In-Space Manufacturing of Janus Base Nano-Matrix Components for Cartilage Repair

Introduction: The development of our Janus Base Nano-Matrix, or JBNm, has shown great potential in the field of cartilage tissue engineering. This novel scaffold is comprised of our proprietary Janus Base Nanotubes (JBNts), Transforming Growth Factor β2 (TGFβ-2), and Matrilin-1. JBNts structurally mimic collagen proteins, allowing them to induce both cell adhesion and chondrogenic differentiation. TGFβ-2 is a bioactive molecule which promotes chondrocyte proliferation. Matrilin-1 is a structural protein found in the extracellular matrix of cartilage which aids in adhesion and mediates proper TGFβ-2 signaling. Together, these molecules self assemble to create a layer-by-layer structure that encourages localized cartilage regeneration. Their self assembly occurs at room temperature in water, separating these materials from traditional nanomaterial-based scaffolds. Due to the lack of certain forces like convection in microgravity, the JBNm has even greater potential in space to form more homogeneous and larger structures. As a result, we have manufactured our JBNm in low Earth orbit aboard the International Space Station (ISS).

Materials and

Methods: JBN monomers were first synthesized via a previously published method. Following the lyophilization of these monomers and the aforementioned TGFβ-2 and Matrilin-1 proteins, our samples were packaged and flown aboard SpaceX’s Falcon 9 rocket on the AX-2 mission. Our samples, along with four crewmembers, then boarded the ISS. Three main groups of Aged, Premade, and Immediate JBNts were created, and each of these were mixed with either Matrilin-1 alone or Matrilin-1 with TGFβ-2 to create various types of JBNms. JBNts were also kept as nanotubes to further study the affect microgravity had on their formation. Repeat JBNts and JBNms were fabricated in tandem on the ground to act as controls. After these operations were complete, the samples were returned to Earth, where the analysis began. The structural characterization of the JBNts and JBNms created in space was investigated and compared to those created on Earth. Similarly, chondrocytes were seeded onto both groups of JBNms, where adhesive ability and cellular architecture were explored via fluorescent staining. Figure 1 depicts the methodology and various structural measurements of JBNm components.

Results, Conclusions, and Discussions: We observed significant improvements in the Flight group (the group that was fabricated in space) compared to the Ground group (the group that was fabricated on Earth). Figure 2 depicts these differences, as well as TEM images to visually show their differences. In the Matrilin-1 alone group, there was a significant increase in zeta potential for the Flight samples, which likely resulted in the greater attraction of cells to the matrix. The average width of the JBNm strands also greatly increased, along with the cell area of those that bound to the matrix. This trend continued for the JBNms made with Matrilin-1 and TGFβ-2, although for these samples, the zeta potential decreased. This could be due to a higher degree of TGFβ-2 incorporation into the matrix. Overall, a high degree of improvement was realized by manufacturing these materials in a microgravity environment. In conclusion, we observed significant improvements in the fabrication of our JBNms aboard the ISS, both in terms of structural qualities as well as their impact on bioactivity. We will be returning to space with these materials in September of 2024, with a scaled up set of samples and more rigorous analysis planned, including in vivo application of in-space manufactured JBNms.

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