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. Extract the aqueous layer with ethyl acetate (3 x 250 mL), and wash with 100 mL of brine. Dry the organic layer over anhydrous sodium sulfate, filter and evaporate volatiles under vacuum to get a pale-yellow solid.</li>
		<li>Subject the yellow solid to silica gel flash column chromatography (3% methanol/dichloromethane) to yield 15.61 g of compound A1 as a white powder.</li>
	</ol>
	</li>
	<li>Synthesis of compound A2
	<ol>
		<li>Mix 3.12 g of compound A1 and 2.75 g of potassium carbonate in 50 mL of N, N-dimethylformamide. Stir at room temperature for 2 h.</li>
		<li>Add 2.6 mL of carbon disulfide to the reaction suspension. Keep stirring for 4 h.</li>
		<li>Add absolute ethanol to the reaction mixture at 0 &#176;C. Filter the yellow precipitate out and wash with diethyl ether. Dry under vacuum overnight to yield 6.18 g of compound A2 as pale-yellow solid.</li>
	</ol>
	</li>
	<li>Synthesis of compound A3
	<ol>
		<li>Add 2.7 mL of methyl iodide in 15 mL of acetonitrile. Also, separately prepare a solution of compound A2 by adding 6.18 g of A2 to 80 mL of water/acetonitrile (7:3 ratio). Mix both the solutions and stir at room temperature for 30 min.</li>
		<li>Heat the reaction mixture to 95 &#176;C and keep stirring for 3 h.</li>
		<li>Cool the reaction mixture to room temperature, and then evaporate the volatiles under vacuum.</li>
		<li>Extract the residue 3x with 100 mL of ethyl acetate and wash with brine. Dry the organic layer over anhydrous sodium sulfate, filter and evaporate the volatiles under vacuum to obtain a yellow solid.</li>
		<li>Subject the yellow solid to silica gel flash column chromatography (30% ethyl acetate/hexanes) to yield 4.52 g of the compound A3 as a light-yellow solid.</li>
	</ol>
	</li>
	<li>Synthesis of compound A4
	<ol>
		<li>Prepare allylamine solution by adding 925 &#181;L of allylamine in 20 mL of ethanol. Add this solution dropwise to the solution of compound A3, prepared by adding 3.14 g of A3 in 75 mL of absolute ethanol, over 30 min. Then heat the reaction mixture to reflux for 16 h.</li>
		<li>Cool the reaction mixture to room temperature and evaporate the volatiles to give a yellow solid.</li>
		<li>Subject the yellow solid to silica gel flash column chromatography (50% ethyl acetate/hexanes to 2% methanol/dichloromethane) to yield 1.80 g of compound A4 as a white crystal.</li>
	</ol>
	</li>
	<li>Synthesis of compound A5
	<ol>
		<li>Add 2.05 g of guanidinium hydrochloride and 7.8 mL of sodium ethoxide (21 wt% in ethanol) in 14 mL of absolute ethanol. Heat the mixture to 45 &#176;C for 15 min.</li>
		<li>Filter off the insoluble material and add the filtrate directly into the solution of compound A4, prepared by adding 2.70 g of A4 in 40 mL absolute ethanol. Heat the reaction mixture to reflux for 16 h.</li>
		<li>Filter the precipitate out to obtain 2.65 g of compound A5 as an off-white solid.</li>
	</ol>
	</li>
	<li>Synthesis of compound A6
	<ol>
		<li>Add 24.9 mL of triethylamine slowly to the slurry mixture obtained by adding 2.11 g of compound A5 and 1.10 g of 4-Dimethylaminopyridine in 120 mL of tetrahydrofuran over 1 min. Stir at room temperature for 2 min.</li>
		<li>Add 20.7 mL of di-tert-butyl dicarbonate dropwise to the reaction mixture and keep stirring at room temperature for 40 h.</li>
		<li>Add 20 mL of water to quench the reaction. Evaporate the volatiles under vacuum.</li>
		<li>Extract the red viscous residue with 500 mL of ethyl acetate. Then, wash it in with 250 mL of water, 75 mL of 10% citric acid, 2x with 200 mL of water, 200 mL of saturated sodium bicarbonate, and 200 mL of brine. Dry the organic layer over anhydrous sodium sulfate, filter and evaporate the volatiles under vacuum to give a red/orange solid.</li>
		<li>Subject the solid to silica gel flash column chromatography (0-20% ethyl acetate/hexanes) to yield 3.87 g of compound A6 as a white foam.</li>
	</ol>
	</li>
	<li>Synthesis of compound A7
	<ol>
		<li>Add N-methylmorpholine N-oxide (50 wt.% in water, 1.64 mL) dropwise to the solution of compound A6 (2.93 g) in acetone/water (8:1, 58.5 mL) and stir at room temperature for 5 min.</li>
		<li>Add osmium tetraoxide (4% aqueous solution) dropwise to the mixture over a period of 3 min and keep stirring at room temperature for 24 h.</li>
		<li>Quench the reaction with 1.0 M aqueous sodium sulfite until the solution turns colorless. Remove the volatiles under vacuum to result in a white slurry in water. Extract the residue with 350 mL of dichloromethane and then wash with 150 mL of water followed by a wash with 150 mL of brine.</li>
		<li>Dry the organic layer over anhydrous sodium sulfate, filter and evaporate the volatiles under vacuum to yield 2.45 g of compound A7 as a white solid.</li>
	</ol>
	</li>
	<li>Synthesis of compound A8
	<ol>
		<li>Add 760 g of sodium periodide to the mixture of 1.36 g of compound A7 in 35 mL of dichloromethane/water (6:1, 35 mL) and stir at room temperature for 42 h.</li>
		<li>Filter the insoluble material off and concentrate the filtrate under vacuum. Extract the resulting residue with 250 mL of dichloromethane, and then wash with 100 mL of water and 100 mL of brine. Dry the organic layer over anhydrous sodium sulfate, filter and evaporate the volatiles under vacuum to give the crude product.</li>
		<li>Subject the crude product to silica gel flash column chromatography (5-40% ethyl acetate/hexanes, then 5% methanol/dichloromethane) to yield 1.08 g of compound A8.</li>
	</ol>
	</li>
	<li>Synthesis of compound A9
	<ol>
		<li>Prepare a solution of 830 mg of compound A8 in 15 mL of 1,2-dichloroethane. Add the solution of dropwise to the mixture of 6-benzyloxycarbonylamino-2-L-amino-hexanoic acid trimethylsilyl ethyl ester (649.5 mg) and N,N-diisopropylethylamine (591 &#181;L) in 24 mL of 1,2-dichloroethane over 3 min and stir at room temperature for 15 min.</li>
		<li>Add 360.3 mg of sodium triacetoxyborohydride to the solution and keep stirring at room temperature for 24 h.</li>
		<li>Quench the reaction mixture with 6 mL of water. Extract the reaction mixture 3x with 200 mL of dichloromethane and wash with 200 mL of 10% citric acid in water, 2x with 200 mL of water, 200 mL of saturated sodium bicarbonate, and 200 mL of brine in that order. Dry the organic layer over anhydrous sodium sulfate, filter and evaporate the volatiles under vacuum to obtain the crude product.</li>
		<li>Subject the crude product to silica gel flash column chromatography (5-30% ethyl acetate/hexanes) to yield 885 mg compound A9.</li>
	</ol>
	</li>
	<li>Synthesis of JBNT monomer
	<ol>
		<li>Dissolve 0.54 g of compound A9 (0.54 g) in 10 mL of 94% trifluoroacetic acid/thioanisole solution and stir at room temperature for 72 h.</li>
		<li>Add diethyl ether (80 mL) to the reaction mixture and centrifuge the precipitate down. Pour the clear trifluoroacetic acid containing supernatant out and wash the white precipitate with diethyl ether and methanol to yield 168.6 mg crude JBNT monomer (Supplemental File 1).</li>
		<li>Purify the crude JBNT monomer by a high-performance liquid chromatography (HPLC) with a reverse phase column. The isolation program is listed below: Solvent A: 100% water; solvent B: 100% acetonitrile; solvent C: pH=1 HCl water solution; flow rate: 3 mL/min (see Table 1).</li>
		<li>Collect the largest peak at about 7.2 min.</li>
	</ol>
	</li>
</ol>
2. Fabrication for JBNT/FN
<ol>
	<li>Add 80 &#181;L of 1 mg/mL JBNT aqueous solution to 40 &#181;L of 1 mg/mL FN aqueous solution and pipette several times.</li>
	<li>Check for the presence of white solid suspension after pipetting for about 10 s. 
	NOTE: A video was shot to capture the self-assembly process of NM (Supplemental File 2).</li>
</ol>
3. Observation of lyophilized specimens
NOTE: This step is performed to show the scaffold structure of NM made from JBNTs and FN.
<ol>
	<li>Prepare FN, JBNT and FN/JBNT NM solutions for lyophilized materials observation. Dilute 10 &#181;L of 100 &#181;g/mL of FN with 40 &#181;L of distilled water to make a 20 &#181;g/mL solution of FN.</li>
	<li>Prepare 100 mg/mL solution of JBNTs by diluting 5 &#181;L of 1 mg/mL JBNTs in 45 &#181;L of DI water.</li>
	<li>Mix 10 &#181;L of 100 &#181;g/mL FN and 5 &#181;L of 1 mg/mL of JBNTs in 35 &#181;L of DI water to form the NM solution.</li>
	<li>Coat three wells of 96-well clear round bottom microplates with the FN, JBNT solution and NM solution, respectively. Each well receives 50 &#181;L of the solution.</li>
	<li>Perform lyophilization, as described below, to visualize the scaffold structure of the NM.
	<ol>
		<li>Freeze the plate at -80 &#176;C overnight.</li>
		<li>Turn on the lyophilized instrument and vacuum pump.</li>
		<li>Press the cool down button to reduce the temperature of the system to -50 &#176;C.</li>
		<li>Put the frozen plate into the lyophilized instrument and close all openings of the instruments.</li>
		<li>Click the start button to reduce the system pressure to 0.9 mbar.</li>
		<li>After 4 h, press the aerate button and open the valve to increase the system pressure to the atmospheric pressure.</li>
		<li>Take the plate out of the lyophilized instrument.</li>
		<li>Use a microscope to observe the FN, JBNTs and the resulting self-assembled NM. Take several photographs with a camera for the materials.</li>
	</ol>
	</li>
</ol>
4. Absorption spectra measurement
NOTE: Use the change of spectra for FN and JBNTs to present the self-assembled JBNT/FN NM12.
<ol>
	<li>Mix 30 &#181;L of 100 &#181;g/mL FN with 15 &#181;L of distilled water and 5 &#181;L of 1 mg/mL JBNTs to prepare the JBNT/FN NM solution. White solid suspension can be seen after pipetting the solution several times. 
	NOTE: The final concentrations of JBNTs and FN in the solution are 100 &#181;g/mL and 60 &#181;g/mL, respectively. JBNTs and FN solutions at the same concentration to the one used in the NM solution were also prepared as control solutions.</li>
	<li>Dilute 5 &#181;L of 1 mg/mL of JBNTs with 45 &#181;L of distilled water to prepare JBNT control solution.</li>
	<li>Dilute 30 &#181;L of 100 &#181;g/mL FN with 20 &#181;L of distilled water to prepare FN control solution.</li>
	<li>Measure the UV-vis absorption spectra of FN, JBNT and JBNT/FN NM solutions with the spectrophotometer (see Table of Materials) to characterize the assembly of NM.
	<ol>
		<li>Click the UV-Vis button. Clean the test surface of the spectrophotometer and drop 2 &#181;L of distilled water on it. Measure it as blank from 190-850 nm.</li>
		<li>Clean the test surface of the spectrophotometer and drop 2 &#181;L of FN solution on it. Measure the UV-Vis absorption spectra of FN solution from 190 nm to 850 nm.</li>
		<li>Clean the test surface of the spectrophotometer and drop 2 &#181;L of JBNT solution on it. Measure the absorption spectra of JBNT solution from 190 nm to 850 nm.</li>
		<li>Clean the test surface of the spectrophotometer and drop 2 &#181;L of JBNT/FN NM solution on it. Measure the absorption spectra of JBNT/FN NM solution from 190 nm to 850 nm.</li>
	</ol>
	</li>
</ol>
5. Preparation of JBNT/FN NM for transmission electronic microscopy (TEM)
<ol>
	<li>Clean several grids before negative staining with a basic plasma cleaner (see Table of Materials).</li>
	<li>Perform the negative staining for the specimens following two different processes.
	<ol>
		<li>Drop 3 &#181;L of 1 mg/mL of JBNTs aqueous solution and 3 &#181;L of JBNT/FN NM solution on separated grids and leave it for 2 min. Then rinse each grid with 100 &#181;L of 0.5% uranyl acetate (UA) solution. Drain the excess solution with filter paper and air dry the grids.</li>
		<li>Mix 9 &#181;L of JBNT/FN NM solution with 3 &#181;L of 2.0% UA solution and pipette it several times. Pipette the mixed solution on the grids and keep it on the grids for 2 min. Use a filter paper to remove the mixed solution from the grids and dry the grids at room temperature.</li>
	</ol>
	</li>
	<li>Finally, characterize the specimen for JBNTs, JBNT/FN NM stained with step 5.2.1, and JBNT/FN NM stained with step 5.2.2 with the TEM.</li>
</ol>
6. In vitro biological function assay
<ol>
	<li>Add 200 &#181;L of negative controls (NC, PBS), JBNTs, FN and the JBNT/FN NM solutions into one well of an 8-well chambered slide, respectively.</li>
	<li>Freeze dry the 8-well chambered coverglass with the lyophilized instrument to coat materials on the bottom of wells. The protocol for freeze-drying is the same as step 3.5.</li>
	<li>Then add 10,000 cells/wells hMSCs (Passage 3) into the wells and incubate them for 24 h at 37 &#176;C with 5% CO2.</li>
	<li>After incubation, pipette out the cell culture medium from the wells and rinse the culture with 1x PBS solution.</li>
	<li>Add 100 &#181;L of the fixative solution to each well with cells and leave for 5 min. Rinse cells gently with 1x PBS twice.</li>
	<li>Dilute 1% Triton-X 100 to 0.1%. Add 100 &#181;L of 0.1% Triton-X 100 to each well with cells and leave for 10 min. Rinse cells gently with 1x PBS twice.</li>
	<li>Dilute the Rhodamine Phalloidin to 1.65 &#181;M. Add 100 &#181;L of Rhodamine Phalloidin to each well with cells and incubate the chambered coverglass keeping from light at room temperature for 30 min. Rinse cells gently with 1x PBS twice.</li>
	<li>Capture the cell images with a fluorescent microscope.</li>
	<li>Count the cell numbers on fluorescent images for each group. Calculate the cell adhesion density by averaging of three random areas in each well.</li>
	<li>Present all data as average &#177; standard deviation (SD). Perform statistical analyses using a one-tailed t-test, followed by analysis of variance (ANOVA) with p&#60;0.05, which is considered statistically significant.</li>
</ol>

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Dr. Yupeng Chen is a co-founder of NanoDe Therapeutics, Inc.

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.
The NM has shown great potential to serve as a novel tissue engineering scaffold although a lot of efforts have been made to fabricate tissue regenerative scaffolds, which were used to facilitate hMSCs adhesion and differentiation. However, most of these materials are pre-made with a specific shape, such as a 3D printed scaffold18,19. As such, they are not easy to be implanted into injured sites that are deep in the joint. However, the NM solution here can be injected as a liquid and then serve as a scaffold for cell adhesion.
One limitation is that the NM is not mechanically strong. Unlike polymers, they are fabricated via self-assembly of noncovalent interactions, so they cannot bear a lot of mechanical loading or tension. On the other hand, the noncovalent structure also presents very good biodegradability and biocompatibility suitable for biological functions8,20,21.
The injectable NM has shown great potential to provide a target location and a scaffold for MSC homing to enhance bone fracture healing in the body. However, when injected into the injured site, the noncovalent NM may not stay in place for a long time, which may reduce the therapeutic effect. In the future, we will explore to incorporate other materials (such as hydrogels) or to alternate the NM structures to further optimize the properties of the NM scaffold.

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&lt;sup&gt;c&lt;/sup&gt; 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 crosslinking13.
We captured and analyzed the camera images of FN, JBNTs, and the NM (Figure 3). For the FN group, except for short white spots, no long fibers were observed. The short clusters were the protein agglomerations consisting of FN, which indicates that the scaffold structure cannot be formed with FN alone without JBNTs (Figure 3A). As shown in Figure 3B, the long and thinner fibers indicate the existence of JBNTs. The width of the white strands of the NM is wider compared to JBNTs, indicating that the JBNTs and FN crosslinked with each other forming NM. The NM fiber can grow up to several centimeters in length (Figure 3C).
We obtained the UV-Vis spectra to indicate the assembly between JBNTs and FN. As shown in Figure 4, JBNTs exhibits two absorption peaks at 220 nm and 280 nm, respectively. Compared to JBNTs, the JBNT/FN NM has two absorption peaks at the same location with a significantly reduced value, which was from JBNTs but affected by the non-covalent self-assembled JBNTs and FN in between.
TEM was used to characterize the morphology of the JBNTs and NM. As shown in Figure 5A, the JBNTs are slender tubes with uniform diameters. Under neutral conditions, JBNTs are positively charged while FN are negatively charged. When mixed, they formed long fibroid NM via charge interactions (Figure 5B). When pH is lower than the isoelectric point of the FN (pI of 5.5-6.0), the NM bundles present self-release due to the positively charged FN. As shown in Figure 5C, when preserved in a solution with low pH (4.0), the NM was dissembled, and a lot of FN was released from the NM. The FN distributed alongside the nanotube is also a strong evidence that the NM was fabricated by JBNTs and FN10.
We explored the effect of the JBNT/FN NM on cell adhesion. As shown in Figure 6, the cell adhesion density of the NM group showed significant difference (p-value &#60;0.05) value compared to the negative control. The difference may be because the NM are designed to morphologically mimic ECM, which provide a scaffold for cell adhesion14. The ECM was composed of collagens and cell adhesive glycoproteins, such as fibronectin15. Collagen has been presented with the ability to promote higher adhesion and proliferation of hMSCs16. Additionally, fibronectin has been shown to enhance the hMSCs adhesion in the injured site as well15. Fluorescence imaging also demonstrated that the hMSCs adherence of the NM group increased significantly compared to the negative control group (Figure 7)15,17.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61317/61317fig01.jpg" /> 
Figure 1: Schematic illustration of the hierarchical self-assembly of JBNTs with a lysine side chain. 
This figure is reprinted with permission from the previous publication12. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61317/61317fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61317/61317fig02.jpg" /> 
Figure 2: Demonstration for the self-assembled process of the NM. 
(A) FN solution. (B) JBNTs mixed with FN. This figure is reprinted with permission from the previous publication12. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61317/61317fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61317/61317fig03.jpg" /> 
Figure 3: Brightfield photographs. 
Brightfield photographs of (A) FN (B) JBNTs and (C) NM formed by JBNTs and FN. Each area has a diameter of 2 cm. This figure is reprinted with permission from the previous publication12. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61317/61317fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61317/61317fig04.jpg" /> 
Figure 4: Absorption spectra of FN, JBNTs, and JBNT/FN NM. 
This figure is reprinted from the previous publication12. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61317/61317fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61317/61317fig05.jpg" /> 
Figure 5: TEM images. 
TEM images of (A) JBNTs (B) JBNT/FN NM, and (C) released JBNT/FN NM. This figure is reprinted with permission from the previous publication12. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61317/61317fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61317/61317fig06.jpg" /> 
Figure 6: Statistical analysis of cellular adhesion. 
Cell adhesion density was recorded in this experiment. *p&#60;0.01 compared to negative controls. **p&#60;0.05 compared to JBNT alone. N=3. This figure is reprinted with permission from the previous publication12. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61317/61317fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61317/61317fig07.jpg" /> 
Figure 7: Fluorescence images. 
Fluorescence images of (A) the hMSCs and (B) the hMSC incubated with the JBNTs. (C) the hMSC incubated with the FN. (D) the hMSC incubated with the JBNT/FN NM. Scale bar: 100 &#956;m. This figure is reprinted with permission from the previous publication12. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61317/61317fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Time</td>
			<td>A%</td>
			<td>B%</td>
			<td>C%</td>
		</tr>
		<tr>
			<td>0.00 min</td>
			<td>98</td>
			<td>0</td>
			<td>2</td>
		</tr>
		<tr>
			<td>15.00 min</td>
			<td>68</td>
			<td>30</td>
			<td>2</td>
		</tr>
		<tr>
			<td>25.00 min</td>
			<td>8</td>
			<td>90</td>
			<td>2</td>
		</tr>
		<tr>
			<td>26.00 min</td>
			<td>0</td>
			<td>100</td>
			<td>0</td>
		</tr>
	</tbody>
</table>
Table 1: Gradient elution time.
Supplemental File 1: Scheme for the synthesis of JBNT monomer. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61317/61317supfig01.jpg" target="_blank">Please click here to download this figure.</a>
Supplemental File 2: Video for self-assembly of the FN/JBNT NM. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61317/Supplemental_File_2.mp4" target="_blank">Please click here to download this video.</a>

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 ...

fabrication-biomimetic-nano-matrix-with-janus-base-nanotubes

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

4.0K Views.  University of Connecticut. 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.

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

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations having a major effect. Here we present a method to obtain large-scale uneven nanopatterns of arginine-glycine-aspartic acid (RGD)-functionalized dendrimers that permit the nanoscale control of local RGD surface density. Nanopatterns are formed by surface adsorption of dendrimers from solutions at different initial concentrations and are characterized by water contact angle (CA), X-ray photoelectron spectroscopy (XPS), and scanning probe microscopy techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The local surface density of RGD is measured using AFM images by means of probability contour maps of minimum interparticle distances and then correlated with cell adhesion response and differentiation. The nanopatterning method presented here is a simple procedure that can be scaled up in a straightforward manner to large surface areas. It is thus fully compatible with cell culture protocols and can be applied to other ligands that exert concentration-dependent effects on cells.

A method to obtain dendrimer-based uneven nanopatterns that permit the nanoscale control of local arginine-glycine-aspartic acid (RGD) surface density is described and applied for the study of cell adhesion and chondrogenic differentiation.

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular reactions. However, it is difficult to determine the relation between the size of a nanostructure and the responses of cells owing to the lack of proper screening tools. Here, we show the development of reproducible and cost-effective gradient nanopattern plates for the manipulation of cellular responses. Using anodic aluminum oxide (AAO) as a master mold, gradient nanopattern plates with nanopillars of increasing diameter ranges [120-200&#8239;nm (GP 120/200), 200-280&#8239;nm (GP 200/280), and 280-360&#8239;nm (GP 280/360)] were fabricated by a thermal imprinting technique. These gradient nanopattern plates were designed to mimic the various sizes of nanotopography in the ECM and were used to screen the responses of human endothelial colony-forming cells (hECFCs). In this protocol, we describe the step-by-step procedure of fabricating gradient nanopattern plates for cell engineering, techniques of cultivating hECFCs from human peripheral blood, and culturing hECFCs on nanopattern plates.

Here, we present a protocol for the fabrication of gradient nanopattern plates via thermal nanoimprinting and the method of screening responses of human endothelial progenitor cells to the nanostructures. By using the described technology, it is possible to produce a scaffold that can manipulate cell behavior by physical stimuli.

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular ...

Fabrication and Characterization of Layer-By-Layer Janus Base Nano-Matrix to Promote Cartilage Regeneration

Various biomaterial scaffolds have been developed to guide cell adhesion and proliferation in hopes to promote specific functions for in vitro and in vivo uses. The addition of growth factors into these biomaterial scaffolds is generally done to provide an optimal cell culture environment, mediating cell differentiation and its subsequent functions. However, the growth factors in a conventional biomaterial scaffold are typically designed to be released upon implantation, which could result in unintended side effects on surrounding tissue or cells. Here, the DNA-inspired Janus base nano-matrix (JBNm) has successfully achieved a highly localized microenvironment with a layer-by-layer structure for self-sustainable cartilage tissue constructs. JBNms are self-assembled from Janus base nanotubes (JBNts), matrilin-3, and transforming growth factor beta-1 (TGF-&#946;1) via bioaffinity. The JBNm was assembled at a TGF-&#946;1:matrilin-3:JBNt ratio of 1:4:10, as this has been the determined ratio at which proper assembly into the layer-by-layer structure could occur. First, the TGF-&#946;1 solution was added to the matrilin-3 solution. Then, this mixture was pipetted several times to ensure sufficient homogeneity before the addition of the JBNt solution. This formed the layer-by-layer JBNm, after pipetting several times again. A variety of experiments were performed to characterize the layer-by-layer JBNm structure, JBNts alone, matrilin-3 alone, and TGF-&#946;1 alone. The formation of JBNm was studied with UV-Vis absorption spectra, and the structure of the JBNm was observed with transmission electron microscopy (TEM). As the innovative layer-by-layer JBNm scaffold is formed on a molecular scale, the fluorescent dye-labeled JBNm could be observed. The TGF-&#946;1 is confined within the inner layer of the injectable JBNm, which can prevent the release of growth factors to surrounding areas, promote localized chondrogenesis, and promote an anti-hypertrophic microenvironment.

This protocol describes the assembly of a layer-by-layer Janus base nano-matrix (JBNm) scaffold by adding Janus base nanotubes (JBNts), matrilin-3, and Transforming Growth Factor Beta-1 (TGF-&#946;1) sequentially. The JBNm was fabricated and characterized; additionally, it displayed excellent bioactivity, encouraging cell functions such as adhesion, proliferation, and differentiation.

Various biomaterial scaffolds have been developed to guide cell adhesion and proliferation in hopes to promote specific functions for in vitro and in vivo ...

Fabricating Engineered Nanofibrillar Collagen with Directed Patterning

Production of Nanofibrillar Patterned Collagen for Tissue Engineering

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are designed to emulate the biophysical properties of native tissue, providing cellular phenotypic and morphological guidance that contributes to the restoration of the regenerative tissue niche. Collagen, a prevalent extracellular matrix protein, is a common component of these regenerative biomaterials due to its biocompatibility and other favorable properties. The current study describes a novel and straightforward method for the fabrication of engineered nanofibrillar collagen with directed fibril patterning. Through the manipulation of shear stress, temperature, and pH, collagen fibrillogenesis and alignment are precisely controlled without requiring specialized equipment. This approach allows for the creation of nanofibrillar collagen biomaterials that mimic the native structure of tissues exhibiting either anisotropic or isotropic characteristics. The flexibility in collagen nanofibril patterning not only facilitates the study of nanoscale patterning on cell behavior but also offers diverse possibilities for patterned tissue engineering applications.

The following protocol presents a shear-based extrusion method for the fabrication of collagen hydrogels with nanoscale patterned fibrils, which can be applied to a broad range of tissue engineering applications.

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are ...