This work is supported by NIH grants 7R01AR072027 and 7R03AR069383, NSF Career Award 1905785, NSF 2025362, and the University of Connecticut. This work is also supported in part by NIH grant S10OD016435.

1. Synthesis of JBNts
<ol>
	<li>Prepare the JBNt monomer utilizing previously published methods, involving the synthesis of a variety of compounds12.</li>
	<li>Purify crude JBNt monomer after it has been synthesized with high-performance liquid chromatography (HPLC) using a reverse-phase column. Use solvent A: 100% water, solvent B: 100% acetonitrile, and solvent C: HCl water solution with pH = 1. Use a flow rate of 3 mL/min. Collect the largest peak obtained in the HPLC at 7.2 min.</li>
</ol>
2. Fabrication for JBNt/Matn1/TGF-&#946;1 (Video 1)
NOTE: Video 1 shows that the JBNm is an injectable solid formed in a physiological environment (water solution, no UV light, no chemical additives, and no heating), which is also biologically inspired.
<ol>
	<li>Add 8 &#181;L of 1 mg/mL TGF-&#946;1 suspended in H2O to 32 &#181;L of 1 mg/mL matrilin-3 suspended in H2O. Pipette to ensure proper mixing of these proteins.</li>
	<li>Add 80 &#181;L of 1 mg/mL JBNts suspended in H2O to the TGF-&#946;1/matrilin-3 solution. Pipette repeatedly to ensure proper blending. The presence of white floccules can be observed immediately after the addition of JBNts, indicating the formation of the JBNm (Video 1).</li>
</ol>
3. Observation of specimens with ultraviolet-visible (UV-Vis) absorption
NOTE: The UV-Vis absorption spectra were studied to characterize the assembly of the JBNm. This measurement was analyzed for four categories: JBNts alone, matrilin-3 alone, TGF-&#946;1 alone, and the full layer-by-layer JBNm, comprised of all three parts. All initial concentrations are suspended in H2O.
<ol>
	<li>For the JBNt group, add 5 &#181;L of 1 mg/mL JBNts to 50 &#181;L of H2O to make a 90.9 &#181;g/mL&#160;solution.</li>
	<li>For the matrilin-3 group, add 40 &#181;L of 10 &#181;g/mL matrilin-3 to 15 &#181;L of H2O to make a 7.3 &#181;g/mL solution.</li>
	<li>For the TGF-&#946;1 group, add 10 &#181;L of 10 &#181;g/mL&#160;TGF-&#946;1 to 45 &#181;L of H2O to make a 1.8 &#181;g/mL solution.</li>
	<li>For the JBNm group, add 40 &#181;L of 10 &#181;g/mL matrilin-3 to 10 &#181;L of 10 &#181;g/mL&#160;TGF-&#946;1. Agitate to ensure proper mixing, and then add 5 &#181;L of 1 mg/mL JBNts to the solution, pipetting repeatedly to mix the sample.</li>
	<li>Using a spectrophotometer, measure the absorption spectrum of each group.</li>
</ol>
4. Zeta-potential measurement of specimens
NOTE: The zeta-potential was analyzed to better predict how the JBNm would interact with in vivo tissue. Three groups were measured: matrilin-3 alone, matrilin-3 with TGF-&#946;1, and the full layer-by-layer JBNm.
<ol>
	<li>For the matrilin-3 group, add 160 &#181;L of 10 mg/mL matrilin-3 to 640 &#181;L of H2O to make an 800 &#181;L solution.</li>
	<li>For the matrilin-3/TGF-&#946;1 compound, add 160 &#181;L of 10 &#181;g/mL matrilin-3 to 40 &#181;L of 10 &#181;g/mL TGF-&#946;1. Pipette repeatedly to ensure proper homogeneity. Then, add it to 600 &#181;L of H2O resulting in an 800 &#181;L solution.</li>
	<li>For the JBNm group, add 160 &#181;L of 10 &#181;g/mL&#160;matrilin-3 to 40 &#181;L of 10 &#181;g/mL TGF-&#946;1. Pipette multiple times to mix the proteins. Then, add 20 &#181;L of 1 mg/mL JBNts to the solution and pipette to ensure homogeneity. Finally, add the JBNm solution to 580 &#181;L of H2O to produce an 800 &#181;L solution.</li>
	<li>Measure the zeta-potential values for the three groups.</li>
</ol>
5. Preparation of JBNt/matrilin-3 nano-matrix for transmission electron microscopy (TEM)
NOTE: TEM characterization is performed to characterize the morphology of JBNts and JBNm.
<ol>
	<li>Insert two grids into a plasma cleaner to properly clean the grids before the negative staining of the JBNts and JBNm according to the published protocols and manufacturer&#39;s protocols12.</li>
	<li>Create a 200 &#181;g/mL JBNt solution by mixing 10 &#181;L of 1 mg/mL JBNts with 40 &#181;L of distilled water.</li>
	<li>Mix 30 &#181;L of 100 &#181;g/mL matrilin-3 with 20 &#181;L of 100 &#181;g/mL TGF-&#946;1 and pipette several times. Add 10 &#181;L of 1 mg/mL JBNts to the matrilin-3/TGF-&#946;1 mixture to prepare the JBNm sample. Again, pipette repeatedly.</li>
	<li>Add 3 &#181;L of the JBNt solution (200 &#181;g/mL) and 3 &#181;L of the JBNm solution on separate grids, and leave them for 2 min.</li>
	<li>Rinse each grid with 100 &#181;L of uranyl acetate solution (0.5%). Use filter papers to remove the excess solution and allow the grids to air dry.</li>
	<li>Operate a transmission electron microscope to properly observe and characterize the samples, as published previously11,12.</li>
</ol>
6. Absorption spectra measurement of fluorescent-labeled proteins
NOTE: The structure of JBNm is verified by observing the structures of the JBNm with absorption spectral analysis.
<ol>
	<li>Label the TGF-&#946;1 by using a protein labeling kit following the manufacturer&#39;s instructions. Add 20 &#181;L of 20 &#181;g/mL labeled TGF-&#946;1 to 25 &#181;L of H2O to make an 8.9 &#181;g/mL test solution.</li>
	<li>Label the matrilin-3 by using a separate labeling kit. Add 20 &#181;L of 80 &#181;g/mL labeled matrilin-3 to 25 &#181;L of H2O to make a 36 &#181;g/mL test solution. 
	NOTE: The fluorescent dye labeling of the TGF-&#946;1 resulted in a final concentration of 20 &#181;g/mL, while the labeling of the matrilin-3 resulted in a final concentration of 80 &#181;g/mL.</li>
	<li>Mix 20 &#181;L of 80 &#181;g/mL labeled matrilin-3 with 20 &#181;L of 20 &#181;g/mL labeled TGF-&#946;1 and 5 &#181;L of H2O, resulting in a labeled TGF-&#946;1/matrilin-3 compound. Pipette the mixture several times to mix.</li>
	<li>Add 5 &#181;L of 1 mg/mL JBNts to 40 &#181;L of H2O, resulting in a 111 &#181;g/mL solution.</li>
	<li>Add 20 &#181;L of 20 &#181;g/mL labeled TGF-&#946;1 to 20 &#181;L of 80 &#181;g/mL labeled matrilin-3 and pipette several times. Add 5 &#181;L of 1 mg/mL JBNts to the labeled TGF-&#946;1/ matrilin-3 solution, and pipette repeatedly to properly mix the compound. 
	NOTE: The final concentrations of the JBNt, labeled TGF-&#946;1, and labeled matrilin-3 samples were 111 &#181;g/mL, 8.9 &#181;g/mL, and 36 &#181;g/mL, respectively.</li>
	<li>Transfer each sample group (i.e., labeled TGF-&#946;1 (step 6.1), labeled matrilin-3 (step 6.2), labeled TGF-&#946;1/matrilin-3 (step 6.3), JBNts (step 6.4), JBNm (step 6.5)) to their own well of a black 384-well plate.</li>
	<li>Load the black 384-well plate into a multi-mode microplate reader and take measurements at excitation wavelengths of 488 nm and 555 nm following the manufacturer&#39;s protocol.</li>
</ol>
7. In vitro biological function assay
<ol>
	<li>Cell adhesion test on pre-coated coverglass chambers
	<ol>
		<li>Prepare two chambered coverglasses with five groups of samples, as one coverglass is used for each cell type.</li>
		<li>Add 1.25 &#181;L of 1 mg/mL&#160;JBNts to 198.75 &#181;L of distilled water, resulting in a 6.25 &#181;g/mL solution.</li>
		<li>Add 10 &#181;L of 10 &#181;g/mL&#160;matrilin-3 to 190 &#181;L of distilled water, resulting in a 0.5 &#181;g/mLsolution.</li>
		<li>Add 2.5 &#181;L of 10 &#181;g/mL&#160;TGF-&#946;1 to 197.5 &#181;g/mLof distilled water, resulting in a 0.125 &#181;g/mLsolution.</li>
		<li>For the JBNm group, add 10 &#181;L of 10 &#181;g/mL&#160;matrilin-3 to 2.5 &#181;L of 10 &#181;g/mLTGF-&#946;1 and pipette repeatedly. Add 1.25 &#181;L of JBNts with a concentration of 1 mg/mLto the mixture solution and pipette. Finally, add 186.25 &#181;L of distilled water to result in a 200 &#181;L JBNm solution.
		<ol>
			<li>For the control group, use 200 &#181;L of distilled water.</li>
		</ol>
		</li>
		<li>Add each sample group into their own well of a No. 1.5 chambered coverglass. Place the coverglass into a -80 &#176;C freezer for 1 h, and then freeze-dry it with a lyophilizer instrument.</li>
		<li>Seed human mesenchymal stem cells (hMSCs) and human chondrocyte cells (10,000 cells per well) into each well of the prepared chambered coverglasses.</li>
		<li>Incubate the two coverglasses for 4 h in a 37 &#176;C incubator. Then, aspirate the cell culture medium and rinse twice with PBS.</li>
		<li>Fix cells with 4% paraformaldehyde for 5 min, and then remove and rinse samples with PBS. Rinse the sample a second time to completely remove the 4% paraformaldehyde.</li>
		<li>Incubate cells with 100 &#181;L of 0.1% Triton-X for 10 min and wash with PBS. Add 100 &#181;L of 0.165 &#181;M rhodamine-phalloidin to each well. Wait for 30 min and then wash with PBS again.</li>
		<li>Add 0.1 &#181;g/mL&#160;DAPI to each well to stain the nuclei. Wait for 5 min and rinse the wells with PBS twice.</li>
		<li>Use a spectral confocal microscope to observe cell morphology and capture fluorescent images.</li>
		<li>Further, analyze the cell number and morphology using image processing software. Open the software and load the images. Add a scale bar and calibrate the software. Count the number of cells in a given area for each sample. Then, use the measuring tool to collect data on cell morphology for each sample.</li>
	</ol>
	</li>
	<li>Cell proliferation
	<ol>
		<li>Prepare three untreated 96-well plates, adding five groups of samples to each.
		<ol>
			<li>For the JBNt group, add 3.75 &#181;L of 1 mg/mL JBNts to 596.25 &#181;L of distilled water, resulting in a 600 &#181;L JBNt solution with a concentration of 6.25 &#181;g/mL.</li>
			<li>For the TGF-&#946;1 group, add 7.5 &#181;L of 10 &#181;g/mL TGF-&#946;1 to 592.5 &#181;L of distilled water, resulting in a 0.125 &#181;g/mL test solution.</li>
			<li>For the matrilin-3 group, add 30 &#181;L of 10 &#181;g/mL matrilin-3 to 570 &#181;L of distilled water, resulting in a 0.5 &#181;g/mL test solution.</li>
			<li>For the JBNm group, mix 7.5 &#181;L of 10 &#181;g/mL TGF-&#946;1 with 30 &#181;L of 10 &#181;g/mL matrilin-3 and pipette several times, followed by adding 3.75 &#181;L of 1 mg/mL JBNts to the solution.</li>
			<li>Dilute JBNm solution with 558.75 &#181;L of distilled water to obtain the final concentrations of 6.25 &#181;g/mL, 0.125 &#181;g/mL, and 0.5 &#181;g/mL, respectively, for JBNt, TGF-&#946;1, and matrilin-3 samples.</li>
			<li>For the control group, use 600 &#181;L of distilled water.</li>
		</ol>
		</li>
		<li>Divide each sample group into six wells (100 &#181;L sample per well).</li>
		<li>Place the three plates containing all samples into a -80 &#176;C freezer for 1 h and then freeze-dry with a lyophilizing instrument.</li>
		<li>Seed hMSCs onto these plates, each receiving a 100 &#181;L cell suspension (per well) containing 5,000 cells. Incubate the three plates at 37 &#176;C for 1 day, 3 days, or 5 days at 5% CO2.</li>
		<li>Add 10 &#181;L of CCK-8 solution to each well with the cells and incubate at 37 &#176;C for another 2 h.</li>
		<li>Measure the absorption values of each well with multi-mode microplate readers at 450 nm.</li>
		<li>Seed a known gradient of hMSCs onto a 96-well plate and incubate for 4 h. Use the CCK-8 assay to measure the absorption values of the known number of cells and generate a standard curve.</li>
		<li>Calculate cell proliferation by using the absorption standard curve.</li>
	</ol>
	</li>
	<li>Stability test 
	NOTE: A human TGF-&#946;1 targeted ELISA kit was used to determine the percentage release of TGF-&#946;1 from the JBNm in an agarose hydrogel.
	<ol>
		<li>Prepare 2% agarose by adding 400 mg of agarose powder to 20 mL of PBS and heating it up to 100 &#176;C to completely dissolve the agarose powder.</li>
		<li>Prepare the JBNm inside the cooled 2% agarose hydrogel.
		<ol>
			<li>Combine 10 &#181;L of 10 &#181;g/mL TGF-&#946;1, 40 &#181;L of 10 &#181;g/mL matrilin-3, 5 &#181;L of 1 mg/mL JBNts, and 195 &#181;L of PBS. Mix this solution with 250 &#181;L of 2% agarose, creating the JBNm hydrogel.</li>
		</ol>
		</li>
		<li>Add 500 &#181;L of PBS, using it as a release solution, and ensure to change it every 3 days. Keep every released solution, and let the sample sit for 15 days.</li>
		<li>Utilize an ELISA kit to test each of the captured release solutions, by following the manufacturer&#39;s instructions. 
		NOTE: By subtracting the amount of TGF-&#946;1 released in the PBS from the theoretical loading value of 100%, the remaining amount of TGF-&#946;1 can be determined.</li>
	</ol>
	</li>
	<li>Cell differentiation analysis with PCR26.
	<ol>
		<li>Prepare seven groups of samples with each sample containing 20 &#181;L of cell suspension (4 x 104 cells), 30 &#181;L of the specific solution (or PBS, depending on the sample group), and 50 &#181;L of 2 wt % agarose.
		<ol>
			<li>For the TGF-&#946;1 group, add 5 &#181;L of 10 &#181;g/mL TGF-&#946;1 to 25 &#181;L of PBS, resulting in a 1.6 &#181;g/mL solution.</li>
			<li>For the matrilin-3 group, add 20 &#181;L of 10 &#181;g/mL matrilin-3 to 10 &#181;L of PBS, resulting in a 6.7 &#181;g/mL solution.</li>
			<li>For the JBNt group, add 2.5 &#181;L of 1 mg/mL JBNts to 27.5 &#181;L of PBS, resulting in an 83.3 &#181;g/mL solution.</li>
			<li>For the J/T/M JBNm group, add 10 &#181;L of 10 &#181;g/mL matrilin-3 to 5 &#181;L of 10 &#181;g/mL TGF-&#946;1 and pipette up and down to mix the solution. Then, add 2.5 &#181;L of 1 mg/mL JBNts and pipette again. Finally, dilute with 2.5 &#181;L of PBS.</li>
			<li>For each control group, use 30 &#181;L of PBS as the sample.</li>
		</ol>
		</li>
		<li>Add 0.5 mL of cell culture medium to each well, making sure to replace it every 3 days.
		<ol>
			<li>For the positive control group, use a commercially available hMSC chondrogenic medium containing added TGF-&#946;1. This additional TGF-&#946;1 is equal to the amount in both the TGF-&#946;1 and J/T/M JBNm groups.</li>
			<li>For one of the negative control groups, use a commercial hMSC chondrogenic medium that does not contain TGF-&#946;1. For the other negative control group, use a DMEM cell culture medium containing 10% fetal bovine serum (FBS).</li>
			<li>For every other group, use a commercial hMSC chondrogenic cell culture medium without TGF-&#946;1.</li>
		</ol>
		</li>
		<li>Culture the samples for 15 days.</li>
		<li>Extract the samples&#39; RNA and perform PCR to study the differentiation of the samples as described previously26.</li>
	</ol>
	</li>
	<li>Immunostaining for type X collagen expression assay
	<ol>
		<li>Prepare seven agarose-based samples in the same way as the cell differentiation with the PCR method section (step 7.4).</li>
		<li>Culture the samples for 15 days.</li>
		<li>Fix the samples with 4% formaldehyde for 1 day, soak in 30% sucrose solution overnight, and then freeze overnight at -80 &#176;C with liquid nitrogen. Fix the samples using the optimal cutting temperature compound reagent (OCT), a blend of water-soluble glycols and resins, at -10 &#176;C.</li>
		<li>Operate a cryostat microtome to obtain 20 &#181;m thick frozen sections of each sample.</li>
		<li>Stain each section with an Anti-Collagen X antibody with 1:800 dilution and a fluorescent labeling secondary antibody diluted with PBS, according to the manufacturer&#39;s protocols.</li>
		<li>Observe each section with a confocal microscope, using an excitation of 488 nm and a magnification of 40x on the eyepiece.</li>
	</ol>
	</li>
</ol>

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T., Chen, Y., Liu, W., Chen, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+tissue+microenvironment+on+stem+cell-based+cartilage+repair.">The influence of tissue microenvironment on stem cell-based cartilage repair.</a> Annals of the New York Academy of Sciences. 1383 (1), 21-33 (2016).</li><li>Chen, Y., 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=Deficient+mechanical+activation+of+anabolic+transcripts+and+post-traumatic+cartilage+degeneration+in+matrilin-1+knockout+mice.">Deficient mechanical activation of anabolic transcripts and post-traumatic cartilage degeneration in matrilin-1 knockout mice.</a> PLoS One. 11 (6), 0156676 (2016).</li><li>Zhou, L., Zhang, W., Lee, J., Kuhn, L., Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+self-assembly+of+DNA-mimicking+nanotubes+to+form+a+layer-by-layer+scaffold+for+homeostatic+tissue+constructs.">Controlled self-assembly of DNA-mimicking nanotubes to form a layer-by-layer scaffold for homeostatic tissue constructs.</a> ACS Applied Material Interfaces. 13 (43), 51321-51332 (2021).</li><li>Belluoccio, D., Schenker, T., Baici, A., Trueb, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+human+matrilin-3+(MATN3).">Characterization of human matrilin-3 (MATN3).</a> Genomics. 53 (3), 391-394 (1998).</li><li>Yau, A., Yu, H., Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+detection+with+fluorescence-base+imaging+techniques+for+arthritis+diagnosis.">mRNA detection with fluorescence-base imaging techniques for arthritis diagnosis.</a> Journal of Rheumatology Research. 1 (2), 39-46 (2019).</li><li>Lee, J., Sands, I., Zhang, W., Zhou, L., Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-inspired+nanomaterials+for+enhanced+endosomal+escape.">DNA-inspired nanomaterials for enhanced endosomal escape.</a> Proceedings of the National Academy of Sciences. 118 (19), (2021).</li><li>Zhang, W., Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+engineering+of+DNA-inspired+Janus+base+nanomaterials.">Molecular engineering of DNA-inspired Janus base nanomaterials.</a> Juniper Online Journal Material Science. 5 (4), 555670 (2019).</li><li>Yau, A., Sands, I., Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-scale+surface+modifications+to+advance+current+treatment+options+for+cervical+degenerative+disc+disease+(CDDD).">Nano-scale surface modifications to advance current treatment options for cervical degenerative disc disease (CDDD).</a> Journal of Orthopedic Research and Therapy. 4 (9), 1147 (2019).</li><li>Mello, M. A., Tuan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+TGF-beta1+and+triiodothyronine+on+cartilage+maturation:+in+vitro+analysis+using+long-term+high-density+micromass+cultures+of+chick+embryonic+limb+mesenchymal+cells.">Effects of TGF-beta1 and triiodothyronine on cartilage maturation: in vitro analysis using long-term high-density micromass cultures of chick embryonic limb mesenchymal cells.</a> Journal of Orthopaedic Research. 24 (11), 2095-2105 (2006).</li><li>Shi, Y., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+TGF-beta+signaling+from+cell+membrane+to+the+nucleus.">Mechanisms of TGF-beta signaling from cell membrane to the nucleus.</a> Cell. 113 (6), 685-700 (2003).</li><li>Sands, I., Lee, J., Zhang, W., Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+delivery+via+DNA-inspired+janus+base+nanotubes+for+extracellular+matrix+penetration.">RNA delivery via DNA-inspired janus base nanotubes for extracellular matrix penetration.</a> MRS Advances. 5 (16), 815-823 (2020).</li><li>Zhou, L., Rubin, L. E., Liu, C., Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+interfering+RNA+(siRNA)-based+therapeutics+for+cartilage+diseases.">Short interfering RNA (siRNA)-based therapeutics for cartilage diseases.</a> Regenerative Engineering and Translational Medicine. 7 (3), 283-290 (2020).</li><li>Bi, H., 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=Deposition+of+PEG+onto+PMMA+microchannel+surface+to+minimize+nonspecific+adsorption.">Deposition of PEG onto PMMA microchannel surface to minimize nonspecific adsorption.</a> Lab on a Chip. 6 (6), 769-775 (2006).</li><li>Chen, Y., Webster, T. 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Part A. 91 (1), 296-304 (2009).</li><li>Sun, M., Lee, J., Chen, Y., Hoshino, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+nanoparticle+delivery+with+in+vitro+bio-engineered+microtissues.">Studies of nanoparticle delivery with in vitro bio-engineered microtissues.</a> Bioactive Materials. 5 (4), 924-937 (2020).</li><li>Yau, A., Lee, J., Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomaterials+for+protein+delivery+in+anticancer+applications.">Nanomaterials for protein delivery in anticancer applications.</a> Pharmaceutics. 13 (2), 155 (2021).</li></ol>

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

The goal of this study is to develop a biomimetic scaffold platform, the JBNm, to overcome the limitations of conventional tissue constructs that rely on cell culture environments to mediate cell differentiation. The JBNm is a layer-by-layer structure scaffold for a self-sustainable cartilage tissue construct. The innovative design is based on novel DNA-inspired nanomaterials, the JBNts. The JBNm, composed of JBNts30, TGF-&#946;1, and matrilin-3, is assembled through a novel layer-by-layer technique where the self-assembly of the scaffold was controlled at the molecular level. The assembly of JBNm was observed and characterized with zeta-potential measurement, UV-Vis absorption, TEM, and fluorescence spectral analysis.
The UV-Vis spectra in Figure 2B (step 3) have demonstrated the formation of the hierarchical layer-by-layer interior of the JBNm. A difference in absorbance peaks can be observed due to a difference in binding affinity. More major interactions occur between JBNts and matrilin-3 than between JBNts and TGF-&#946;1 indicating that JBNts mimic collagen proteins in terms of their fibrous morphology and lysine surface chemistry. This technique is necessary to observe the binding of JBNts to matrilin-3 rather than TGF-&#946;1, allowing for the encapsulation of TGF-&#946;1 and therefore providing a slow release of TGF-&#946;1 into the surrounding tissue. The most critical step in the development of the JBNm is the combination of proteins with the JBNts. TGF-&#946;1 and matrilin-3 must be added prior to the addition of JBNts to observe the full effect of the FRET phenomenon between the labeled matrilin-3 and TGF-&#946;1. The limitation of this technique stems from the incorrect addition sequence of proteins and nanotubes, resulting in varied measurements. This is an important step for the formation of JBNm for future studies, and, therefore, will be applied to future JBNm fabrications and studies.
Matrilin-3 is a highly negative protein, whereas TGF-&#946;1 is a slightly neutral protein, as seen in Figure 2A. The charge difference was observed to determine the binding of these proteins. From the negatively charged state of matrilin-3, zeta-potential increased to a near-neutral value after the addition of TGF-&#946;1 (Figure 2A). This step is important to determine the first inner layer of the biomimetic scaffold, and to indicate that the combination of both proteins is through charge interaction. After the addition of JBNts, which is the second step of the JBNm fabrication, the zeta-potential of JBNm was measured at ~10 &#177; 5 mV (Figure 2A). Because JBNts are highly positive, the charge of JBNm was observed to increase as expected. Determination of charges with zeta-potential is thus important to observe the formation of JBNm step-by-step via charge interactions. Similar to UV-Vis spectroscopy, the sequence of addition is important in this step, and incorrect addition order may result in varied measurements. Similarly, it is important to pipette the mixture up and down prior to measurement.
Next, TGF-&#946;1 and matrilin-3 are labeled so that the development of JBNm can be observed with confocal microscopy and fluorescence spectral analysis with multimode microplate readers. The samples were excited with a 488 nm laser and showed emission peaks at 520 nm and 570 nm (Figure 3). No emission peak was observed for JBNts alone because they are not labeled. FRET occurred from the TGF-&#946;1 donor to the matrilin-3 acceptor, thereby reducing the emission peak at 520 nm and increasing the emission peak at 570 nm; the two components are 10 nm apart in distance, allowing the FRET phenomenon to occur. Therefore, the assembly of JBNm is based on spatial (i.e., physical distance) processes together with charge interactions. The most critical step in the development of JBNm is the addition sequence; TGF-&#946;1 and matrilin-3 must be added prior to the addition of JBNts to observe the full effect of the FRET phenomenon. Magnification of at least 40x on the eyepiece is required to observe the fluorescent JBNm. The limitation of this technique is that the JBNts are not labeled and, therefore, could not be observed with confocal microscopy. However, the protein labeling technique can be applied to label JBNt with some modifications for future applications.
JBNm can aid cell functions, such as cell adhesion and cell proliferation. In this study, hMSCs were cultured (starting with 5,000 cells) on different materials (JBNm, JBNts, TGF-&#946;1, matrilin-3, and negative controls without additives) to compare the cell numbers after incubation for 1, 3, and 5 days using CCK-8 solution, following the exact manufacturer&#39;s protocols to determine cell proliferation. JBNm, especially in the presence of TGF-&#946;1, and TGF-&#946;1 alone showed significant cell proliferation compared to the other three groups. JBNts mimic collagen in the ECM morphologically while matrilin-3 is a cartilage-specific protein, resulting in increased cell proliferation activity after incubation at 37 &#176;C for 3 and 5 days (Figure 5). The JBNm has shown great potential to serve as an injectable and biomimetic tissue engineering scaffold to overcome the limitations of traditional cell constructs for various future applications as biomaterials29,31. JBNm mimics the cartilage ECM morphologically, providing adhesion sites and allowing the release of pro-chondrogenic differentiative factors into the microenvironment, such as TGF-&#946;132,33. The ability of the JBNm to incorporate TGF-&#946;1 in the inner layer of the scaffold prevents it from leaking to undesired areas, thus improving the effectiveness of the scaffold (Figure 4 and Figure 5). Many hMSCs are seen to cluster along the JBNm scaffold, while the cells are sparsely distributed in matrilin-3, TGF-&#946;1, and negative control groups (Figure 4). The morphology of the cells was also observed, indicating excellent affinity with JBNm surfaces. These precisely designed biomaterials prevent the rapid release of incorporated bioactive molecules into the cell culture environment and improve the self-sustainability of the tissue constructs within the matrix34. A limitation of this technique is that the starting cell numbers are critical for determining the proliferation of cells. A starting cell number of 5,000 was found most optimal for this study. A standard curve is also needed to determine the cell number by plotting a known number of cells and measuring them in the multimode microplate reader. This technique can be applied in the future as a standardized method to determine cell proliferation across all studies relating to JBNm scaffolds.
hMSCs were cultured in 3D agarose pellets with and without JBNm to determine the long-term function study for 15 days. After 15 days, total RNA was extracted from the hMSCs in the agarose pellets, containing positive control (pellets were supplied with fresh TGF-&#946;1 every time medium was changed), JBNm, JBNts, matrilin-3, TGF-&#946;1, and no additives. Real-time qPCR was carried out for gene analysis, testing out for chondrogenic differentiation markers, Aggrecan (ACAN), and hypertrophy marker type X collagen (COL X). ACAN expression in the JBNm group increased significantly compared to other groups, demonstrating that JBNm promotes stem cell chondrogenic differentiation significantly while inhibiting COL X. Meanwhile, the positive control has enhanced chondrogenesis, as noted in the increase in ACAN, and also hypertrophy of the differentiated cell26. A further limitation of this study is that the RNA extraction is from the cells embedded in a hydrogel. The total RNA obtained in this protocol, therefore, was low in concentration and purity. To overcome this limitation, more samples were cultured and extracted to obtain the optimal concentration and purity for the next step, real-time qPCR. While qPCR is important for the study, minor modification of this technique is necessary for future studies to ensure efficient sample extraction without sacrificing a large number of samples.
A stability test was performed with a human TGF-&#946;1 ELISA kit to test the release of protein from the JBNm hydrogel. In this study, TGF-&#946;1 is expected to be encapsulated in the J/T/M JBNm, and therefore no TGF-&#946;1 release should be observed (i.e., the theoretical loading value is 100%). A limitation of this step is that all TGF-&#946;1 is assumed to be encapsulated into the JBNm layer-by-layer scaffold. After 15 days, solutions released from the hydrogel are collected and tested with the kit, following the manufacturer&#39;s protocols. Then, the amount of TGF-&#946;1 was calculated by subtracting the amount of the released TGF-&#946;1 in PBS from the theoretical loading value. Because TGF-&#946;1 was encapsulated in the inner layer of the JBNm, the slow release of the protein was anticipated. The study demonstrated that the TGF-&#946;1 is localized within the JBNm scaffold and does not release rapidly into the surrounding areas26.
This innovative layer-by-layer JBNm cartilage tissue construct was realized by highly organized and controlled self-assembly on the molecular level. The TGF-&#946;1 is confined in the inner layer of the matrix fibers, preventing its leakage to undesired locations, and promoting localized chondrogenesis simultaneously. In addition, matrilin-3 is localized in the outer layer of the matrix fibers, creating an anti-hypertrophic microenvironment35. JBNts have been shown to not only serve as a structural scaffold backbone but also enhance stem cell anchorage and adhesion to localize cells along the matrix fibers30,36. As for future work, the layer-by-layer design of the JBNt-based scaffold will be customized for applications in various tissues37,38,39.

Scaffolds in tissue engineering play a vital role in providing structural support for cell attachment and subsequent tissue development1. Typically, conventional tissue constructs without any scaffolding rely on the cell culture environment and added growth factors to mediate cell differentiation. Furthermore, this addition of bioactive molecules into scaffolds is often the preferred approach in guiding cell differentiation and function2,3. Some scaffolds can mimic the biochemical microenvironment of native tissues independently, while others can directly influence cell functions via growth factors. However, researchers often encounter challenges in selecting scaffolds that could positively affect cell adhesion, growth, and differentiation, while providing optimal structural support and stability over a long period4,5. The bioactive molecules are often loosely bound to the scaffold leading to rapid release of these proteins upon implantation, resulting in their release in undesired locations. This culminates in side effects on tissues or cells that were not intentionally targeted6,7.
Scaffolds are typically made of polymeric materials. The Janus base nano-matrix (JBNm) is a biomimetic scaffold platform created with a novel layer-by-layer method for self-sustainable cartilage tissue construct8. These novel DNA-inspired nanotubes have been named Janus base nanotubes (JBNts), as they properly mimic the structure and surface chemistry of collagen found in the extracellular matrix (ECM). With the addition of bioactive molecules, such as matrilin-3 and Transforming Growth Factor Beta-1 (TGF-&#946;1), the JBNm can create an optimal microenvironment which can then stimulate desired cell and tissue functionality9.
JBNts are novel nanotubes derived from synthetic versions of the nucleobase adenine and thymine. The JBNts are formed through self-assembly10; six synthetic nucleobases bond to form a ring, and these rings undergo &#960;-&#960; stacking interactions to create a nanotube 200-300 &#181;m in length11. These nanotubes are structurally similar to collagen proteins; by mimicking an aspect of the native cartilage microenvironment, JBNts have been shown to provide a favorable attachment site for chondrocytes and human mesenchymal stem cells (hMSCs)11,12,13,14. Because the nanotubes undergo self-assembly and do not require any sort of initiator (such as UV-light), they show exciting potential as an injectable scaffold for hard-to-reach defect areas15.
Matrilin-3 is a structural extracellular matrix protein found in cartilage. This protein plays a significant role in chondrogenesis and proper cartilage function16,17. Recently, it has been included in biomaterial scaffolds, encouraging chondrogenesis without hypertrophy9,18,19. By including this protein in the JBNm, cartilage cells are attracted to a scaffold that contains similar components to that of its native microenvironment. Additionally, it has been shown that matrilin-3 is needed for proper TGF-&#946;1 signaling within chondrocytes20. Growth factors function as signaling molecules, causing specific growth of a certain cell or tissue. Thus, to achieve optimal cartilage regeneration, matrilin-3 and TGF-&#946;1 are essential components within the JBNm. The addition of TGF-&#946;1 into the layer-by-layer scaffold can further promote cartilage regeneration in a tissue construct. TGF-&#946;1 is a growth factor employed to encourage the healing process of osteochondral defects, encouraging chondrocyte and hMSC proliferation and differentiation21,22. Thus, TGF-&#946;1 plays a key role in the cartilage regeneration JBNm (J/T/M JBNm)23, encouraging proper growth especially when it is localized within the JBNm layers.
As mentioned previously, growth factors are typically assembled on the outside of scaffolds with no specific methods of incorporation. Here, with the precisely designed nano-architecture of the biomaterials, the JBNm was developed for specific targeting of intended cells and tissues. The JBNm is composed of TGF-&#946;1 adhered on JBNt surfaces in the inner layer and matrilin-3 adhered on JBNt surfaces in the outer layer24,25. The incorporation of TGF-&#946;1 in the inner layer of the layer-by-layer structure allows for a highly localized microenvironment along the JBNm fibers, creating a homeostatic tissue construct with a much slower release of the protein12. The injectability of the JBNm makes it an ideal cartilage tissue construct for various future biomaterial applications26.

<table><tbody><tr><td>10 % Normal Goat Serum</td><td>Thermo Fisher</td><td>50062Z</td><td>Agent used to block nonspecific antibody binding actions during staining.</td></tr><tr><td>24-well plate</td><td>Corning</td><td>07-200-740</td><td>24-well plate used for comparative cell culture.</td></tr><tr><td>384-Well Black Untreated Plate</td><td>Thermo Fisher</td><td>262260</td><td>384-well plate used for absorption measurements.</td></tr><tr><td>8-well chambered coverglass</td><td>Thermo Fisher</td><td>155409PK</td><td>8-well coverglass used for comparative cell culture.</td></tr><tr><td>96-well flat bottom</td><td>Corning</td><td>07-200-91</td><td>96-well plate used for comparative cell culture.</td></tr><tr><td>96-Well Plate non- treated</td><td>Thermo Fisher</td><td>260895</td><td>96-well plate used for comparative cell culture and analysis.</td></tr><tr><td>Agarose Gel</td><td>Sigma-Aldrich</td><td>A9539</td><td>Hydrogel used for cell culture.</td></tr><tr><td>Agarose Gel</td><td>Sigma Aldrich</td><td>A9539</td><td>Hydrogel used as an environment for cell culture.</td></tr><tr><td>Alexa Fluor Microscale Protein Labeling Kit</td><td>Thermo Fisher</td><td>A30006 (488) and A30007 (555)</td><td>Fluorescent dye used to label proteins.</td></tr><tr><td>Anti-Collagen X Antibody</td><td>Thermo Fisher</td><td>41-9771-82</td><td>Antibody used to stain collagen-X.</td></tr><tr><td>Bio-Rad PCR Machine</td><td>Bio-Rad</td><td></td><td>Equipment used to perform PCR on samples.</td></tr><tr><td>C28/I2 Chondrocyte Cell Line</td><td></td><td></td><td>Cells used to analyze proliferative abilities of various samples.</td></tr><tr><td>Cell Counting Kit 8</td><td>Milipore Sigma</td><td>96992</td><td>Cell proliferation assay.</td></tr><tr><td>Cell Profiler</td><td>Broad Institute</td><td></td><td>Software used to analyze cell images.</td></tr><tr><td>Cryostat Microtome</td><td></td><td></td><td>Equipment used to produce thin segments of samples for use in staining and microscopy.</td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>D1306</td><td>Blue fluorescent stain that binds to adenine-thymine DNA regions.</td></tr><tr><td>Disposable cuvettes</td><td>FISHER Scientific</td><td>14-955-128</td><td>Container used for spectrophotometry.</td></tr><tr><td>DMEM Cell Culture Medium</td><td>Thermo Fisher</td><td>10566032</td><td>Media used to support cellular growth.</td></tr><tr><td>Fetal Bovine Serum</td><td>GIBCO</td><td>A4766801</td><td>Serum used in cell culture medium to support cell growth.</td></tr><tr><td>Fluoromount-G Mounting Medium</td><td>Thermo Fisher</td><td>00-4958-02</td><td>Solution used to mount slides for immunostaining.</td></tr><tr><td>Formaldehyde</td><td></td><td></td><td>Compound used to fix samples prior to microtoming.</td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody</td><td>Thermo Fisher</td><td>A16110</td><td>Antibody used for protein staining.</td></tr><tr><td>Human Mesenchymal Stem Cells</td><td>LONZA</td><td>PT-2501</td><td>Cells used to analyze differentiative abilities of various samples.</td></tr><tr><td>Human Mesenchymal Stem Chondrogenic Medium</td><td>LONZA</td><td>PT-3003</td><td>Cell medium used to promote chondrogenic differentiation.</td></tr><tr><td>ImageJ</td><td>National Institutes of Health</td><td></td><td>Image analysis software used in conjunction with microscopy.</td></tr><tr><td>itaq Universal SYBR Green One-Step Kit</td><td>BioRad</td><td>1725150</td><td>Kit used for PCR.</td></tr><tr><td>Janus-base nanotubes (JBNts)</td><td></td><td></td><td>Nanotube made from synthetic nucleobases to act as cell scaffolding tool.</td></tr><tr><td>LaB6 20-120 kV Transmission Electronic Microscope</td><td>Tecnai</td><td></td><td>Equipment used to perform transmission electron microscopy on a sample.</td></tr><tr><td>MATLAB</td><td>MathWorks</td><td></td><td>Statistical software used for modeling and data analysis.</td></tr><tr><td>Matrilin-3</td><td>Fisher Scientific</td><td>3017MN050</td><td>Structural protein used as adhesion sites for chondrocytes.</td></tr><tr><td>NanoDrop Spectrophotometer</td><td>Thermo Fisher</td><td></td><td>Equipment used to measure absorption values of a sample.</td></tr><tr><td>Nikon A1R Spectral Confocal Microscope</td><td>Nikon</td><td>A1R HD25</td><td>Confocal microscope used to analyze samples.</td></tr><tr><td>Number 1.5 Chamber Coverglass</td><td>Thermo Fisher</td><td>152250</td><td>Environment for sterile cell culture and imaging.</td></tr><tr><td>Optimal Cutting Temperature Compound Reagent</td><td></td><td></td><td>Compound used to embed cells prior to microtoming.</td></tr><tr><td>Paraformaldehyde</td><td>Thermo Scientific</td><td>AAJ19943K2</td><td>Compound used to fix cells.</td></tr><tr><td>PDC-32G Plasma Cleaner</td><td>Harrick Plasma</td><td></td><td>Cleaner used to prepare grids prior to transmission electron microscopy.</td></tr><tr><td>penicillin-streptomycin</td><td>GIBCO</td><td>15-140-148</td><td>Antibiotic agent used to discourage bacterial growth during cell culture.</td></tr><tr><td>Phosphate Buffered Saline</td><td>Thermo Fisher</td><td>10010023</td><td>Solution used to wash cell medium and act as a buffer during experimentation.</td></tr><tr><td>Rhodamine-phalloidin</td><td>Invitrogen</td><td>R415</td><td>F-Actin red fluorescent dye.</td></tr><tr><td>Rneasy Plant Mini Kit</td><td>QIAGEN</td><td>74904</td><td>Kit used to filter and homogenize samples during RNA extraction.</td></tr><tr><td>Sucrose Solution</td><td></td><td></td><td>Solution used to process samples prior to microtoming.</td></tr><tr><td>TGF beta-1 Human ELISA Kit</td><td>Invitrogen</td><td>BMS249-4</td><td>Assay kit used to determine the presence of TGF-&amp;beta;1 in a sample.</td></tr><tr><td>TGF-&amp;beta;1</td><td>PEPROTECH</td><td>100-21C</td><td>Growth factor used for the stimulation of chondrogenic differentiation and proliferation.</td></tr><tr><td>Triton-X</td><td>Invitrogen</td><td>HFH10</td><td>Compound used to lyse cells not fixed during staining process.</td></tr><tr><td>TRIzol Reagent</td><td>Thermo Fisher</td><td>15596026</td><td>Reagent used to isolate RNA.</td></tr><tr><td>Zetasizer Nano ZS</td><td>Malvern Panalytical</td><td></td><td>Equipment used to measure zeta-potential values of a sample.</td></tr></tbody></table>

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.

Following protocol, JBNts were successfully synthesized and characterized with UV-Vis absorption and TEM. The JBNm is an injectable solid scaffold that undergoes a rapid biomimetic process. After JBNts were added to a mixture of TGF-&#946;1/matrilin-3 solution in a physiological environment, a solid white-mesh scaffold was formed indicating the successful assembly of JBNm, as seen in Figure 1. This was demonstrated in the characterization methods.
Under physiological conditions, matrilin-3 is negatively charged due to its isoelectric point27 (Figure 2A). After the addition of the TGF-&#946;1 solution to the matrilin-3 solution, the zeta-potential of the TGF-&#946;1/matrilin-3 compound increased to a near-neutral value, indicating that the two proteins were bound via charge interactions. As seen in Figure 2A, the zeta-potential of JBNm is the highest among the three groups owing to the JBNts, as the isoelectric point of the lysine side chain is around 9.74 in a physiological environment. The increase in zeta-potential values of the JBNm indicates the successful assembly of its layer-by-layer structure.
UV-Vis absorption spectra (Figure 2B) clarify the formation of the hierarchical layer-by-layer interior structure of the JBNm. The aromatic rings of the lysine side chains and the JBNts contributed to the two absorption peaks at 220 nm and 280 nm, respectively. The decrease in absorption value was observed after the addition of TGF-&#946;1, indicating that binding occurs between TGF-&#946;1 and JBNts. After the addition of JBNts to matrilin-3, a more obvious decrease in the absorption intensity of the peaks was observed, once again indicating successful binding between matrilin-3 and JBNts. Similarly, after the addition of JBNts to a mixture of TGF-&#946;1/matrilin-3, the JBNm was formed, and the absorption peaks decreased in intensity. The absorption peak of JBNm is closer to the matrilin-3/JBNts line than the TGF-&#946;1/JBNts line, indicating that the JBNts prefer to bind to matrilin-3, forming a layer-by-layer outer structure with matrilin-3 while TGF-&#946;1 resides in the inner layer. TEM is used to characterize the morphology of the JBNts and JBNm (Figure 2C). After combining with proteins, thick bundles of JBNm were observed forming a scaffold structure.
Fluorescence microscopy has confirmed the presence of the layer-by-layer structure (Figure 3A) and demonstrated the cross-section of the JBNm. After labeling TGF-&#946;1 and matrilin-3, it was observed that the red fluorescent matrilin-3 envelops the JBNt bundles, forming the outer layer of the JBNm. In Figure 3B, the green-fluorescent TGF-&#946;1 formed an inner layer, contributing to the capability of storing growth factors and allowing the localization of TGF-&#946;1. Figure 3C displays the fluorescence resonance energy transfer (FRET) process between the fluorescent dye-labeled proteins as characterized by fluorescence spectra, with emission peaks at 520 nm and 570 nm for labeled TGF-&#946;1 and matrilin-3 groups, respectively28. After the addition of JBNts according to the protocol, the layer-by-layer structure is formed; peaks were observed at both 520 nm and 570 nm for the JBNm group, indicating a successful binding and assembly of the proteins and JBNts.
Additionally, the effect of the JBNm on hMSCs adhesion and cell proliferation was explored. As shown in Figure 4, the cell adhesion density of the JBNm coated on the surface of the chambered coverglasses was determined. The JBNm showed hMSCs clustered along itself (Figure 4A), whereas fewer cells adhered to JBNts. However, for the other groups, the hMSCs were evenly distributed without alignment as compared to the JBNm group. The alignment of cells, as well as the size of the cells on the JBNm, demonstrated that JBNts played a role in cell adhesion, while the proteins in the JBNm increased the affinity of cell adhesion (Figure 4B,C).
After day 1 of cell culture with the JBNm, JBNts, matrilin-3 alone, TGF-&#946;1 alone, and negative control, the JBNm and TGF-&#946;1 groups showed significant cell proliferation when compared to the other groups. When the cell culture duration was increased to 3 and 5 days, the JBNm and TGF-&#946;1 groups demonstrated even more increased cell proliferation, as seen in Figure 5. A long-term function study was performed to determine the differentiation of hMSCs with JBNm and without JBNm (negative control). After 15 days, an enhanced Alcian blue stain was observed, indicating chondrogenesis of hMSCs growing alongside JBNm26. Thus, the cells preferred the JBNts of the JBNm. This was likely due to its DNA-mimicking structure and ability to be disassembled into small-molecule units, the latter of which can be triggered by a low pH or sufficient enzymatic activity (such as uptake by cells)14,29.
Video 1: Video recording of JBNm assembly. This recording depicts the formation of the JBNts, matrilin-3, and TGF-&#946;1 into the JBNm. This figure has been modified from Zhou et al. (2021)26. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63984/am1c13345_si_002.zip" target="_blank">Please click here to download this Video.</a>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63984/63984fig01.jpg" /> 
Figure 1: Schematic illustration of the chemical structure of JBNts, the J/T/M JBNm, and the J/T/M JBNm&#39;s chondrogenic ability. (A) The chemical structure of the JBNts, highlighting their formation from monomer to rosette ring to JBNt. (B) The components that make up the J/T/M JBNm, as well as how they assemble into the JBNm. (C) Schematic of culturing human mesenchymal stem cells on the J/T/M JBNm. This figure has been modified from Zhou et al. (2021)26. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63984/63984fig01large.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/63984/63984fig02.jpg" /> 
Figure 2: Material characterization of the J/T/M JBNm. (A) Zeta-potential spectra of matrilin-3 alone, the TGF-&#946;1/matrilin-3 compound, and the J/T/M JBNm. (B) UV-Vis absorption spectra of JBNts alone, matrilin-3 alone, the TGF-&#946;1 alone, matrilin-3/JBNts compound, the TGF-&#946;1/JBNts compound, and the J/T/M JBNm. (C) Images of JBNts alone and the J/T/M JBNm obtained from transmission electron microscopy (TEM). This figure has been modified from Zhou et al. (2021)26. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63984/63984fig02large.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/63984/63984fig03.jpg" /> 
Figure 3: Fluorescent confocal imaging and fluorescence spectra of J/T/M JBNm. (A) 3D confocal image of the J/T/M JBNm, with red-fluorescent-labeled matrilin-3 and green-fluorescent-labeled TGF-&#946;1. (B) 2D confocal images of J/T/M JBNm, with red-fluorescent-labeled matrilin-3, green-fluorescent-labeled TGF-&#946;1, and a merged version. (C) Fluorescence spectra of various compounds to characterize the FRET process between the labeled proteins. This figure has been modified from Zhou et al. (2021)26. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63984/63984fig03large.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/63984/63984fig04.jpg" /> 
Figure 4: Analysis of human mesenchymal stem cell (hMSC) culture. (A) Optical microscopy images of hMSCs with TGF-&#946;1, matrilin-3, and JBNts, cultured on an agarose gel. (B) Confocal microscopy images of hMSCs cultured on the chambered cover glass coated with a variety of JBNm components. (C) Graph of the number of cells adhered per square millimeter for each group. Error bars denote standard deviations. (D) Graph of cell major axis length in &#181;m per group. Error bars denote standard deviations. Note: N &#8805; 3, *P&#60; 0.05, **P&#60; 0.01, ***P&#60; 0.001, ****P&#60; 0.0001 (compared against negative controls, NC). This figure has been modified from Zhou et al. (2021)26. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63984/63984fig04large.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/63984/63984fig05.jpg" /> 
Figure 5: Comparison of cell numbers for various groups between days 1, 3, and 5. Cell number statistics of hMSCs after being cultured with different materials on days 1, 3, and 5. Error bars denote standard deviations. Note: N = 6, *P &#60; 0.05, **P &#60; 0.01, ***P &#60; 0.001, ****P &#60; 0.0001. This figure has been modified from Zhou et al. (2021)26. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63984/63984fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Fabrication and Characterization of Layer-By-Layer Janus Base Nano-Matrix to Promote Cartilage Regeneration at JoVE.com

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

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 ...

fabrication-characterization-layer-layer-janus-base-nano-matrix-to

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

2.0K Views.  University of Connecticut. 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-β1) sequentially. The JBNm was fabricated and characterized; additionally, it displayed excellent bioactivity, encouraging cell functions such as adhesion, proliferation, and differentiation. 

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

Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated cartilage lesions lead to ongoing pain, negatively affect the quality of life and predispose for osteoarthritis. During the last decades, several surgical techniques have been developed to treat such lesions. However, until now it was not possible to achieve a full repair in terms of covering the defect with hyaline articular cartilage or of providing satisfactory long-term recovery 2-4. Therefore, articular cartilage injuries remain a prime target for regenerative techniques such as Tissue Engineering. In contrast to other surgical techniques, which often lead to the formation of fibrous or fibrocartilaginous tissue, Tissue Engineering aims at fully restoring the complex structure and properties of the original articular cartilage by using the chondrogenic potential of transplanted cells. Recent developments opened up promising possibilities for regenerative cartilage therapies.
The first cell based approach for the treatment of full-thickness cartilage or osteochondral lesions was performed in 1994 by Lars Peterson and Mats Brittberg who pioneered clinical autologous chondrocyte implantation (ACI) 5. Today, the technique is clinically well-established for the treatment of large hyaline cartilage defects of the knee, maintaining good clinical results even 10 to 20 years after implantation 6. In recent years, the implantation of autologous chondrocytes underwent a rapid progression. The use of an artificial three-dimensional collagen-matrix on which cells are subsequently replanted became more and more popular 7-9.
MACT comprises of two surgical procedures: First, in order to collect chondrocytes, a cartilage biopsy needs to be performed from a non weight-bearing cartilage area of the knee joint. Then, chondrocytes are being extracted, purified and expanded to a sufficient cell number in vitro. Chondrocytes are then seeded onto a three-dimensional matrix and can subsequently be re-implanted. When preparing a tissue-engineered implant, proliferation rate and differentiation capacity are crucial for a successful tissue regeneration 10. The use of a three-dimensional matrix as a cell carrier is thought to support these cellular characteristics 11.
The following protocol will summarize and demonstrate a technique for the isolation of chondrocytes from cartilage biopsies, their proliferation in vitro and their seeding onto a 3D-matrix (Chondro-Gide, Geistlich Biomaterials, Wollhusen, Switzerland). Finally, the implantation of the cell-matrix-constructs into artificially created chondral defects of a rabbit's knee joint will be described. This technique can be used as an experimental setting for further experiments of cartilage repair.

An experimental technique for the treatment of chondral defects in the rabbit's knee joint is described. The implantation of autologous chondrocytes seeded on a matrix is a well-accepted method for the remodeling and repair of articular cartilage lesions providing satisfying long-term results. Matrix-assisted autologous chondrocyte transplantation (MACT) offers a standardized and clinically established implantation method.

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated ...

Medicine

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not occur. With conventional templates, the absence of internal micro-channels results in the lack of cell infiltration, distribution, and inhabitance deep inside the templates. Hence, a highly porous and uniformly interconnected trabecular-bone-like template with micro-channels (biogenic microenvironment template; BMT) has been developed to address these obstacles. The novel BMT was created by innovative concepts (capillary action) and fabricated with a sponge-template coating technique. The BMT consists of several structural components: inter-connected primary-pores (300-400 &#181;m) that mimic pores in trabecular bone, micro-channels (25-70 &#181;m) within each trabecula, and nanopores (100-400 nm) on the surface to allow cells to anchor. Moreover, the BMT has been documented by mechanical test study to have similar mechanical strength properties to those of human trabecular bone (~3.8 MPa)12.
The BMT exhibited high absorption, retention, and habitation of cells throughout the bridge-shaped (&#928;) templates (3 cm height and 4 cm length). The cells that were initially seeded into one end of the templates immediately mobilized to the other end (10 cm distance) by capillary action of the BMT on the cell media. After 4 hr, the cells homogenously occupied the entire BMT and exhibited normal cellular behavior. The capillary action accounted for the infiltration of the cells suspended in the media and the distribution (active migration) throughout the BMT. Having observed these capabilities of the BMT, we project that BMTs will absorb bone marrow cells, growth factors, and nutrients from the periphery under physiological conditions.
The BMT may resolve current limitations via rapid infiltration, homogenous distribution and inhabitance of cells in large, volumetric templates to repair massive skeletal defects.

A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels.

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not ...

Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to the success of this approach is a scaffold that is able to conformally fit within an irregular defect while also having the requisite biodegradability, pore interconnectivity and bioactivity. By nature of their shape recovery and fixity properties, shape memory polymer (SMP) scaffolds could achieve defect &#8220;self-fitting.&#8221; In this way, following exposure to warm saline (~60 &#186;C), the SMP scaffold would become malleable, permitting it to be hand-pressed into an irregular defect. Subsequent cooling (~37 &#186;C) would return the scaffold to its relatively rigid state within the defect. To meet these requirements, this protocol describes the preparation of SMP scaffolds prepared via the photochemical cure of biodegradable polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method. A fused salt template is utilized to achieve pore interconnectivity. To realize bioactivity, a polydopamine coating is applied to the surface of the scaffold pore walls. Characterization of self-fitting and shape memory behaviors, pore interconnectivity and in vitro bioactivity are also described.

Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Novel Process for 3D Printing Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1,2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 &#176;C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 &#176;C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...

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.

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 ...