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IRCT20181121041713N5 IRCT 2023-10-22 Shiraz University of Medical Sciences Antibacterial effects of elastomeric ligatures coated with bacterial nanocellulose bacterial nanocellulose = BNC Antibacterial effects of elastomeric ligatures coated with Kombucha-synthesized bacterial nanocellulose against streptococcus mutans Randomized Clinical 2023-10-28 anticipated 72 Complete https://irct.ir/trial/73141 interventional Randomization: Randomized, Blinding: Triple blinded, Placebo: Used, Assignment: Parallel, Purpose: Diagnostic, Randomization description: The selected 72 patients will be randomly divided into test and control groups (n=36). Block randomization method will be used with block size of 6, used online software RANDOM.ORG to allocate patients to intervention and control groups with a ۱:۱ ratio. Subsequently, the random sequences to the study groups will be concealed in opaque envelopes and shuffled before the intervention to increase the unpredictability of the random allocation sequence. Each patient will be asked to pick a sealed envelope to assign to either test or control .Allocation concealment will be done to avoid selection bias, Blinding description: triple-blind clinical trial Due to the color change in elastomeric ligatures from gray to gold following BNC coating, the clinician could not be blind to the study groups. However, the patients, the assessor who collect the biofilm samples and the statistician will be blind to the study groups. 3 Antibacterial effects of elastomeric ligatures coated with Kombucha-synthesized bacterial nanocellulose against streptococcus mutans. Intervention 1: Intervention group: A minimum of ۲۰ brackets will be used for patients. fixed pre adjusted edgewise appliances (Mini Master Bracket, ۰.۰۲۲-in MBT prescription; American Orthodontics, USA) with a slot size of ۰.۰۲۲x۰.۰۲۸ inch will be used in all patients. The same type of composite and adhesive will be used for bracket bonding in all patients. After primary leveling and alignment and at placing of steel wire ۱۸, baseline saliva samples will be collected.ntervention group receive coated elastomeric ligatures.Dental plaque sample (T۰) will be obtained from the upper lateral incisor site bilaterally using a sterile periodontal probe. manually delete the supra-gingival plaque , dries and then inter the sterile paper points with tweezers in selected sites. The tip left in situ for about one minute , then removed and inserted into a sterile container for sending in the laboratory (۱۵). The plaque sample will be transferred into a microtube containing ۱ mL saline. After obtaining the plaque samples in T۰, BNC releasing ligatures will be used for patients in the test group while conventional ligatures will be used for control patients. The samples will be placed next to dry ice and will be sent to a microbiology lab in less than ۲ h. Saliva and plaque samples will be collected again at ۷, ۱۴, ۲۱ and ۲۸ days after placement of ligatures (T۱, T۲, T۳, T۴)Preparation of coating solution and Preparation of elastomeric ligatures:The Kombucha SCOBY starter culture was obtained from an Iranian origin and activated every ۱۴ days. The raw cellulose material in the study was Kombucha membranes, an outgrowth of a symbiotic culture of bacteria and yeast fermenting tea broth. The process of making tea infusion involved steeping ۵ grams of green tea (Basilur green tea, Ceylon) in boiling sterile water for ۱۵ min followed by adding ۱۲% high fructose corn syrup as sweetener (Hungrasweet F۵۰, HungranaKft., Ipartelep, Hungary). The sweetened infusion, amounting of two liters, was aseptically poured into a ۳-liter sterile brown glass bottle. Then, ۱۰% of Kombucha tea previously fermented was added as inoculum. The symbiotic culture of bacteria and yeast (SCOBY), originating from a Romanian culture, contained acetobacteria from Komagataeibacter and Gluconobacter genera, as well as yeasts from several genera such as Zygosaccharomyces, Brettanomyces/Dekkera and Pichia, and lactobacteria. A cotton towel was wrapped around the brown glass bottle and secured at the neck of the bottle. The Kombucha culture was allowed to ferment at around ۲۳ °C for ۳۰ days in order to yield Kombucha vinegar. The initial brown Kombucha membranes, referred to as K_۰ sample, weighed ۴۶۱ g. NaOH was tested in ۱ and ۴ M concentrations (۱:۲ solid:liquid ratio) to extract KM from proteins, saccharides, and amino acids, resulting in samples encoded K_۱M and K_۴M. With respect to the thickness of initial membranes, they were washed between ۱۰ to ۳۰ times (۱۰ min each time) by using ۱ M NaOH solutions. The washing process was made more effective by using an Elmasonic P ultrasonic bath (Elma, Singen, Germany). Following the alkaline treatment and ultrasonication process, the purified KMs underwent multiple washes with distilled water until reaching a neutral pH. The beige white membranes (K_۱M), weighing ۳۴۷ g, underwent additional treatment with ۴ M NaOH before being neutralized with distilled water to produce K_۴M. Purified K_۴M never-dried membranes, weighing ۳۳۰ g (۱% w dried cellulose, all concentrations will refer to dry-cellulose content), were ground with a blender (۱۰۰۰ W, ۱۰ series of ۵ min) and diluted with ۵.۵ L water (۵.۷ × ۱۰-۲% w/v) to produce the K_B samples. The K_B samples were subjected to ۳ hours of wet deep grinding by a recirculating colloidal mill (۲۵۰ µm blade space and ۲۵ L/min flow rate), representing about ۸۰۰ passes. Then, the following approach weas experimented:The colloidal mill sample was diluted ۱۰× (۵.۷ × ۱۰-۳% w/v) and then atomized (or spray-dried) by using a Mini Spray dryer B-۲۹۰ (Buchi, Flawil, Switzerland) to attain BNC in dried form. The dried form of nanocellulose is crucial for various bionanocomposites. The optimized operating parameters for nano-atomization were a flow rate of ۴ mL/min for the cellulose suspension, an air flow of ۵۰۰ L/h, with inlet and outlet temperatures of respectively ۱۷۵ °C and ۹۰ °C. Intervention 2: Control group: A minimum of ۲۰ brackets will be used for patients. fixed pre adjusted edgewise appliances (Mini Master Bracket, ۰.۰۲۲-in MBT prescription; American Orthodontics, USA) with a slot size of ۰.۰۲۲x۰.۰۲۸ inch will be used in all patients. The same type of composite and adhesive will be used for bracket bonding in all patients. After primary leveling and alignment and at placing of steel wire ۱۸, baseline saliva samples will be collected from both the control group receive non coated elastomeric ligatures. Yes - There is a plan to make this available What will be shared: General data including age, sex, medical and dental status, along with outcomes and results can be shared. When: Access starts 6 months after results are published To whom: Researchers working in academic and scientific institutions Conditions: Access for researchers and people who intend to continue scientific research on this matter. Where to obtain: Dr.neda babnouri +98 71 3626 3193 babanouri@sums.ac.ir How to obtain: The information will reach them within one month at the latest after the request and review of the request. Comments:
public Dr. Neda Babanouri
Mehr St.,Qasr al-Dasht St. Faculty of Dentistry
Shiraz Iran (Islamic Republic of) 7195615878 +98 71 3626 3193 babanouri@sums.ac.ir Shiraz University of Medical Sciences scientific Dr. Neda Babanouri
Mehr St.,Qasr al-Dasht St. Faculty of Dentistry
Shiraz Iran (Islamic Republic of) 7195615878 +98 71 3626 3193 hajmohammadi1996@yahoo.com Shiraz University of Medical Sciences Iran (Islamic Republic of) Male and female patients Age 15 to 30 years non pregnant No significant medical history or drug use No anti-inflammatory or antibiotic medications taken within ۳ months prior to the study No chewing gum or mouthwash use in the week before and during the study habit of brushing twice daily with fluoride toothpaste No history of periodontal therapy or current periodontal disease Probing depth less than ۴ mm across the entire dentition No gingivitis or active carious lesion Gingival index and plaque index (Silness-Löe) value <1 15 years 30 years Both Diseases and medication use likely to affect dental biofilm Pregnancy Poor oral hygiene Active periodontal disease Severe crowding of anterior teeth or malposition of the lateral incisor likely to interfere with the cleaning of the tooth surface Smoking History of mouth breathing Prevention Placebo Intervention group: A minimum of ۲۰ brackets will be used for patients. fixed pre adjusted edgewise appliances (Mini Master Bracket, ۰.۰۲۲-in MBT prescription; American Orthodontics, USA) with a slot size of ۰.۰۲۲x۰.۰۲۸ inch will be used in all patients. The same type of composite and adhesive will be used for bracket bonding in all patients. After primary leveling and alignment and at placing of steel wire ۱۸, baseline saliva samples will be collected.ntervention group receive coated elastomeric ligatures.Dental plaque sample (T۰) will be obtained from the upper lateral incisor site bilaterally using a sterile periodontal probe. manually delete the supra-gingival plaque , dries and then inter the sterile paper points with tweezers in selected sites. The tip left in situ for about one minute , then removed and inserted into a sterile container for sending in the laboratory (۱۵). The plaque sample will be transferred into a microtube containing ۱ mL saline. After obtaining the plaque samples in T۰, BNC releasing ligatures will be used for patients in the test group while conventional ligatures will be used for control patients. The samples will be placed next to dry ice and will be sent to a microbiology lab in less than ۲ h. Saliva and plaque samples will be collected again at ۷, ۱۴, ۲۱ and ۲۸ days after placement of ligatures (T۱, T۲, T۳, T۴)Preparation of coating solution and Preparation of elastomeric ligatures:The Kombucha SCOBY starter culture was obtained from an Iranian origin and activated every ۱۴ days. The raw cellulose material in the study was Kombucha membranes, an outgrowth of a symbiotic culture of bacteria and yeast fermenting tea broth. The process of making tea infusion involved steeping ۵ grams of green tea (Basilur green tea, Ceylon) in boiling sterile water for ۱۵ min followed by adding ۱۲% high fructose corn syrup as sweetener (Hungrasweet F۵۰, HungranaKft., Ipartelep, Hungary). The sweetened infusion, amounting of two liters, was aseptically poured into a ۳-liter sterile brown glass bottle. Then, ۱۰% of Kombucha tea previously fermented was added as inoculum. The symbiotic culture of bacteria and yeast (SCOBY), originating from a Romanian culture, contained acetobacteria from Komagataeibacter and Gluconobacter genera, as well as yeasts from several genera such as Zygosaccharomyces, Brettanomyces/Dekkera and Pichia, and lactobacteria. A cotton towel was wrapped around the brown glass bottle and secured at the neck of the bottle. The Kombucha culture was allowed to ferment at around ۲۳ °C for ۳۰ days in order to yield Kombucha vinegar. The initial brown Kombucha membranes, referred to as K_۰ sample, weighed ۴۶۱ g. NaOH was tested in ۱ and ۴ M concentrations (۱:۲ solid:liquid ratio) to extract KM from proteins, saccharides, and amino acids, resulting in samples encoded K_۱M and K_۴M. With respect to the thickness of initial membranes, they were washed between ۱۰ to ۳۰ times (۱۰ min each time) by using ۱ M NaOH solutions. The washing process was made more effective by using an Elmasonic P ultrasonic bath (Elma, Singen, Germany). Following the alkaline treatment and ultrasonication process, the purified KMs underwent multiple washes with distilled water until reaching a neutral pH. The beige white membranes (K_۱M), weighing ۳۴۷ g, underwent additional treatment with ۴ M NaOH before being neutralized with distilled water to produce K_۴M. Purified K_۴M never-dried membranes, weighing ۳۳۰ g (۱% w dried cellulose, all concentrations will refer to dry-cellulose content), were ground with a blender (۱۰۰۰ W, ۱۰ series of ۵ min) and diluted with ۵.۵ L water (۵.۷ × ۱۰-۲% w/v) to produce the K_B samples. The K_B samples were subjected to ۳ hours of wet deep grinding by a recirculating colloidal mill (۲۵۰ µm blade space and ۲۵ L/min flow rate), representing about ۸۰۰ passes. Then, the following approach weas experimented:The colloidal mill sample was diluted ۱۰× (۵.۷ × ۱۰-۳% w/v) and then atomized (or spray-dried) by using a Mini Spray dryer B-۲۹۰ (Buchi, Flawil, Switzerland) to attain BNC in dried form. The dried form of nanocellulose is crucial for various bionanocomposites. The optimized operating parameters for nano-atomization were a flow rate of ۴ mL/min for the cellulose suspension, an air flow of ۵۰۰ L/h, with inlet and outlet temperatures of respectively ۱۷۵ °C and ۹۰ °C Control group: A minimum of ۲۰ brackets will be used for patients. fixed pre adjusted edgewise appliances (Mini Master Bracket, ۰.۰۲۲-in MBT prescription; American Orthodontics, USA) with a slot size of ۰.۰۲۲x۰.۰۲۸ inch will be used in all patients. The same type of composite and adhesive will be used for bracket bonding in all patients. After primary leveling and alignment and at placing of steel wire ۱۸, baseline saliva samples will be collected from both the control group receive non coated elastomeric ligatures Bacterial colony counting. Timepoint: The beginning of the study and days 7, 14, 21 and 28. Method of measurement: Counting the number of bacterial colonies after sampling. Shiraz University of Medical Sciences Approved 2023-08-13 Ethics committee of Shiraz University of Dentistry Mehr St.- Qasr Al-Dasht St.- Faculty of Dentistry Shiraz Fars Iran (Islamic Republic of)