Revolutionary Bioplastic: The Future of Eco-Friendly Dental Care!
Ethics
Our commitment to ethical standards is unwavering. Every procedure in this study was conducted in strict compliance with relevant ethical regulations. The analysis of oral microbial biofilm growth utilizing human saliva was sanctioned by the Institutional Review Board of Yonsei University Dental Hospital in South Korea (Approval No. 2-2019-0049). Furthermore, our in vivo animal experiments received the green light from the Institutional Animal Care and Use Committee at Yonsei Medical Center (Approval No. 2022-0190).
Template-Mediated Entanglement and MSB Preparation
Silk fibroin was meticulously extracted from B. mori silkworm cocoons through a degumming process using 0.02 M sodium carbonate, followed by thorough washing and drying. These cocoons were sourced from a local market. For the template synthesis, we combined 0.425 M hexamethylene diisocyanate isocyanurate trimer and a quaternary ammonium compound in propylene carbonate, heating the mixture to 80 °C. The addition of polyethylene glycol, crucial for inducing entanglement within the MA structure, enhances its antimicrobial properties. This mixture was then cast into a polystyrene mold and baked at 80 °C for a week to create the template. The dissolving medium was prepared with formic acid and calcium chloride, allowing for the silk fibroin to dissolve before being cast into the template. After an entanglement period of three days, the material was treated with an ethanol-deionized water co-solvent, detached, stored in a glycerol solution, and finally dried. The silk fibroin was also subjected to a native renaturation process to prepare the native silk fibroin.
Bio-Based Carbon Content Measurement
The Korea Apparel Testing & Research Institute utilized accelerator mass spectrometry to analyze the 14C radiocarbon content and determine the percent modern carbon in the native silk fibroin and MSB samples, each prepared at a weight of 2.0 g, following ASTM D 6866-22 Method B.
Small-Angle X-Ray Scattering
We conducted X-ray scattering experiments at the 9 A beamline of the Pohang Light Source. The synchrotron X-ray beam was calibrated to a wavelength of 0.626 Å with a typical beam size of 0.8 × 0.8 mm2. Scattered beam intensities were captured using a 2D Mar CCD detector at a sample-to-detector distance of 2.5 m, with exposure times ranging from 10 to 30 seconds.
Differential Scanning Calorimetry
The glass transition temperatures of the MSB and silk fibroin post-renaturation were assessed using differential scanning calorimetry (DSC 4000). The initial sample weight hovered between 1 to 2 mg, with the chamber purged with nitrogen gas. Measured heat flow ranged from 25 to 300 °C, with heating rates of 5, 10, and 20 °C per minute.
Mechanical Testing
The MSB was fabricated into a 4 cm2 beam shape, 400 μm thick, and subjected to tensile experiments using a universal testing machine. A uniaxial tension of 5.0 mm/min was applied until failure. Cyclic bending tests were conducted at 1%, 3%, and 5% strains, recovering after each bend, repeated for 100 cycles at each strain point. Subsequently, twisting tests followed at 10°, 30°, and 60° at a rate of 10° per minute, also repeated for 100 cycles.
Creep-Recovery Experiments
MSB and PETG were shaped into 1.0 cm2 films, 1.0 mm thick, and placed under initial compression until a force of 5.0 N was reached. Creep stress was applied at incremental values, monitored during recovery phases.
Stress Relaxation Tests
Beam-shaped samples of MSB and PETG were prepared and subjected to predisplacement before monitoring their relaxation profiles for 30 minutes.
In Silico Finite-Element Analysis
Utilizing advanced simulations, we modeled the first stage of orthodontic clear aligner treatment to address gaps between teeth, specifically focusing on a 0.2 mm movement of the central incisors. Detailed 3D models were generated using advanced laser scanning technology, ensuring every element was accounted for in the analysis.
In Vivo Rabbit Incisor Orthodontic Treatment
We utilized New Zealand white rabbits for our orthodontic assessments, selecting male specimens for their documented periodontal health. Each rabbit was acclimatized before undergoing a midline diastema model to evaluate iterative orthodontic movements, employing a band-like MSB appliance aligned closely to the rabbits’ incisors. With a control group in place, we monitored tooth movements meticulously every 48 hours.
Direct Contact Test of Cell Viability
A suspension of L-929 cells was cultured under strict conditions, followed by a cytotoxicity assessment using a neutral red staining method. The viability of test samples was compared to controls to determine cytotoxic effects.
Bacterial Proliferation Assay
The antibacterial properties of MSB were evaluated against Streptococcus mutans and Staphylococcus aureus. Bacterial cultures were initiated, and the proliferation was assessed by measuring optical densities and counting colony-forming units after incubation.
Bacterial Attachment Assay
To quantify bacterial attachment, pre-sterilized samples were cultured with both bacterial strains, followed by a thorough washing procedure to remove unattached bacteria. Scanning electron microscopy was employed for detailed surface interaction analysis.
Human Saliva Biofilm Growth
In our exploration of oral health, we evaluated biofilm growth on MSB and PETG surfaces using human saliva samples, following strict ethical protocols. Saliva was pooled from healthy donors, cultivated, and analyzed to assess the microbial composition.
Human Saliva Biofilm Microbiome Analysis
DNA sequencing was performed to unravel the complex composition of the biofilm. Following meticulous protocols, we prepared and sequenced samples, ensuring detailed taxonomic insights into the microbial landscape.
Downstream Analysis of Microbiome Data
Data processing employed advanced tools within RStudio for diversity analyses and statistical comparisons, ensuring a rigorous examination of microbial populations and their variations.
Cell Culture and Extract Preparation
Various cell lines were cultured under optimal conditions, and extracts from MSB were prepared for subsequent viability assessments, ensuring high standards of experimental integrity.
Cell Viability Assay
Cell viability was evaluated through an MTT assay, providing a quantitative measure of the extracts’ effects on cell health and proliferation.
Quantitative Real-Time Polymerase Chain Reaction (qPCR) Assay
The pro-inflammatory responses induced by various materials were assessed using qPCR, yielding insights into cellular behavior under different experimental conditions.
ESF and Template Extraction
Extraction processes for the MSB were carefully conducted to isolate silk fibroin, ensuring purity and integrity for further applications.
Solid-State 13C NMR
High-resolution spectra were obtained using solid-state NMR techniques, providing crucial information about the molecular structure of our samples.
Pyrolysis Gas Chromatography-Mass Spectrometry
We employed advanced pyrolysis gas chromatography-mass spectrometry to analyze the extracted templates, ensuring a detailed understanding of their composition and extraction efficiencies.
Reporting Summary
For further insights into our research design, please refer to the comprehensive Nature Portfolio Reporting Summary.
