A Modular Strategy to Engineer Complex Tissues and Organs.

Currently, there are no synthetic or biologic materials suitable for long-term treatment of large tracheal defects. A successful tracheal replacement must (1) have radial rigidity to prevent airway collapse during respiration, (2) contain an immunoprotective respiratory epithelium, and (3) integrate with the host vasculature to support epithelium viability. Herein, biopolymer microspheres are used to deliver chondrogenic growth factors to human mesenchymal stem cells (hMSCs) seeded in a custom mold that self-assemble into cartilage rings, which can be fused into tubes. These rings and tubes can be fabricated with tunable wall thicknesses and lumen diameters with promising mechanical properties for airway collapse prevention. Epithelialized cartilage is developed by establishing a spatially defined composite tissue composed of human epithelial cells on the surface of an hMSC-derived cartilage sheet. Prevascular rings comprised of human umbilical vein endothelial cells and hMSCs are fused with cartilage rings to form prevascular-cartilage composite tubes, which are then coated with human epithelial cells, forming a tri-tissue construct. When prevascular- cartilage tubes are implanted subcutaneously in mice, the prevascular structures anastomose with host vasculature, demonstrated by their ability to be perfused. This microparticle-cell self-assembly strategy is promising for engineering complex tissues such as a multi-tissue composite trachea.

Mutations in the bacterial cell division protein FtsZ highlight the role of GTP binding and longitudinal subunit interactions in assembly and function.

BACKGROUND: Assembly of the tubulin-like GTPase, FtsZ, at the future division site initiates the process of bacterial cytokinesis. The FtsZ ring serves as a platform for assembly of the division machinery and constricts at the leading edge of the invaginating septum during cytokinesis. In vitro, FtsZ assembles in a GTP-dependent manner, forming straight filaments that curve upon GTP hydrolysis. FtsZ binds but cannot hydrolyze GTP as a monomer. Instead, the active site for GTP hydrolysis is formed at the monomer-monomer interface upon dimerization. While the dynamics of GTP hydrolysis and assembly have been extensively studied in vitro, significantly less is known about the role of GTP binding and hydrolysis in vivo. ftsZ84, a GTPase defective allele of Escherichia coli ftsZ, provides a striking example of the disconnect between in vivo and in vitro FtsZ assembly. RESULTS: Although ftsZ84 mutants are defective for FtsZ ring formation and division under nonpermissive conditions, they are near wild type for ring formation and division under permissive conditions. In vitro, however, purified FtsZ84 is defective in GTP binding, hydrolysis and assembly under standard reaction conditions. To clarify the nature of the FtsZ84 assembly defect, we isolated and characterized three intragenic suppressors of ftsZ84. All three suppressor mutations increased the apparent affinity of FtsZ84 for GTP, consistent with improved subunit-subunit interactions along the longitudinal interface. Although kinetic analysis indicates that the suppressor mutations increase the affinity of FtsZ84 for GTP, all three exhibit reduced rates of GTP hydrolysis and fail to support assembly in vitro. CONCLUSION: Together, our data suggest that FtsZ, and potentially other enzymes whose assembly is similarly regulated, can compensate for defects in catalysis through increases in substrate binding and subunit-subunit interactions. In addition, these results highlight the dichotomy between commonly used in vitro assembly conditions and FtsZ ring formation in the complex intracellular milieu.

Engineered cartilaginous tubes for tracheal tissue replacement via self-assembly and fusion of human mesenchymal stem cell constructs.

There is a critical need to engineer a neotrachea because currently there are no long-term treatments for tracheal stenoses affecting large portions of the airway. In this work, a modular tracheal tissue replacement strategy was developed. High-cell density, scaffold-free human mesenchymal stem cell-derived cartilaginous rings and tubes were successfully generated through employment of custom designed culture wells and a ring-to-tube assembly system. Furthermore, incorporation of transforming growth factor-ß1-delivering gelatin microspheres into the engineered tissues enhanced chondrogenesis with regard to tissue size and matrix production and distribution in the ring- and tube-shaped constructs, as well as luminal rigidity of the tubes. Importantly, all engineered tissues had similar or improved biomechanical properties compared to rat tracheas, which suggests they could be transplanted into a small animal model for airway defects. The modular, bottom up approach used to grow stem cell-based cartilaginous tubes in this report is a promising platform to engineer complex organs (e.g., trachea), with control over tissue size and geometry, and has the potential to be used to generate autologous tissue implants for human clinical applications.