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

Summary

In this article, we describe a protocol for fabricating an amelogenin-chitosan hydrogel for superficial enamel reconstruction. Organized in situ growth of apatite crystals in the hydrogel formed a dense enamel-restoration interface, which will improve the effectiveness and durability of restorations.

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 provides^1-2. Unlike other mineralized tissues, such as bone and dentin, mature enamel is acellular and cannot regenerate itself after substantial mineral loss^1-2, 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 format....

Protocol

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.

1. Preparation of Acid-etched Tooth Slice

1. Select a human third molar without any restored caries.
2. Remove the root portion of the molar, and cut the crown of the molar longitudinally into slices 2 mm thick using a water-cooled low-speed diamond saw. Ultrasonically clean these slices for 2 min, and then rinse them with deionized water 3 times.
3. To simulate erosive lesions, etch the tooth slices ....

Results

The effectiveness of the protocol described here is demonstrated by scanning electron microscopy (SEM), selected area electron diffraction (SAED) and X-ray diffraction (XRD) analyses. After repair by amelogenin-chitosan (CS-AMEL) hydrogel for 7 days, an enamel-like layer with a thickness of 15 μm was formed on the etched enamel surface. The newly-grown layer was made of highly ordered arrays of crystals with a diameter of ~50 nm, growing preferentially along the c-axis, perpendicular to the surface (arrows in Fig.......

Discussion

While the mineral content of enamel is high making it the hardest mineralized tissue in the human body, this bioceramic is susceptible to demineralization processes, which often occur as dental caries or erosion. Gene mutations can also cause thin or soft enamel leading to a series of inherited diseases of enamel malformation called Amelogenesis Imperfecta²⁰. Oral health-care products containing fluoride or CPP-ACP have been on the market for several years (i.e., varnishes, tooth pas.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
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