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.
