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.

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
<ol>
	<li>Select a human third molar without any restored caries.</li>
	<li>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.</li>
	<li>To simulate erosive lesions, etch the tooth slices with 30% phosphoric acid for 30 sec then immediately rinse them with deionized water.</li>
	<li>Ultrasonically clean the etched slices for 2 min, and then rinse them with deionized water 3 times.</li>
</ol>
2. Preparation of Amelogenin-chitosan Hydrogel
<ol>
	<li>Preparation of chitosan stock solution
	<ol>
		<li>Dissolve 1% (w/v) chitosan (medium molecular weight, 75-85% deacetylated) in a 1% (v/v) acetic acid solution followed by stirring at 80 &#176;C O/N.</li>
		<li>After cooling the solution to RT, filter the chitosan solution using a 0.45 &#956;m filter. Adjust the pH value to 5.0 by adding 1 M NaOH solution.</li>
	</ol>
	</li>
	<li>Preparation of calcium and phosphate stock solution (0.1 M)
	<ol>
		<li>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.</li>
		<li>Weigh out dibasic sodium phosphate (Na2HPO4) and prepare a 0.1 M solution, then mix using a vortex until the solution is clear.</li>
	</ol>
	</li>
	<li>Expression and purification of recombinant amelogenin17-18 
	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.
	<ol>
		<li>Add 20 g of Lysogeny broth (LB) Agar powder into 500 ml deionized water, autoclave the solution at 121 &#176;C (liquid setting) for 20 min. Let agar cool to ~ 55 &#176;C, add ampicillin (100 &#956;g/ml) into agar solution.</li>
		<li>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 &#176;C.</li>
		<li>Streak the LB agar plate with recombinant cells (E.coli&#8211;BC21) from the glycerol stock (-80 &#176;C). Incubate the plates O/N at 37 &#176;C.</li>
		<li>Prepare 50 ml of NZCYM media and autoclave the media at 121 &#176;C for 30 min. Allow the media to cool down, add 50 &#181;l of 100 &#181;g/ml ampicillin to the 50 ml media.</li>
		<li>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 &#176;C.</li>
		<li>Prepare 1 L of NZCYM media and autoclave the media at 121 &#176;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.</li>
		<li>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.</li>
		<li>Induce with 700 &#181;l of Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG, 1 M) when the bacterial growth reaches to about 0.75~0.8 OD at 595 nm.</li>
		<li>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 &#176;C. Keep the cell pellet in the -20 &#176;C O/N.</li>
		<li>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.</li>
		<li>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 &#176;C. Keep the supernatant. Add 20% Saturated ammonium sulfate using the saturated stock solution and stir in the cold room, O/N.</li>
		<li>Centrifuge for 30 min, 8,554 x g at 4 &#176;C and keep the pellet. Reconstitute the pellets in 0.1% trifluoroacetic acid (TFA), load onto a C4 column (10 x 250 mm, 5 &#181;m), 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&#183;min-1.</li>
		<li>Freeze the protein solution on dry ice and lyophilize the frozen sample O/N.</li>
	</ol>
	</li>
	<li>Preparation of amelogenin-chitosan (CS-AMEL) hydrogel (Figure 1A)
	<ol>
		<li>Add 200 &#181;g of rp172 into a tube containing 960 &#181;l of 1% chitosan solution, then mix using a vortex until the solution is clear.</li>
		<li>Add 25 &#181;l of 0.1 M CaCl2 solution followed by 15 &#181;l of 0.1 M Na2HPO4 solution to the amelogenin-containing chitosan solution, then mix using a vortex for 5 min.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Enamel Regrowth in Amelogenin-chitosan Hydrogel
<ol>
	<li>Preparation of artificial saliva solution
	<ol>
		<li>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.</li>
		<li>Adjust pH to 7.0 with 1 M NaOH and store the artificial saliva solution at 4 &#176;C.</li>
		<li>Before using the solution, add sodium fluoride (NaF, 300 ppm).</li>
	</ol>
	</li>
	<li>Application of CS-AMEL hydrogel
	<ol>
		<li>Apply about 20 &#181;l CS-AMEL hydrogel to the etched tooth slice using a syringe (Figure 1B).</li>
		<li>Dry the hydrogel-covered tooth slice in a desiccator at RT for 2 hr.</li>
		<li>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 &#176;C for 7 days.</li>
		<li>Remove the tooth slice and dry it in a desiccator at RT.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>Deposit a carbon layer (15 &#215; 3 &#181;m) on the sample to protect the underlying structure (Figure 2A).</li>
	<li>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).</li>
	<li>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.</li>
	<li>Lift out the Pt tip with the sample slowly, and mount the lamellar sample onto a lift-out TEM grid.</li>
	<li>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.</li>
</ol>
5. Assessment of Binding and Mechanical Properties of Newly Grown Layer
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
6. Immunofluorescence Staining
<ol>
	<li>Wash the tooth slice with Tris buffer saline (TBS) for 15 min.</li>
	<li>Block with 1% bovine serum albumin (BSA) in TBS for 15 min.</li>
	<li>Remove the liquid and incubate the sample O/N with 1&#176;Ab (1:500 Chicken Anti-Amelogenin) diluted in TBS (0.1% BSA, 0.3% Triton X-100).</li>
	<li>Wash the sample with TBS for 30 min and incubate O/N with 2&#176;Ab (1:100 Anti-chicken FITC) diluted in TBS.</li>
	<li>Wash the sample with TBS for 30 min and leave to dry for another 30 min.</li>
	<li>Observe by fluorescence microscope.</li>
</ol>

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The authors have nothing to disclose.

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...

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.

<table><tbody><tr><td>Human third molar&amp;nbsp;</td><td>Ostrow School of Dentistry of the University of Southern California&amp;nbsp;</td><td>N/A</td><td>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><td>Recombinant pocine amelogenin</td><td>Expression and purification&amp;nbsp; in lab</td><td>N/A</td><td>rP172, full-length&amp;nbsp;</td></tr><tr><td>Chitosan&amp;nbsp;</td><td>Sigma-Aldrich</td><td>448877</td><td>Medium molecular weight, 75-85% deacetylated</td></tr><tr><td>Phosphoric acid&amp;nbsp;</td><td>VWR</td><td>AA033266</td><td></td></tr><tr><td>Acetic acid glacial</td><td>VWR</td><td>A036289</td><td></td></tr><tr><td>Sodium hydroxide</td><td>VWR</td><td>BDH9292</td><td></td></tr><tr><td>Calcium chloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>223506</td><td></td></tr><tr><td>Dibasic sodium phosphate anhydrous</td><td>VWR</td><td>BDH0316</td><td></td></tr><tr><td>BL21-CodonPlus (DE3)-RP&amp;nbsp;</td><td>Agilent Technologies Inc.</td><td>230255</td><td></td></tr><tr><td>Ammonium sulfate</td><td>VWR</td><td>BDH8001</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>VWR</td><td>AAAL06374</td><td></td></tr><tr><td>Acetonitrile</td><td>VWR</td><td>BDH1103</td><td></td></tr><tr><td>Magnesium chloride&amp;nbsp;</td><td>VWR</td><td>BDH0244</td><td></td></tr><tr><td>Potassium dihydrogen phosphate&amp;nbsp;</td><td>VWR</td><td>BDH9268</td><td></td></tr><tr><td>Potassium chloride&amp;nbsp;</td><td>VWR</td><td>BDH0258</td><td></td></tr><tr><td>Ammonium chloride&amp;nbsp;</td><td>VWR</td><td>AAAA15000</td><td></td></tr><tr><td>HEPES (4-(2-Hydroxyethyl)piperazine-1-ethane-sulfonic acid)</td><td>VWR</td><td>AAA14777</td><td></td></tr><tr><td>Sodium fluoride&amp;nbsp;</td><td>VWR</td><td>AA11561</td><td></td></tr><tr><td>Tris-buffered saline</td><td>Bio-Rad</td><td>170-6435</td><td>10&amp;times; TBS</td></tr><tr><td>Bovine serum albumin</td><td>EMD Millipore&amp;nbsp;</td><td>12659</td><td>CalBioChem, Albumin, Bovine Serum, Fraction V, Low Heavy Metals&amp;nbsp;</td></tr><tr><td>Triton X-100</td><td>EMD Millipore&amp;nbsp;</td><td>TX1568-1</td><td></td></tr><tr><td>Chicken Anti-Amelogenin</td><td>N/A</td><td>N/A</td><td>A gift from Prof. Malcolm Snead, University of Southern California</td></tr><tr><td>Bovine Anti-Chicken IgY-FITC</td><td>Santa Cruz Biotechnology</td><td>Sc-2700</td><td></td></tr><tr><td>High Performance Liquid Chromatography System</td><td>Agilent Technologies Inc.</td><td>Varian Prostar 210</td><td></td></tr><tr><td>C4 column</td><td>Phenomenx&amp;nbsp;</td><td>Jupiter 5&amp;mu; 300A</td><td></td></tr><tr><td>Scanning Electron Microscopy&amp;nbsp;</td><td>JEOL&amp;nbsp;</td><td>JSM-7001</td><td></td></tr><tr><td>FIB-SEM&amp;nbsp;</td><td>JEOL&amp;nbsp;</td><td>JIB-4500</td><td></td></tr><tr><td>Transmission Electron Microscopy&amp;nbsp;</td><td>JEOL</td><td>JEM-2100F</td><td></td></tr><tr><td>Digital low speed diamond saw</td><td>MTI Corporation</td><td>SYJ-150</td><td></td></tr><tr><td>Fluorescence microscopy</td><td>Leica</td><td>DMI3000 B</td><td></td></tr><tr><td>Ultrasonic cleaner&amp;nbsp;</td><td>Branson&amp;nbsp;</td><td>2510</td><td>42 kHz, 100 W</td></tr><tr><td>Nano-indenter&amp;nbsp;</td><td>Agilent Technologies Inc.</td><td>MTS XP</td><td></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.

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 &#956;m 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 <stron...

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

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.

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 ...

development-amelogenin-chitosan-hydrogel-for-vitro-enamel-regrowth

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

14.9K Views.  University of Southern California. 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.

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

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is prepared by mixing solutions of glycol chitosan with a synthesized benzaldehyde terminated polymer gelator, and hydrogels are efficiently obtained in several minutes at room temperature. By varying ratios between glycol chitosan, polymer gelator, and water contents, versatile hydrogels with different gelation times and stiffness are obtained. When damaged, the hydrogel can recover its appearances and modulus, due to the reversibility of the dynamic imine bonds as crosslinkages. This self-healable property enables the hydrogel to be injectable since it can be self-healed from squeezed pieces to an integral bulk hydrogel after the injection process. The hydrogel is also multi-responsive to many bio-active stimuli due to different equilibration statuses of the dynamic imine bonds. This hydrogel was confirmed as bio-compatible, and L929 mouse fibroblast cells were embedded following standard procedures and the cell proliferation was easily assessed by a 3D cell cultivation process. The hydrogel can offer an adjustable platform for different research where a physiological mimic of a 3D environment for cells is profited. Along with its multi-responsive, self-healable, and injectable properties, the hydrogels can potentially be applied as multiple carriers for drugs and cells in future bio-medical applications.

Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel&#8217;s mechanical strength and its application in 3D cell culture are presented.

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

Chemistry

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) substrates such as tissue culture polystyrene (TCPS). However, characteristic changes of PDL cells have been observed during in vitro culture. This phenomenon is probably because the 2D TCPS differs from the in vivo three-dimensional (3D) microenvironment. Compared to cells cultured on 2D substrates, cells grown in a 3D microenvironment exhibit more similarities to in vivo cells. Therefore, 3D cell culture models provide a promising alternative for conventional 2D monolayer cell culture. To improve conventional PDL cell culture models, we have recently developed a 3D cell culture method, which is based on spheroid formation of PDL cells on chitosan films. Here, we present detailed cell spheroid culture protocols based on chitosan films. The 3D culture system of PDL cellular spheroids overcome some of the limitations related to conventional 2D monolayer cell culture, and thus may be suitable for producing PDL cells with an enhanced therapeutic efficacy for future periodontal tissue regeneration.

Here, we present protocols of culturing human periodontal ligament (PDL) cell spheroids by chitosan films. The culture of three-dimensional (3D) cellular spheroids provides an alternative to conventional tissue culture polystyrene (TCPS) culture system.

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) ...

Developmental Biology

Establishing Organoids from Human Tooth as a Powerful Tool Toward Mechanistic Research and Regenerative Therapy

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development and biology is scarce. In particular, not much is known about the tooth's epithelial stem cells and their function. We succeeded in developing a novel organoid model starting from human tooth tissue (i.e., dental follicle, isolated from extracted wisdom teeth). The organoids are robustly and long-term expandable and recapitulate the proposed human tooth epithelial stem cell compartment in terms of marker expression as well as functional activity. In particular, the organoids are capable to unfold an ameloblast differentiation process as occurring in vivo during amelogenesis. This unique organoid model will provide a powerful tool to study not only human tooth development but also dental pathology, and may pave the way toward tooth-regenerative therapy. Replacing lost teeth with a biological tooth based on this new organoid model could be an appealing alternative to the current standard implantation of synthetic materials.

We present a protocol to develop epithelial organoid cultures starting from human tooth. The organoids are robustly expandable and recapitulate the tooth's epithelial stem cells, including their ameloblast differentiation capacity. The unique organoid model provides a promising tool to study human dental (stem cell) biology with perspectives for tooth-regenerative approaches.

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development ...

Propagation of Dental and Respiratory Cells and Organs in Microgravity

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) mimic the loss of gravity as it occurs in space and instead provide a microgravity environment through continuous rotation of cultured cells or tissues. These RCCSs ensure an un-interrupted supply of nutrients, growth and transcription factors, and oxygen, and address some of the shortcomings of gravitational forces in motionless 2D (two dimensional) cell or organ culture dishes. In the present study we have used RCCSs to co-culture cervical loop cells and dental pulp cells to become ameloblasts, to characterize periodontal progenitor/scaffold interactions, and to determine the effect of inflammation on lung alveoli. The RCCS environments facilitated growth of ameloblast-like cells, promoted periodontal progenitor proliferation in response to scaffold coatings, and allowed for an assessment of the effects of inflammatory changes on cultured lung alveoli. This manuscript summarizes the environmental conditions, materials, and steps along the way and highlights critical aspects and experimental details. In conclusion, RCCSs are innovative tools to master the culture and 3D (three dimensional) growth of cells in vitro and to allow for the study of cellular systems or interactions not amenable to classic 2D culture environments.

This protocol presents a method for the culture and 3D growth of ameloblast-like cells in microgravity to maintain their elongated and polarized shape as well as enamel-specific protein expression. Culture conditions for the culture of periodontal engineering constructs and lung organs in microgravity are also described.

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) ...

Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result from altered activity of cells responsible for enamel synthesis named ameloblasts, which present in enamel organ. During amelogenesis, ameloblasts follow a specific and precise sequence of events of proliferation, differentiation, and death. A rat continually growing incisors is a suitable experimental model to study ameloblast activity and differentiation stages in physiological and pathological conditions. Here, we describe a reliable and consistent method to micro-dissect enamel organ of rats exposed to environmental toxicants. The micro-dissected dental epithelia contain secretion- and maturation-stage ameloblasts that may be used for qualitative experiments, such as immunohistochemistry assays and in situ hybridization, as well as for quantitative analyses such as RT-qPCR, RNA-seq, and Western blotting.

Understanding enamel formation and possible alterations requires the study of ameloblast activity. Here, we describe a reliable and consistent method to micro-dissect enamel organs containing secretion- and maturation-stage ameloblasts that may be used for further quantitative and qualitative experimental procedures.

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result ...

Medicine

Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, chitosan microgels are commonly fabricated using emulsion methods that require organic solvent rinses, which are toxic and harmful to the environment. The present protocol presents a rapid, non-cytotoxic, non-emulsion-based method for fabricating chitosan-genipin microgels without the need for organic solvent rinses. The microgels described herein can be fabricated with precise size control. They exhibit sustained release of biomolecules, making them highly relevant for tissue engineering, biomaterials, and regenerative medicine. Chitosan is crosslinked with genipin to form a hydrogel network, then passed through a syringe filter to produce the microgels. The microgels can be filtered to create a range of sizes, and they show pH-dependent swelling and degrade over time enzymatically. These microgels have been employed in a rat growth plate injury model and were demonstrated to promote increased cartilage tissue repair and to show complete degradation at 28 days in vivo. Due to their low cost, high convenience, and ease of fabrication with cytocompatible materials, these chitosan microgels present an exciting and unique technology in tissue engineering.

The present protocol describes a non-emulsion-based method for the fabrication of chitosan-genipin microgels. The size of these microgels can be precisely controlled, and they can display pH-dependent swelling, degrade in vivo,&#160;and be loaded with therapeutic molecules that release over time in a sustained manner, making them highly relevant for tissue engineering applications.

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Biology

Accessing the Cytotoxicity and Cell Response to Biomaterials

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by several methods, but a high discrepancy exists between the approaches used, compromising the reproducibility and the comparison among the obtained results. In this paper, we propose a protocol to evaluate biomaterials cytotoxicity using soluble extracts, which we use for dental biomaterials. The extracts preparation is detailed, from pellets production to its extraction in a culture medium. The biomaterials cytotoxicity evaluation is based on metabolic activity using the MTT assay, cell viability using the Sulphorhodamine B (SBR) assay, cell death profile by flow cytometry, and cell morphology using May-Gr&#252;nwald Giemsa. Additional to cytotoxicity evaluation, a protocol to evaluate cell function is described based on the expression of specific markers assessed by immunocytochemistry and PCR. This protocol provides a comprehensive guide for biomaterials cytotoxicity and cellular effects evaluation, using the extracts methodology, in a reproducible and robust manner.

This methodology aims to evaluate biomaterial cytotoxicity through the preparation of soluble extracts, using viability assays and phenotypic analysis, including flow cytometry, RT-PCR, immunocytochemistry, and other cellular and molecular biology techniques.

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by ...

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

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Rozdziały w tym wideo

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films

Establishing Organoids from Human Tooth as a Powerful Tool Toward Mechanistic Research and Regenerative Therapy

Propagation of Dental and Respiratory Cells and Organs in Microgravity

Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants

Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Accessing the Cytotoxicity and Cell Response to Biomaterials