cementoenamel junction to assure that cavosurface line angles were prepared within enamel.6
All teeth were randomly divided into five experimental groups (n=8) according to the cavity disinfection protocol.
Herbal extract preparation:
-Propolis
Propolis extract was purchased from www.thehealthshop.com (Brazilian green bee Propolis, Uniflora, FDA Reg.#10760930494, Brazil).
-Liquorice
Liquorice collection:
Liquorice was purchased from a commercial herbal company (Al Fahd Apothecary, Egypt) and identified at the Pharmacognosy Department, Misr University for Science and Technology after permission number PG7. A voucher specimen of Liquorice under number 16.9.2020 was deposited in Pharmacognosy Laboratory, Faculty of Pharmacy, Cairo.
Liquorice extract preparation:
200 gm of Liquorice leaves were chopped into pieces and inserted into an electric oven at 45±5°C for 24 hours until complete dryness. Then it was ground using an electric miller to produce a fine powder. Each 150 gm powder was immersed in a flask containing 96% ethanol (300 mL) for 72 hours at room temperature. The solution was filtered and evaporated using a rotary flask evaporator to obtain the well-concentrated extract. Finally, the extract was stored in the refrigerator at 4°C until needed.
Nanogel preparation
Preparation of CHX, Propolis, and Liquorice nanocrystals(NCs):
Both CHX (C22H30CL2N10, Nerol, Egypt) and Propolis nanocrystals NCs were separately prepared through antisolvent precipitation method followed by high-shear homogenization to obtain NCs of the required size.
To prepare the solvent phase, 2% CHX, and 10% of each of the Propolis and Liquorice extracts were first dissolved in a suitable quantity of methanol separately. The prepared solvent phase was added dropwise into the antisolvent phase with a ratio of 1:10 solvent to antisolvent containing pre-screened surfactant which is the lipid phase containing Compritol 888 ATO, Span60 solid lipid nanoparticles (4% of total concentration) with a stirring speed of 1200 rpm at room temperature.
Each of the prepared formulas was subjected to rotary evaporation for 20 min at 100 rpm and 70 ◦C to evaporate the organic phase. Then they were subjected to a homogenizer at a speed of 7000 rpm for 20 min. The developed formulations were further freeze-dried to obtain CHX, Propolis, and Liquorice Nanocrystals dried powder.23
Each formula was assessed for entrapment efficiency percentage (EE %), Particle size (PS), Zeta potential (ZP), and Polydispersity index (PDI) (Malvern Instruments, Ltd., UK). The prepared solid lipid nanoparticles (SLNs) were incorporated into the gel using 1% noveon. The prepared nanoparticles showed particle sizes ranging from 80 to 100 nm, all with a negative charge and PDI below 0.5. The EE% ranged from 85-90% using Cooling Centrifuge (Sigma 3 K 30, Germany)
Restoration
After cavity disinfection protocols, all cavities were etched using HV Etch (Bisco, USA) followed by bond application; Gluma Bond Universal (Heraeus Kulzer, Germany) according to the manufacturer’s instructions [24]. Then all cavities were restored with Charisma composite (Kulzer, Germany) and light cured according to the manufacturer’s instructions.
Thermocycling process
All teeth were immersed in distilled water at 37 °C for 24 hours. Then they were incubated for 78 s (30 s at 55 °C, 10 s stop, 30 s at 5 °C, and 8 s to return the cycle to the starting point). A total of 500 thermal cycles were conducted (TC-300 Vafai Factory Thermocycler).25
Microleakage test
-Samples preparation for dye Penetration:
After thermocycling, all teeth were dried and their apices were blocked with sticky wax to prevent dye penetration. Afterward, they were coated with two layers of nail varnish within approximately 2mm of the tooth-restoration interface except for 1mm gingivally in order to prevent dye penetration except at tooth-restoration interface.25 Then all teeth were immersed in 2% methylene blue dye (Sparks, USA) for 24 hours at room temperature. After dye penetration, teeth were rinsed and dried then sectioned longitudinally in buccolingual direction using a microtome (MTI Corporation, Richmond CA).26
-Microleakage assessment:
Assessment of linear dye penetration at tooth- restoration interface both occlusal and gingival was done using the stereomicroscope at 100X magnification (MA 100 Nikon stereomicroscope Japan with Omnimet image analysis software) to study the degree of microleakage in millimeters. The depth of dye penetration was measured by Omnimet image analysis software.27,28
Statistical analysis
Data management and statistical analysis were performed using the Statistical Package for Social Sciences (SPSS) version 18. Data were explored for normality by checking the data distribution and using Kolmogorov-Smirnov and Shapiro-Wilk tests which revealed that most data were normally distributed (Table 2). Numerical data were summarized using mean, standard deviation, and confidence intervals. Comparisons between groups with respect to normally distributed numeric variables were compared by one-way analysis of variance (ANOVA) test, followed by Bonferroni’s and Tukey’s post hoc test. All p-values are two-sided. P-values ≤0.05 were considered significant.