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

We developed a biomimetic nano-matrix with great promise to serve as a tissue engineering scaffold. This protocol demonstrates how to assemble the nano-matrix from Janus base nanotubes synthesized in my lab, as well as the fibronectin protein. This nano-matrix can self-assemble in a few seconds without using any additional assistance.

When co-cultured with human mesenchymal stem cells, the nano-matrix exhibited excellent bioactivity in encouraging cell adhesion. The nano-matrix mimics the extracellular matrix morphologically. Therefore, it has the potential to serve as an injectable scaffold to repair bone fractures by promoting cell adhesion and function at the target location.

Begin by fabricating the nano-matrix. Add 80 microliters of one-milligram-per-milliliter Janus base nanotubes to 40 microliters of one-milligram-per-milliliter fibronectin, and pipette the mixture gently and slowly several times. After 10 seconds, visually inspect the tube for the presence of a white solid suspension.

Next, prepare fibronectin, JBNT, and Fibronectin/JBNT nano-matrix solutions for lyophilized materials observation according to manuscript directions. Coat three wells of a 96-well, clear, round-bottom microplate with solutions, adding 50 microliters to each well. Freeze the plate at minus 80 degrees Celsius overnight.

On the next day, turn on the lyophilized instrument and vacuum pump, and press the cool down button to reduce the temperature of the system to minus 50 degrees Celsius. Put the frozen plate into the instrument, and close all openings. Click the start button to reduce the system pressure to 0.9 millibar.

After four hours, press the aerate button, and open the valve to increase the system pressure to the atmospheric pressure. Then, take the plate out of the lyophilized instrument. Prepare the JBNT/fibronectin nano-matrix solution as described in the text manuscript, and dilute the JBNT and fibronectin solutions with distilled water to make control solutions.

Measure the absorption spectra of the solutions with the spectrophotometer to characterize the assembly of nano-matrices. Click the UV-vis button. Then, clean the test surface of the spectrophotometer, and drop two microliters of distilled water on it.

Measure the water as blank from 190 to 850 nanometers. For each sample, clean the test surface, and drop two microliters of the solution on it. Then, measure the UV-vis absorption spectra from 190 to 850 nanometers.

Add 200 microliters of negative controls, JBNTs, fibronectin, and the nano-matrix solutions into the wells of an eight-well chambered slide. Freeze-dry the eight-well chambered coverglass with the lyophilized instrument to coat materials onto the bottom of the wells. Then, add 10, 000 human mesenchymal stem cells to each well of the coverglass, and incubate it for 24 hours at 37 degrees Celsius with 5%carbon dioxide.

After the incubation, remove the cell culture medium from the wells, and rinse them with PBS. Add 100 microliters of the fixative solution to each well, and leave it for five minutes at room temperature. Then, gently rinse the cells with PBS twice, and add 100 microliters of 0.1%Triton X-100 to each well.

After 10 minutes, repeat the rinses with PBS. Add 100 microliters of rhodamine phalloidin to each well, and incubate the chambered coverglass for 30 minutes, protecting it from light. Rinse the cells twice with PBS, and image them with a fluorescent microscope.

Nano-matrix formation is fast and completely biomimetic. White floccule was visible 10 seconds after the JBNT solution was mixed with the fibronectin solution. Camera images of fibronectin, JBNTs, and the nano-matrix were captured and analyzed.

No long fibers were observed in the fibronectin group, indicating that the scaffold structure can't form without JBNTs. JBNTs appear as long and thin fibers. When crosslinked with fibronectin, the nano-matrix fiber can grow up to several centimeters in length.

The assembly of the nano-matrix was also confirmed by obtaining UV-vis spectra. The two absorption peaks of JBNTs alone diminish after nano-matrix formation. Transmission electron microscopy was used to characterize the morphology of the JBNTs and nano-matrices.

The JBNTs are slender tubes with uniform diameters. When mixed with fibronectin, they form a long fibroid nano-matrix via charge interactions. When the pH is lower than the isoelectric point of the fibronectin, the nano-matrix bundles self-release due to the positively charged fibronectin.

The effect of the JBNT/fibronectin nano-matrix on cell adhesion was also explored. Since nano-matrices are designed to morphologically mimic the extracellular matrix, they provide a scaffold and increase cell adhesion. Fluorescence microscopy confirmed that incubation with the nano-matrices increased cell adhesion.

In the following procedure, cell migration, differentiation, and the long-term function can be shown to further explore the effect of nano-matrix on human mesenchymal stem cell growth and functions.