Name: fabrication of a biomimetic nano-matrix with janus base nanotubes and fibronectin for stem cell adhesion
Uploaded: 2020-05-10T14:45:00
Description: Watch this Scientific Journal Video about Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion at JoVE.com

This work is financially supported by NIH (Grants 1R01AR072027-01, 1R03AR069383-01), NSF Career Award (1653702) and University of Connecticut.

1. Synthesis of JBNTs
NOTE: JBNT monomer was prepared as published previously11.
<ol>
	<li>Synthesis of compound A1
	<ol>
		<li>Prepare a solution containing 8.50 g of 2-cyanoacetic acid and 9.80 g of ethylcarbamate in 25 mL of toluene and 2.5 mL of N, N-dimethylformamide. Add 4.90 mL of phosphoryl chloride dropwise. Then heat the mixture to 70 &#176;C and keep stirring for 1.5 h.</li>
		<li>Cool the reaction mixture to room temperature and pour in 100 g of ice water....

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> U. S. Patent. , (2017).</li><li>Zhou, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembled+biomimetic+Nano-Matrix+for+stem+cell+Anchorage.">Self-assembled biomimetic Nano-Matrix for stem cell Anchorage.</a> Journal of Biomedical Materials and Research. 108, 984-991 (2020).</li><li>Jones, M., Leroux, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymeric+micelles+-+a+new+generation+of+colloidal+drug+carriers.">Polymeric micelles - a new generation of colloidal drug carriers.</a> European Journal of Pharmaceutics and Biopharmaceutics. 48 (2), 101-111 (1999).</li><li>Singh, P., Schwarzbauer, J. 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In this study, we developed a self-assembled biomimetic NM, which was formed with DNA-inspired JBNTs and FN. When preparing the JBNT solution, the JBNT lyophilized powder should be dissolved into the water instead of PBS because PBS will cause agglomeration of JBNTs, which inhibits their assembly. Moreover, the NM should also be assembled in water if we want to observe the nano-fibril structures of the NM, because the salt in PBS will bundle with NM fibers, which can greatly reduce the resolution of the images.
<p cl...

Human mesenchymal stem cells (hMSCs) have shown the potential for self-renewal and self-differentiation along different mesenchymal lineages, which helps in the regeneration and maintenance of tissues1. Based on the differentiation potential, hMSCs are considered as candidates for mesenchymal tissue injuries and hematopoietic disorder therapy2. hMSCs have shown the ability to promote wound healing by increasing tissue repair, angiogenesis, and reducing inflammation3. However, without biochemical or biomaterials assistance, the efficiency for the hMSCs to reach a target tissue and function at the desired location is low4. Although various engineered scaffolds have been utilized to attract hMSCs to adhere onto the lesions, some sites such as growth plate fracture, in the middle of a long bone, are not easily accessible by the conventional pre-fabricated scaffolds, which may not fit perfectly into an irregularly shaped injured site.
Here, we have developed a biomimetic nanomaterial that can self-assemble in situ and be injected to a hard to reach target area. The injectable bio-scaffold NM is composed of Janus base nanotubes (JBNTs) and fibronectin (FN). JBNTs, also known as the Rosette Nanotubes (RNTs), are derived from DNA base pairs, specifically thymine and adenine, here5,6,7. As seen in Figure 1, the nanotubes are formed when six molecules of the derived DNA base pairs self-assemble via hydrogen bonds to form a plane6. Six molecules are then stacked onto each other in a plane via a strong pi-stacking interaction7, which can be up to 200-300 &#181;m in length. The JBNTs are designed to morphologically mimic collagen fibers so that FN will react with them.
FN is a high molecular weight adhesive glycoprotein, which can be found in the extracellular matrix (ECM)9. These can mediate the attachment of stem cells to other components of the ECM, particularly collagen10. We designed JBNTs to morphologically mimic collagen fibers so FN can react with them to form NM in a few seconds via noncovalent bonding. Therefore, NM is a promising bio-scaffold to be injected into a bone fracture site that could not be accessible by the conventionally fabricated scaffolds. Here, the injectable NM presents an excellent ability to enhance hMSC anchorage in vitro, exhibiting their potential to serve as a scaffold for tissue regeneration.

<table>
	<tbody>
		<tr>
			<td>1,2-dichloroethane</td>
			<td>Alfa Aesar</td>
			<td>39121</td>
			<td></td>
		</tr>
		<tr>
			<td>2-cyanoacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>C88505</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Dimethylaminopyridine</td>
			<td>TCI America</td>
			<td>D1450</td>
			<td></td>
		</tr>
		<tr>
			<td>8 wells Chambered Coverglass</td>
			<td>Thermo Fisher</td>
			<td>155409</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Corning</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>absolute ethanol</td>
			<td>Thermo Fisher</td>
			<td>BP2818500</td>
			<td></td>
		</tr>
		<tr>
			<td>acetone</td>
			<td>Sigma-Aldrich</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr>
			<td>acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>34851</td>
			<td></td>
		</tr>
		<tr>
			<td>allylamine</td>
			<td>Sigma-Aldrich</td>
			<td>145831</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC32G</td>
			<td></td>
		</tr>
		<tr>
			<td>citric acid</td>
			<td>Sigma-Aldrich</td>
			<td>251275</td>
			<td></td>
		</tr>
		<tr>
			<td>concentrated hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td>Thermo Fisher</td>
			<td>15230147</td>
			<td></td>
		</tr>
		<tr>
			<td>dichloromethane</td>
			<td>Sigma-Aldrich</td>
			<td>270997</td>
			<td></td>
		</tr>
		<tr>
			<td>diethyl ether</td>
			<td>Sigma-Aldrich</td>
			<td>296082</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-tert-butyl dicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>361941</td>
			<td></td>
		</tr>
		<tr>
			<td>ethyl acetate</td>
			<td>Sigma-Aldrich</td>
			<td>319902</td>
			<td></td>
		</tr>
		<tr>
			<td>ethylcarbamate</td>
			<td>Sigma-Aldrich</td>
			<td>U2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Thermo Fisher</td>
			<td>PHE0023</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixative Solution (4 % formaldehyde prepared in PBS)</td>
			<td>Thermo Fisher</td>
			<td>R37814</td>
			<td></td>
		</tr>
		<tr>
			<td>guanidinium hydrochloride</td>
			<td>Alfa Aesar</td>
			<td>A13543</td>
			<td></td>
		</tr>
		<tr>
			<td>hexanes</td>
			<td>Sigma-Aldrich</td>
			<td>227064</td>
			<td></td>
		</tr>
		<tr>
			<td>Human mesenchymal stem cells</td>
			<td>Lonza</td>
			<td>PT-2501</td>
			<td></td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>methyl iodide</td>
			<td>Sigma-Aldrich</td>
			<td>289566</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Diisopropylethylamine</td>
			<td>Alfa Aesar</td>
			<td>A17114</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-dimethylformamide</td>
			<td>Sigma-Aldrich</td>
			<td>227056</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Methylmorpholine N-oxide</td>
			<td>Alfa Aesar</td>
			<td>A19802</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetraoxide</td>
			<td>Alfa Aesar</td>
			<td>45385</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher</td>
			<td>15140163</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Solution</td>
			<td>Thermo Fisher</td>
			<td>20012050</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphoryl chloride</td>
			<td>Sigma-Aldrich</td>
			<td>201170</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>347825</td>
			<td></td>
		</tr>
		<tr>
			<td>reverse phase column</td>
			<td>Thermo Fisher</td>
			<td>25305-154630</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine Phalloidin</td>
			<td>Thermo Fisher</td>
			<td>R415</td>
			<td></td>
		</tr>
		<tr>
			<td>silica gel</td>
			<td>TCI America</td>
			<td>S0821</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium ethoxide</td>
			<td>Alfa Aesar</td>
			<td>L13083</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium periodide</td>
			<td>Sigma-Aldrich</td>
			<td>71859</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>239313</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium sulfite</td>
			<td>Sigma-Aldrich</td>
			<td>S0505</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium triacetoxyborohydride</td>
			<td>Alfa Aesar</td>
			<td>B22060</td>
			<td></td>
		</tr>
		<tr>
			<td>spectrophotometer(NanoDrop One/One&#7580; UV-Vis)</td>
			<td>Thermo Fisher</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>Stem Cell Growth Medium BulletKit</td>
			<td>Lonza</td>
			<td>PT-3001</td>
			<td></td>
		</tr>
		<tr>
			<td>tetrahydrofuran</td>
			<td>Sigma-Aldrich</td>
			<td>401757</td>
			<td></td>
		</tr>
		<tr>
			<td>thioanisole</td>
			<td>Sigma-Aldrich</td>
			<td>T28002</td>
			<td></td>
		</tr>
		<tr>
			<td>toluene</td>
			<td>Sigma-Aldrich</td>
			<td>179418</td>
			<td></td>
		</tr>
		<tr>
			<td>triethylamine</td>
			<td>Alfa Aesar</td>
			<td>A12646</td>
			<td></td>
		</tr>
		<tr>
			<td>trifluoroacetic acid</td>
			<td>Alfa Aesar</td>
			<td>A12198</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Thermo Fisher</td>
			<td>HFH10</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Thermo Fisher</td>
			<td>25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of a biomimetic nano-matrix with janus base nanotubes and fibronectin for stem cell adhesion

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed from JBNTs and FN through self-assembly in an aqueous solution. JBNTs measure 200-300 &#181;m in length with inner hydrophobic hollow channels and outer hydrophilic surfaces. JBNTs are positively charged and FNs are negatively charged. Therefore, when injected into a neutral aqueous solution, they are bonded together via noncovalent bonding to form the NM bundles. The self-assembly process is completed within a few seconds without any chemical initiators, heat source, or UV light. When the pH of the NM solution is lower than the isoelectric point of FNs (pI 5.5-6.0), the NM bundles will self-release due to the presence of positively charged FN.
NM is known to mimic the extracellular matrix (ECM) morphologically and hence, can be used as an injectable scaffold, which provides an excellent platform to enhance hMSC adhesion. Cell density analysis and fluorescence imaging experiments indicated that the NMs significantly increased the anchorage of hMSCs compared to the negative control.

Our studies discovered that the formation of the NM of JBNTs and FN is fast, which happened in 10 seconds. As shown in Figure 2, white floccule was obtained when the JBNT solution was mixed with the FN solution and pipetted several times. The formation process of NM is completely biomimetic. No external stimuli are needed. The process of fabrication is much easier than that of some emerging biomaterials, which is based on ultraviolet light or chemical initiator for crosslinking<sup class="xr...

Watch this Scientific Journal Video about Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion at JoVE.com

Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion

The goal of this protocol is to show the assembly of a biomimetic nanomatrix (NM) with Janus base nanotubes (JBNTs) and fibronectin (FN). When co-cultured with human mesenchymal stem cells (hMSCs), the NMs exhibit excellent bioactivity in encouraging hMSCs adhesion.

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed ...

We developed a biomimetic nano-matrix with great promise to serve as a tissue engineering scaffold. This protocol demonstrates how to assemble the nano-matrix from Janus base nanotubes synthesized in my lab, as well as the fibronectin protein. This nano-matrix can self-assemble in a few seconds without using any additional assistance.When co-cultured with human mesenchymal stem cells, the nano-matrix exhibited excellent bioactivity in encouraging cell adhesion. The nano-matrix mimics the extracellular matrix morphologically. Therefore, it has the potential to serve as an injectable scaffold to repair bone fractures by promoting cell adhesion and function at the target location.Begin by fabricating the nano-matrix. Add 80 microliters of one-milligram-per-milliliter Janus base nanotubes to 40 microliters of one-milligram-per-milliliter fibronectin, and pipette the mixture gently and slowly several times. After 10 seconds, visually inspect the tube for the presence of a white solid suspension.Next, prepare fibronectin, JBNT, and Fibronectin/JBNT nano-matrix solutions for lyophilized materials observation according to manuscript directions. Coat three wells of a 96-well, clear, round-bottom microplate with solutions, adding 50 microliters to each well. Freeze the plate at minus 80 degrees Celsius overnight.On the next day, turn on the lyophilized instrument and vacuum pump, and press the cool down button to reduce the temperature of the system to minus 50 degrees Celsius. Put the frozen plate into the instrument, and close all openings. Click the start button to reduce the system pressure to 0.9 millibar.After four hours, press the aerate button, and open the valve to increase the system pressure to the atmospheric pressure. Then, take the plate out of the lyophilized instrument. Prepare the JBNT/fibronectin nano-matrix solution as described in the text manuscript, and dilute the JBNT and fibronectin solutions with distilled water to make control solutions.Measure the absorption spectra of the solutions with the spectrophotometer to characterize the assembly of nano-matrices. Click the UV-vis button. Then, clean the test surface of the spectrophotometer, and drop two microliters of distilled water on it.Measure the water as blank from 190 to 850 nanometers. For each sample, clean the test surface, and drop two microliters of the solution on it. Then, measure the UV-vis absorption spectra from 190 to 850 nanometers.Add 200 microliters of negative controls, JBNTs, fibronectin, and the nano-matrix solutions into the wells of an eight-well chambered slide. Freeze-dry the eight-well chambered coverglass with the lyophilized instrument to coat materials onto the bottom of the wells. Then, add 10, 000 human mesenchymal stem cells to each well of the coverglass, and incubate it for 24 hours at 37 degrees Celsius with 5%carbon dioxide.After the incubation, remove the cell culture medium from the wells, and rinse them with PBS. Add 100 microliters of the fixative solution to each well, and leave it for five minutes at room temperature. Then, gently rinse the cells with PBS twice, and add 100 microliters of 0.1%Triton X-100 to each well.After 10 minutes, repeat the rinses with PBS. Add 100 microliters of rhodamine phalloidin to each well, and incubate the chambered coverglass for 30 minutes, protecting it from light. Rinse the cells twice with PBS, and image them with a fluorescent microscope.Nano-matrix formation is fast and completely biomimetic. White floccule was visible 10 seconds after the JBNT solution was mixed with the fibronectin solution. Camera images of fibronectin, JBNTs, and the nano-matrix were captured and analyzed.No long fibers were observed in the fibronectin group, indicating that the scaffold structure can't form without JBNTs. JBNTs appear as long and thin fibers. When crosslinked with fibronectin, the nano-matrix fiber can grow up to several centimeters in length.The assembly of the nano-matrix was also confirmed by obtaining UV-vis spectra. The two absorption peaks of JBNTs alone diminish after nano-matrix formation. Transmission electron microscopy was used to characterize the morphology of the JBNTs and nano-matrices.The JBNTs are slender tubes with uniform diameters. When mixed with fibronectin, they form a long fibroid nano-matrix via charge interactions. When the pH is lower than the isoelectric point of the fibronectin, the nano-matrix bundles self-release due to the positively charged fibronectin.The effect of the JBNT/fibronectin nano-matrix on cell adhesion was also explored. Since nano-matrices are designed to morphologically mimic the extracellular matrix, they provide a scaffold and increase cell adhesion. Fluorescence microscopy confirmed that incubation with the nano-matrices increased cell adhesion.In the following procedure, cell migration, differentiation, and the long-term function can be shown to further explore the effect of nano-matrix on human mesenchymal stem cell growth and functions.

Research

JoVE Journal

Bioengineering

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