Utveckling av Amelogenin-chitosan Hydrogel för<em> In Vitro</em> Emalj Återväxt med en Tät gränssnitt
Summary
I den här artikeln beskriver vi ett protokoll för att tillverka en amelogenin-chitosan hydrogel för ytlig emalj rekonstruktion. Anordnades in situ-tillväxten av apatitkristaller i hydrogelen bildas en tät emalj-restaurering gränssnitt, som kommer att förbättra effektiviteten och varaktigheten för restaurationer.
Abstract
Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.
Introduction
Dental enamel is the hard mineralized surface of human teeth. It is composed of numerous needle-like apatite crystals, which are bundled in organized, parallel prisms to ensure the unique mechanical strength and biological protection that enamel provides1-2. Unlike other mineralized tissues, such as bone and dentin, mature enamel is acellular and cannot regenerate itself after substantial mineral loss1-2, which often occurs as dental caries or erosion. Commercially available products such as fluoride containing varnishes, tooth pastes and mouthwashes are effective in re-mineralizing enamel but none of them have the potential to promote the formation of organized apatite crystals. Clinically, the conventional treatment for enamel repair involves a filling procedure with artificial materials such as amalgam, ceramics, or composite resin 3. However, these materials usually do not interface well with the natural tissue surrounding the lesion, because their structures, components and properties are different from the natural enamel. As a result, secondary caries frequently develops overtime at the interface between the tooth and foreign materials. Therefore, in situ regrowth of enamel with a dense interface is an attractive target for the fields of materials science and stomatology. One particularly promising way to achieve this purpose is biomimetic synthesis of enamel-like material on the enamel surface. Recently, numerous in vitro attempts have been made to prepare enamel-like materials using biomimetic systems that contain nano-apatites or different organic additives 4-12. However, developing the optimal biomimetic strategy to promote remineralization crystals to achieve a perfect dense interface is still a challenge.
During enamel mineralization, the oriented growth and elongation of apatite crystals is regulated by an amelogenin-rich matrix 1,13-14. To mimic the organic matrix in developing enamel, herein we describe a detailed protocol for fabricating an amelogenin-chitosan (CS-AMEL) hydrogel for in situ enamel regrowth on an acid-etched enamel surface used as a model for erosive lesions. As “the most versatile growth media” for crystals 15, hydrogel matrices have an advantage over a solution system since clinically they are easier to handle. Moreover, CS-AMEL is biocompatible, biodegradable, and has unique antimicrobial and adhesion properties that compare favorably with other biomimetic systems for dental applications 16. Importantly, the in situ mineralization of apatite crystals on the enamel surface provides a dense interface between the repaired layer and the natural enamel, which can potentially improve the durability of restorations and prevent the formation of new caries at the margin of the restoration.
Protocol
Representative Results
Discussion
Medan innehållet i emalj mineral hög vilket gör det hårdast mineraliserad vävnad i den mänskliga kroppen, är detta biokeramiskt mottagliga för demineralisering processer, som ofta förekommer som karies eller erosion. Genmutationer kan också orsaka tunna eller mjuka emalj leder till en serie av ärftliga sjukdomar av emalj missbildning kallad Amelogenesis Imperfecta 20. Oral hälso-och sjukvårdsprodukter som innehåller fluor eller CPP-ACP har funnits på marknaden i flera år (dvs,</em…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank Prof. Steven Nutt and Mr. Yuzheng Zhang for assistant with the Focus Ion Beam, and the Center for Electron Microscopy and Microanalysis (CEMMA) at USC for electron microscopy. Research was supported by NIH-NIDCR grants; DE-13414 and DE-020099 to J.M.O.
Materials
| Name | Company | Catalog Number | Comments/Description |
| Material/ Reagent | |||
| Human Third Molar | Ostrow School of Dentistry of the University of Southern California | N/A | The human molars were extracted following the standard procedures for extraction at the Ostrow School of Dentistry of the University of Southern California and handled with the approval of the Institutional Review Board. |
| Recombinant Pocine Amelogenin | Expression and purification in lab | N/A | rP172, full-length |
| Chitosan | Sigma-Aldrich | 448877 | medium molecular weight, 75-85% deacetylated |
| Phosphoric Acid | VWR | AA033266 | |
| Acetic Acid Glacial | VWR | A036289 | |
| Sodium Hydroxide | VWR | BDH9292 | |
| Calcium Chloride | Sigma-Aldrich | 223506 | |
| Dibasic Sodium Phosphate Anhydrous | VWR | BDH0316 | |
| BL21-CodonPlus (DE3)-RP | Agilent Technologies Inc. | 230255 | |
| Ammonium Sulfate | VWR | BDH8001 | |
| Trifluoroacetic Acid | VWR | AAAL06374 | |
| Acetonitrile | VWR | BDH1103 | |
| Magnesium Chloride | VWR | BDH0244 | |
| Potassium Dihydrogen Phosphate | VWR | BDH9268 | |
| Potassium Chloride | VWR | BDH0258 | |
| Ammonium Chloride | VWR | AAAA15000 | |
| HEPES (4-(2-Hydroxyethyl)piperazine-1-ethane-sulfonic acid) | VWR | AAA14777 | |
| Sodium Fluoride | VWR | AA11561 | |
| Tris-Buffered Saline | Bio-Rad | 170-6435 | 10× TBS |
| Bovine Serum Albumin | EMD Millipore | 12659 | CalBioChem, Albumin, Bovine Serum, Fraction V, Low Heavy Metals |
| Triton X-100 | EMD Millipore | TX1568-1 | |
| Chicken Anti-Amelogenin | N/A | N/A | A gift from Prof. Malcolm Snead, University of Southern California |
| Bovine Anti-Chicken IgY-FITC | Santa Cruz Biotechnology | Sc-2700 | |
| Equipments | |||
| High Performance Liquid Chromatography System | Agilent Technologies Inc. | Varian Prostar 210 | |
| C4 column | Phenomenx | Jupiter 5μ 300A | |
| Scanning Electron Microscopy | JEOL | JSM-7001 | |
| FIB-SEM | JEOL | JIB-4500 | |
| Transmission Electron Microscopy | JEOL | JEM-2100F | |
| Digital Low Speed Diamond Saw | MTI Corporation | SYJ-150 | |
| Fluorescence Microscopy | Leica | DMI3000 B | |
| Ultrasonic Cleaner | Branson | 2510 | 42 kHz, 100 W |
| Nano-indenter | Agilent Technologies Inc. | MTS XP |
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