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.
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-β1) via bioaffinity. The JBNm was assembled at a TGF-β1: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-β1 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-β1 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-β1 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.
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-β1), 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 π-π stacking interactions to create a nanotube 200-300 µm 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-β1 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-β1 are essential components within the JBNm. The addition of TGF-β1 into the layer-by-layer scaffold can further promote cartilage regeneration in a tissue construct. TGF-β1 is a growth factor employed to encourage the healing process of osteochondral defects, encouraging chondrocyte and hMSC proliferation and differentiation21,22. Thus, TGF-β1 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-β1 adhered on JBNt surfaces in the inner layer and matrilin-3 adhered on JBNt surfaces in the outer layer24,25. The incorporation of TGF-β1 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.
1. Synthesis of JBNts
2. Fabrication for JBNt/Matn1/TGF-β1 (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.
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-β1 alone, and the full layer-by-layer JBNm, comprised of all three parts. All initial concentrations are suspended in H2O.
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-β1, and the full layer-by-layer JBNm.
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.
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.
7. In vitro biological function assay
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-β1/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-β1 solution to the matrilin-3 solution, the zeta-potential of the TGF-β1/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-β1, indicating that binding occurs between TGF-β1 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-β1/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-β1/JBNts line, indicating that the JBNts prefer to bind to matrilin-3, forming a layer-by-layer outer structure with matrilin-3 while TGF-β1 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-β1 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-β1 formed an inner layer, contributing to the capability of storing growth factors and allowing the localization of TGF-β1. 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-β1 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-β1 alone, and negative control, the JBNm and TGF-β1 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-β1 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-β1 into the JBNm. This figure has been modified from Zhou et al. (2021)26. Please click here to download this Video.
Figure 1: Schematic illustration of the chemical structure of JBNts, the J/T/M JBNm, and the J/T/M JBNm'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. Please click here to view a larger version of this figure.
Figure 2: Material characterization of the J/T/M JBNm. (A) Zeta-potential spectra of matrilin-3 alone, the TGF-β1/matrilin-3 compound, and the J/T/M JBNm. (B) UV-Vis absorption spectra of JBNts alone, matrilin-3 alone, the TGF-β1 alone, matrilin-3/JBNts compound, the TGF-β1/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. Please click here to view a larger version of this figure.
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-β1. (B) 2D confocal images of J/T/M JBNm, with red-fluorescent-labeled matrilin-3, green-fluorescent-labeled TGF-β1, 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. Please click here to view a larger version of this figure.
Figure 4: Analysis of human mesenchymal stem cell (hMSC) culture. (A) Optical microscopy images of hMSCs with TGF-β1, 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 µm per group. Error bars denote standard deviations. Note: N ≥ 3, *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 (compared against negative controls, NC). This figure has been modified from Zhou et al. (2021)26. Please click here to view a larger version of this figure.
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 < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. This figure has been modified from Zhou et al. (2021)26. Please click here to view a larger version of this figure.
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-β1, 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-β1 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-β1, allowing for the encapsulation of TGF-β1 and therefore providing a slow release of TGF-β1 into the surrounding tissue. The most critical step in the development of the JBNm is the combination of proteins with the JBNts. TGF-β1 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-β1. 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-β1 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-β1 (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 ± 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-β1 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-β1 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-β1 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-β1, 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's protocols to determine cell proliferation. JBNm, especially in the presence of TGF-β1, and TGF-β1 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 °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-β132,33. The ability of the JBNm to incorporate TGF-β1 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-β1, 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-β1 every time medium was changed), JBNm, JBNts, matrilin-3, TGF-β1, 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-β1 ELISA kit to test the release of protein from the JBNm hydrogel. In this study, TGF-β1 is expected to be encapsulated in the J/T/M JBNm, and therefore no TGF-β1 release should be observed (i.e., the theoretical loading value is 100%). A limitation of this step is that all TGF-β1 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's protocols. Then, the amount of TGF-β1 was calculated by subtracting the amount of the released TGF-β1 in PBS from the theoretical loading value. Because TGF-β1 was encapsulated in the inner layer of the JBNm, the slow release of the protein was anticipated. The study demonstrated that the TGF-β1 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-β1 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.
The authors have nothing to disclose.
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.
10 % Normal Goat Serum | Thermo Fisher | 50062Z | Agent used to block nonspecific antibody binding actions during staining. |
24-well plate | Corning | 07-200-740 | 24-well plate used for comparative cell culture. |
384-Well Black Untreated Plate | Thermo Fisher | 262260 | 384-well plate used for absorption measurements. |
8-well chambered coverglass | Thermo Fisher | 155409PK | 8-well coverglass used for comparative cell culture. |
96-well flat bottom | Corning | 07-200-91 | 96-well plate used for comparative cell culture. |
96-Well Plate non- treated | Thermo Fisher | 260895 | 96-well plate used for comparative cell culture and analysis. |
Agarose Gel | Sigma-Aldrich | A9539 | Hydrogel used for cell culture. |
Agarose Gel | Sigma Aldrich | A9539 | Hydrogel used as an environment for cell culture. |
Alexa Fluor Microscale Protein Labeling Kit | Thermo Fisher | A30006 (488) and A30007 (555) | Fluorescent dye used to label proteins. |
Anti-Collagen X Antibody | Thermo Fisher | 41-9771-82 | Antibody used to stain collagen-X. |
Bio-Rad PCR Machine | Bio-Rad | Equipment used to perform PCR on samples. | |
C28/I2 Chondrocyte Cell Line | Cells used to analyze proliferative abilities of various samples. | ||
Cell Counting Kit 8 | Milipore Sigma | 96992 | Cell proliferation assay. |
Cell Profiler | Broad Institute | Software used to analyze cell images. | |
Cryostat Microtome | Equipment used to produce thin segments of samples for use in staining and microscopy. | ||
DAPI | Invitrogen | D1306 | Blue fluorescent stain that binds to adenine-thymine DNA regions. |
Disposable cuvettes | FISHER Scientific | 14-955-128 | Container used for spectrophotometry. |
DMEM Cell Culture Medium | Thermo Fisher | 10566032 | Media used to support cellular growth. |
Fetal Bovine Serum | GIBCO | A4766801 | Serum used in cell culture medium to support cell growth. |
Fluoromount-G Mounting Medium | Thermo Fisher | 00-4958-02 | Solution used to mount slides for immunostaining. |
Formaldehyde | Compound used to fix samples prior to microtoming. | ||
Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody | Thermo Fisher | A16110 | Antibody used for protein staining. |
Human Mesenchymal Stem Cells | LONZA | PT-2501 | Cells used to analyze differentiative abilities of various samples. |
Human Mesenchymal Stem Chondrogenic Medium | LONZA | PT-3003 | Cell medium used to promote chondrogenic differentiation. |
ImageJ | National Institutes of Health | Image analysis software used in conjunction with microscopy. | |
itaq Universal SYBR Green One-Step Kit | BioRad | 1725150 | Kit used for PCR. |
Janus-base nanotubes (JBNts) | Nanotube made from synthetic nucleobases to act as cell scaffolding tool. | ||
LaB6 20-120 kV Transmission Electronic Microscope | Tecnai | Equipment used to perform transmission electron microscopy on a sample. | |
MATLAB | MathWorks | Statistical software used for modeling and data analysis. | |
Matrilin-3 | Fisher Scientific | 3017MN050 | Structural protein used as adhesion sites for chondrocytes. |
NanoDrop Spectrophotometer | Thermo Fisher | Equipment used to measure absorption values of a sample. | |
Nikon A1R Spectral Confocal Microscope | Nikon | A1R HD25 | Confocal microscope used to analyze samples. |
Number 1.5 Chamber Coverglass | Thermo Fisher | 152250 | Environment for sterile cell culture and imaging. |
Optimal Cutting Temperature Compound Reagent | Compound used to embed cells prior to microtoming. | ||
Paraformaldehyde | Thermo Scientific | AAJ19943K2 | Compound used to fix cells. |
PDC-32G Plasma Cleaner | Harrick Plasma | Cleaner used to prepare grids prior to transmission electron microscopy. | |
penicillin-streptomycin | GIBCO | 15-140-148 | Antibiotic agent used to discourage bacterial growth during cell culture. |
Phosphate Buffered Saline | Thermo Fisher | 10010023 | Solution used to wash cell medium and act as a buffer during experimentation. |
Rhodamine-phalloidin | Invitrogen | R415 | F-Actin red fluorescent dye. |
Rneasy Plant Mini Kit | QIAGEN | 74904 | Kit used to filter and homogenize samples during RNA extraction. |
Sucrose Solution | Solution used to process samples prior to microtoming. | ||
TGF beta-1 Human ELISA Kit | Invitrogen | BMS249-4 | Assay kit used to determine the presence of TGF-β1 in a sample. |
TGF-β1 | PEPROTECH | 100-21C | Growth factor used for the stimulation of chondrogenic differentiation and proliferation. |
Triton-X | Invitrogen | HFH10 | Compound used to lyse cells not fixed during staining process. |
TRIzol Reagent | Thermo Fisher | 15596026 | Reagent used to isolate RNA. |
Zetasizer Nano ZS | Malvern Panalytical | Equipment used to measure zeta-potential values of a sample. |