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 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

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 with 30% phosphoric acid for 30 sec then immediately rinse them with deionized water.
4. Ultrasonically clean the etched slices for 2 min, and then rinse them with deionized water 3 times.

2. Preparation of Amelogenin-chitosan Hydrogel

1. Preparation of chitosan stock solution
   1. Dissolve 1% (w/v) chitosan (medium molecular weight, 75-85% deacetylated) in a 1% (v/v) acetic acid solution followed by stirring at 80 °C O/N.
   2. After cooling the solution to RT, filter the chitosan solution using a 0.45 μm filter. Adjust the pH value to 5.0 by adding 1 M NaOH solution.
2. Preparation of calcium and phosphate stock solution (0.1 M)
   1. Weigh out calcium chloride (CaCl₂) and prepare a 0.1 M solution, then mix using a vortex until the solution is clear. Adjust the pH value to 11 by adding 1 M NaOH solution.
   2. Weigh out dibasic sodium phosphate (Na₂HPO₄) and prepare a 0.1 M solution, then mix using a vortex until the solution is clear.
3. Expression and purification of recombinant amelogenin^17-18
   The recombinant full-length porcine amelogenin (rP172) used here has 172 amino acids and is an analog of full-length native porcine P173, but lacking the N-terminal methionine as well as a phosphate group on Ser16 ¹⁸.
   1. Add 20 g of Lysogeny broth (LB) Agar powder into 500 ml deionized water, autoclave the solution at 121 °C (liquid setting) for 20 min. Let agar cool to ~ 55 °C, add ampicillin (100 μg/ml) into agar solution.
   2. Transfer the agar solution to Petri dishes, let each plate cool until it is solid, then flip to avoid condensation on the agar. Store plates in plastic bags at 4 °C.
   3. Streak the LB agar plate with recombinant cells (E.coli–BC21) from the glycerol stock (-80 °C). Incubate the plates O/N at 37 °C.
   4. Prepare 50 ml of NZCYM media and autoclave the media at 121 °C for 30 min. Allow the media to cool down, add 50 µl of 100 µg/ml ampicillin to the 50 ml media.
   5. Inoculate a single colony from the LB agar plate into the 50ml NZCYM media supplemented with ampicillin. Incubate the cells O/N in a shaking incubator at 37 °C.
   6. Prepare 1 L of NZCYM media and autoclave the media at 121 °C for 30 min. Add 1 ml of 100 mg/ml ampicillin to 1,000 ml media. Take an optical density (OD) reading as a blank using a UV-Vis spectrophotometer. NOTE: Do not discard this after first reading.
   7. Inoculate 10 ml of the cell culture from step 2.3.5 into 1,000 ml media from step 2.3.6. Read the optical density at 595 nm immediately after inoculation. Take the optical density reading periodically to keep track of the growth. NOTE: Read the blank each time the OD is taken.
   8. Induce with 700 µl of Isopropyl β-D-1-thiogalactopyranoside (IPTG, 1 M) when the bacterial growth reaches to about 0.75~0.8 OD at 595 nm.
   9. Harvest the cells when the OD reaches 1.1. Pour the contents of the flask into 6 plastic bottles. Balance and weigh the bottles before using the centrifuge. Centrifuge for 20 min at 8,554 x g at 4 °C. Keep the cell pellet in the -20 °C O/N.
   10. Take out the cell pellets and combine them in a 50 ml tube. Add 3.5 ml of 4M Guanidine HCl per L of fermentation. Sonicate 2-3 times on ice, 30 sec each time, with 1 min intervals.
   11. Make 6x dilutions of cell volume with 0.5% formic acid and let it stir in the cold room for 2 hr. Centrifuge for 30 min, 8,554 x g at 4 °C. Keep the supernatant. Add 20% Saturated ammonium sulfate using the saturated stock solution and stir in the cold room, O/N.
   12. Centrifuge for 30 min, 8,554 x g at 4 °C and keep the pellet. Reconstitute the pellets in 0.1% trifluoroacetic acid (TFA), load onto a C4 column (10 x 250 mm, 5 µm), and fractionate using a linear gradient of A=0.1% TFA and B=60% acetonitrile in TFA, at a flow rate of 1.5 ml·min^-1.
   13. Freeze the protein solution on dry ice and lyophilize the frozen sample O/N.
4. Preparation of amelogenin-chitosan (CS-AMEL) hydrogel (Figure 1A)
   1. Add 200 µg of rp172 into a tube containing 960 µl of 1% chitosan solution, then mix using a vortex until the solution is clear.
   2. Add 25 µl of 0.1 M CaCl₂ solution followed by 15 µl of 0.1 M Na₂HPO₄ solution to the amelogenin-containing chitosan solution, then mix using a vortex for 5 min.
   3. Adjust the pH of the CS-AMEL solution to 6.5 by carefully adding 1 M NaOH solution. CS-AMEL hydrogel will form when the pH value reaches 6.5.

3. Enamel Regrowth in Amelogenin-chitosan Hydrogel

1. Preparation of artificial saliva solution
   1. Dissolve a certain amount of magnesium chloride (MgCl₂, 0.2 mM), calcium chloride (CaCl₂, 1 mM), potassium dihydrogen phosphate (KH₂PO₄, 4 mM), potassium chloride (KCl, 16 mM) and ammonium chloride (NH₄Cl, 4.5 mM) in 20 mM HEPES (4-(2-Hydroxyethyl)piperazine-1-ethane-sulfonic acid) buffer¹².
   2. Adjust pH to 7.0 with 1 M NaOH and store the artificial saliva solution at 4 °C.
   3. Before using the solution, add sodium fluoride (NaF, 300 ppm).
2. Application of CS-AMEL hydrogel
   1. Apply about 20 µl CS-AMEL hydrogel to the etched tooth slice using a syringe (Figure 1B).
   2. Dry the hydrogel-covered tooth slice in a desiccator at RT for 2 hr.
   3. Transfer the tooth slice to a beaker containing 30 ml of artificial saliva solution. Cover the beaker with aluminum foil, and then keep the beaker in an oven at 37 °C for 7 days.
   4. Remove the tooth slice and dry it in a desiccator at RT.

4. Characterization of Interface between Newly Grown Layer and Enamel

The microstructure of the interface between the newly grown layer and enamel was observed by scanning electron microscopy (SEM) and high revolution transmission electron microscopy (HR-TEM). The TEM sample was prepared using a focused ion beam (FIB) technique, the steps for which are as follows.

1. Load the sample into a FIB-SEM instrument and finish all the alignments according to the operating instructions. NOTE: Fine Z adjustment should be performed at least 2 times.
2. Deposit a carbon layer (15 × 3 µm) on the sample to protect the underlying structure (Figure 2A).
3. Mill the sample carefully at the upper, lower and right sides of carbon layer to prepare a thin piece of sample, and then cut the bottom side of this thin piece. NOTE: Check the Ion beam alignment before each milling step (Figure 2B).
4. Weld a Pt tip on the thin piece and cut the left side to separate the sample piece (Figure 3C). NOTE: Check the Ion beam alignment before the welding step.
5. Lift out the Pt tip with the sample slowly, and mount the lamellar sample onto a lift-out TEM grid.
6. Detach the tip from the sample, and thin the sample until its thickness is less than 100 nm (Figure 3D). Remove the sample from the instrument to perform the TEM observation. NOTE: Check the Ion beam alignment before each thinning process.

5. Assessment of Binding and Mechanical Properties of Newly Grown Layer

1. The binding strength is assessed by ultrasonic treatment. Keep the repaired tooth slice in an ultrasonic cleaner (42 kHz, 100 W) for 10 min, and then observe the interface between the repaired layer and natural enamel with a backscattered-electron SEM analysis.
2. Measure the hardness and elastic modulus at 20 test points on each repaired enamel surface (n=3) by a nano-indenter with a Berkovich tip.

6. Immunofluorescence Staining

1. Wash the tooth slice with Tris buffer saline (TBS) for 15 min.
2. Block with 1% bovine serum albumin (BSA) in TBS for 15 min.
3. Remove the liquid and incubate the sample O/N with 1°Ab (1:500 Chicken Anti-Amelogenin) diluted in TBS (0.1% BSA, 0.3% Triton X-100).
4. Wash the sample with TBS for 30 min and incubate O/N with 2°Ab (1:100 Anti-chicken FITC) diluted in TBS.
5. Wash the sample with TBS for 30 min and leave to dry for another 30 min.
6. Observe by fluorescence microscope.

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