Name: development of amelogenin-chitosan hydrogel for in vitro enamel regrowth with a dense interface
Uploaded: 2014-07-10T15:00:00
Description: Watch this Scientific Journal Video about Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface at JoVE.com

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.

Os molares humanos foram extraídos seguindo os procedimentos padrão para a extração da Escola Ostrow de Odontologia da University of Southern California e manipulados com a aprovação do Conselho de Revisão Institucional. 1. Preparação de dente ácido-gravadas Slice <ol><li> Selecione um terceiro molar humano sem cárie restauradas. </li><li> Retirar a porção de raiz do molar, e cortar a coroa do molar longitudinalmente em fatias de 2 mm de espessura, utilizando uma baixa velocid...

<ol>
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Embora o conteúdo mineral do esmalte é alta tornando-o mais difícil de tecido mineralizado no corpo humano, esta biocerâmica é suscetível a processos de desmineralização, que muitas vezes ocorrem como cárie dentária ou erosão. Mutações no gene, também podem causar esmalte finas ou macias que conduzem a uma série de doenças hereditárias de malformação esmalte chamado Amelogenesis imperfeita 20. Produtos de cuidados de saúde oral que contenham flúor ou CPP-ACP ter sido no mercado h...

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 &#8220;the most versatile growth media&#8221; 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.

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			<td height="21" style="height:21px;width:331px;">Name</td>
			<td style="width:209px;">Company</td>
			<td style="width:164px;">Catalog Number</td>
			<td style="width:516px;">Comments/Description</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Material/ Reagent</td>
			<td style="width:209px;"></td>
			<td style="width:164px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:331px;">Human Third Molar&#160;</td>
			<td style="width:209px;">Ostrow School of Dentistry of the University of Southern California&#160;</td>
			<td style="width:164px;">N/A</td>
			<td style="width:516px;">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.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:331px;">Recombinant Pocine Amelogenin</td>
			<td style="width:209px;">Expression and purification&#160; in lab</td>
			<td style="width:164px;">N/A</td>
			<td style="width:516px;">rP172, full-length&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Chitosan&#160;</td>
			<td style="width:209px;">Sigma-Aldrich</td>
			<td style="width:164px;">448877</td>
			<td style="width:516px;">medium molecular weight, 75-85% deacetylated</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Phosphoric Acid&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AA033266</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Acetic Acid Glacial</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">A036289</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Sodium Hydroxide</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH9292</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Calcium Chloride&#160;</td>
			<td style="width:209px;">Sigma-Aldrich</td>
			<td style="width:164px;">223506</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Dibasic Sodium Phosphate Anhydrous</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH0316</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">BL21-CodonPlus (DE3)-RP&#160;</td>
			<td style="width:209px;">Agilent Technologies Inc.</td>
			<td style="width:164px;">230255</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Ammonium Sulfate</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH8001</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Trifluoroacetic Acid</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AAAL06374</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Acetonitrile</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH1103</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Magnesium Chloride&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH0244</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Potassium Dihydrogen Phosphate&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH9268</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Potassium Chloride&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH0258</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Ammonium Chloride&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AAAA15000</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:331px;">HEPES (4-(2-Hydroxyethyl)piperazine-1-ethane-sulfonic acid)</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AAA14777</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Sodium Fluoride&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AA11561</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Tris-Buffered Saline</td>
			<td style="width:209px;">Bio-Rad</td>
			<td style="width:164px;">170-6435</td>
			<td style="width:516px;">10&#215; TBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Bovine Serum Albumin</td>
			<td style="width:209px;">EMD Millipore&#160;</td>
			<td style="width:164px;">12659</td>
			<td style="width:516px;">CalBioChem, Albumin, Bovine Serum, Fraction V, Low Heavy Metals&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Triton X-100</td>
			<td style="width:209px;">EMD Millipore&#160;</td>
			<td style="width:164px;">TX1568-1</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Chicken Anti-Amelogenin</td>
			<td style="width:209px;">N/A</td>
			<td style="width:164px;">N/A</td>
			<td style="width:516px;">A gift from Prof. Malcolm Snead, University of Southern California</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Bovine Anti-Chicken IgY-FITC</td>
			<td style="width:209px;">Santa Cruz Biotechnology</td>
			<td style="width:164px;">Sc-2700</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:331px;">Equipments</td>
			<td style="width:209px;"></td>
			<td style="width:164px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:331px;">High Performance Liquid Chromatography System</td>
			<td style="width:209px;">Agilent Technologies Inc.</td>
			<td style="width:164px;">Varian Prostar 210</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">C4 column</td>
			<td style="width:209px;">Phenomenx&#160;</td>
			<td style="width:164px;">Jupiter 5&#956; 300A</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Scanning Electron Microscopy&#160;</td>
			<td style="width:209px;">JEOL&#160;</td>
			<td style="width:164px;">JSM-7001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">FIB-SEM&#160;</td>
			<td style="width:209px;">JEOL&#160;</td>
			<td style="width:164px;">JIB-4500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Transmission Electron Microscopy&#160;</td>
			<td style="width:209px;">JEOL</td>
			<td style="width:164px;">JEM-2100F</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Digital Low Speed Diamond Saw</td>
			<td style="width:209px;">MTI Corporation</td>
			<td style="width:164px;">SYJ-150</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Fluorescence Microscopy</td>
			<td style="width:209px;">Leica</td>
			<td style="width:164px;">DMI3000 B</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Ultrasonic Cleaner&#160;</td>
			<td style="width:209px;">Branson&#160;</td>
			<td style="width:164px;">2510</td>
			<td style="width:516px;">42 kHz, 100 W</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Nano-indenter&#160;</td>
			<td style="width:209px;">Agilent Technologies Inc.</td>
			<td style="width:164px;">MTS XP</td>
			<td style="width:516px;"></td>
		</tr>
	</tbody>
</table>

development of amelogenin-chitosan hydrogel for in vitro enamel regrowth with a dense interface

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.

A eficácia do protocolo aqui descrito é demonstrado por microscopia eletrônica de varredura (MEV), difração de elétrons selecionado área (SAED) e difração de raios-X (XRD) analisa. Após o reparo por amelogenina-quitosana (CS-AMEL) hidrogel por 7 dias, uma camada de esmalte com uma espessura de 15 mm foi formada na superfície esmalte condicionado. A camada recentemente cultivadas foi efectuada de matrizes altamente ordenada de cristais com um diâmetro de cerca de 50 nm, de preferência crescente ao longo do e...

Watch this Scientific Journal Video about Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface at JoVE.com

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Desenvolvimento de Amelogenina-quitosana Hidrogel para<em&gt; In Vitro</em&gt; Esmalte Regrowth Com uma interface Densa

Neste artigo, nós descrevemos um protocolo para a fabricação de um hidrogel amelogenina-quitosana para reconstrução de esmalte superficial. Organizado em crescimento situ de cristais de apatita no hidrogel formado uma interface esmalte-restauração densa, o que irá melhorar a eficácia e durabilidade das restaurações.

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Research

JoVE Journal

Bioengineering

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Encapsulamento de Cardiomyocytes em um hidrogel de fibrina para engenharia de tecidos cardíacos

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Bioengenharia

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

Cultivo de células progenitoras neurais em um hidrogel Peptide 3-dimensional de auto-montagem

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Bridging the Bio-Electronic Interface with Biofabrication

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Synthesis of an Intein-mediated Artificial Protein Hydrogel

Síntese de uma proteína artificial intein mediada Hidrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

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Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

Irritação dos olhos Test (EIT) para identificação de perigos de olho irritantes químicos usando reconstruída Humano Córnea-like epitelial (RHCE) Modelo Tissue

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

In Vitro Modelo de fisiológicos e patológicos fluxo de sangue com aplicativos para Investigações de Reformas celular Vascular

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

Development of an In Vitro Ocular Platform to Test Contact Lenses

Development of an In Vitro Ocular Platform to Test Contact Lenses

Desenvolvimento de um<em&gt; In Vitro</em&gt; Ocular Platform para testar as lentes de contato

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Melhorado o hidrogel 3D culturas de células gliais primárias para In Vitro modelagem de Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

Hidrogel Bioprintable alginato/gelatina 3D em Vitro sistemas modelo induzem a formação de esferoide de célula

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...