Name: fabrication and characterization of layer-by-layer janus base nano-matrix to promote cartilage regeneration
Uploaded: 2022-07-06T13:20:00
Description: Watch this Scientific Journal Video about Fabrication and Characterization of Layer-By-Layer Janus Base Nano-Matrix to Promote Cartilage Regeneration at JoVE.com

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 ...

<ol>
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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 techniq...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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-&#946;1 in a sample.</td>
		</tr>
		<tr>
			<td>TGF-&#946;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 physiologi...

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 ...

This protocol shows how we assembled and characterized the layer by layer JBNm at a molecular level. JBNm encapsulates TGF-B1, and prevents its release in the surrounding tissue, thus promoting localized chondrogenesis. The formation of the JBNm is standardized, allowing different JBNms to be formed for future applications.The unique layer by layer structure allows for growth factor encapsulation, creating a steady localized release, which avoids hypertrophy, and sustains growth. JBNm provides a homeostatic microenvironment for cartilage tissue regeneration, within a confined location, due to its injectability, which can be used for different scenarios, like irregularly shaped fractures or cavities. To begin, add eight microliters of one milligram per milliliter TGF-B1, suspended in water to 32 microliters of one milligram per milliliter Matrilin-3 suspended in water.Pipette to ensure proper mixing of these proteins. Add 80 microliters of one milligram per milliliter. Janus Base Nanotubes, or JBNts suspended in water, to the TGF-B1, Matrilin-3 solution.Pipette repeatedly to ensure proper blending. For the JBNt group, add five microliters of one milligram per milliliter, JBNts to 50 microliters of water to make a 90.9 microgram per milliliter solution. For the Matrilin-3 group, add 40 microliters of 10 micrograms per milliliter Matrilin-3 to 15 microliters of water, to make a 7.3 microgram per milliliter solution.For the TGF-B1 group, add 10 microliters of 10 micrograms per milliliter, of TGF-B1 to 45 microliters of water, to make a 1.8 microgram per milliliter solution. For the Janus Base Nano-Matrix or JBNm group, add 40 microliters of 10 micrograms per milliliter, Matrilin-3 to 10 microliters of 10 micrograms per milliliter TGF-B1. Agitate to ensure proper mixing.Then, add five microliters of one milligram per milliliter JBNts to the solution, repeatedly pipetting to mix the sample. Using a spectrophotometer, measure the absorption spectrum of each group. Label the TGF-B1 by using a protein labeling kit, following the manufacturer's instructions.Add 20 microliters of 20 micrograms per milliliter, labeled TGF-B1, to 25 microliters of water, to make an 8.9 microgram per milliliter test solution. Label the Matrilin-3 by using a separate labeling kit. Add 20 microliters of 80 micrograms per milliliter labeled Matrilin-3 to 25 microliters of water, to make a 36 microgram per milliliter test solution.Mix 20 microliters of 80 micrograms per milliliter labeled Matrilin-3 with 20 microliters of 20 micrograms per milliliter labeled TGF-B1, and five microliters of water, resulting in a labeled TGF-B1, Matrilin-3 compound. Pipette the mixture several times to mix. Add five microliters of one milligram per milliliter JBNts to 40 microliters of water, resulting in a 111 microgram per milliliter solution.Then add five microliters of one milligram per milliliter JBNts to the labeled TGF-B1, Matrilin-3 solution, and repeatedly pipette to mix the compound. Transfer each sample group to the well of a black 384 well plate. Load the plate into a multi-mode microplate reader, and take measurements at excitation wavelengths of 488 and 555 nanometers following the manufacturer's protocols.Under physiological conditions, the zeta potential spectrum of Matrilin-3 was negatively charged due to its isoelectric point. After the addition of the TGF-B1 to the Matrilin-3, the zeta potential of the TGF-B1 Matrilin-3 compound increased to a near neutral value. The zeta potential of JBNm was the highest among the three groups.The UV visible absorption spectra confirmed 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 absorption peaks, at 220 and 280 nanometers respectively. TEM was used to characterize the morphology of the JBNts and JBNm. After combining with proteins, thick bundles of JBNm were observed forming a scaffold structure.Fluorescence microscopy confirmed the presence of the layer by layer structure, and demonstrated the cross section of the JBNm. After labeling the red fluorescent Matrilin-3 enveloped the JBNt bundles, and formed the outer layer of the JBNm, while the green fluorescent TGF-B1 formed an inner layer. The fluorescence resonance energy transfer displayed emission peaks at 520 nanometers for labeled TGF-B1 groups, and 570 nanometers for Matrilin-3 groups.The effect of JBNm on HMSC's adhesion and cell proliferation was explored. The JBNm showed HMSCs clustered along with itself, whereas, fewer cells adhered to JBNts. The alignment of cells, and the size of the cells on the JBNm demonstrated that JBNts played a role in cell adhesion, while the proteins increased the affinity of cell adhesion.After day one of cell culture, JBNm and TGF-B1 groups showed significant cell proliferation compared to the other groups. When the cell structure duration was increased to three and five days, the JBNm and TGF-B1 groups demonstrated even more increased cell proliferation. When fluorescently labeling the JBNm, it is important that the TGF-B1 and Matrilin-3 be individually labeled and mixed before adding the JBNts, to allow for proper formation of the layer by layer structure.Further assessment of the scaffold can provide an understanding of how they are effective in therapeutic treatments. More specifically, a high throughput proliferation assay, can determine the optimal dosage in vitro, and later in vivo. This novel NanoMatriX creation allows researchers to utilize a nano-material based injectable scaffold to regenerate cartilage.This poly mimicking approach allows for enhanced androgenesis and counter site proliferation.

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