(I-347) In-Space Fabrication and Computation-Aided Optimization of Janus Base Nano-Matrix for Cartilage Regeneration

Introduction:: Our bio-inspired Janus Base Nano-matrices (JBNms) are injectable scaffolds for tissue regeneration. To create these scaffolds, our innovative Janus base nanotubes (JBNts) are mixed with transforming Growth Factor β (TGFβ), and matrilin-1 on the ISS to form the JBNm scaffold. Matrilin-1, a structural protein, is found on the outside of the matrix, encouraging cell adhesion and providing proper cartilage-specific signaling. TGFβ, a bioactive molecule, signals the bound cells to proliferate. Once added to the JBNts, which mimic collagen proteins, the JBNm is fabricated via self-assembly in water at room temperature. This unique process is biomimetic, eco-friendly, and catalyst-free, but gravity related forces, such as convection, are a limitation for their successful self-assembly into larger scaffolds. Specifically, if convection forces are too strong, the matrix will not form in the necessary hierarchy for proper growth. By reducing the impact of these forces, microgravity offers a much more favorable environment for the JBNms to form. Therefore, supported by NASA, Axiom Space, and Eascra Biotech, we are able to conduct in-space fabrication of JBNms on the International Space Station (ISS) via the Ax-2 mission. To further improve the efficacy of our JBNms, we also investigated a computation-aided method to optimize their formulations. Using response surface methodology (RSM), we varied each component to observe its effect on chondrocyte proliferation (cartilage growth) as well as DNA and GAG production (cartilage function); this allowed us to determine which specific mixture of matrix ingredients shows the best potential to induce cartilage repair.

Materials and Methods:: JBNts were synthesized via a previously published method. Four groups of JBNms were then created: i) on Earth, with JBNts formed on Earth, ii) on the ISS, with JBNts formed in space, iii) on Earth, with JBNts formed in space, and iv) on the ISS, with JBNts formed on Earth. This allowed us to observe the effects on microgravity at each stage of development. Structural characterization methods, such as zeta potential, UV-visible spectroscopy, protein labeling, and TEM were performed. The cell proliferation of the JBNms was also analyzed, using a CCK-08 assay. For the optimization with RSM, cell proliferation and GAG production were measured in a variety of component combinations, using a Central Composite Design. Both chondrocytes and mesenchymal stem cells were analyzed, utilizing a 3D culture in fibrin gel. The top performing ratios also underwent structural characterization, similar to the JBNms formed in space.

Results, Conclusions, and Discussions:: We identified a significant peak in cell proliferation of our JBNm, centered around 0.02 μg of TGFβ, 0.1 μg of matrilin-1, and 5 μg of JBNts (ratio of 1:5:250 of TGFβ:matrilin-1:JBNt). This is labeled red in Figure 1. Additionally, another peak around 0.1 μg of TGFβ and 0.4 μg of matrilin-1 (1:4:50, our previously used ratio) was observed. Thus, the JBNm has been seen to be effective in driving proper cartilage growth, encouraging proliferation and functions of chondrocytes that adhere to it. By limiting the effects of convection, which depend on gravity, more proper JBNt and JBNm formation was observed. Additionally, the relationships between its components and the overall scaffold efficacy were examined, with an optimal mixture determined.

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