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

Abstract

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-β1-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 in 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.

A Modular Strategy to Engineer Complex Tissues and Organs

“…Tissue Dimension Measurements and Biomechanical Analysis : Tissue engineered rings composed of different cell numbers ( N = 3) and rat tracheas ( N = 4) were analyzed using uniaxial tension to failure testing and the ring wall thicknesses were measured as previously described 8. Tissue engineered tubes ( N = 3) and rabbit tracheas ( N = 6) (Part Ib and III) were evaluated via luminal collapse and recoil as before with slight modifications 8.…”

“…Tissue engineered tubes ( N = 3) and rabbit tracheas ( N = 6) (Part Ib and III) were evaluated via luminal collapse and recoil as before with slight modifications 8. Freshly harvested five‐ring cartilaginous tubes of 2, 6, and 12 mm inner diameters and prevascular–cartilage 2 mm inner diameter tubes and their cartilage‐only controls were compressed by their respective luminal size at a rate of 0.5 mm min −1 .…”

Cellular Self-Assembly with Microsphere Incorporation for Growth Factor Delivery Within Engineered Vascular Tissue Rings

“…This suggests that microspheres will not adversely affect ring mechanical strength when grown in differentiation medium. Others have reported increases in tissue strength and stiffness with gelatin microsphere incorporation, 13,21 which may be due to improved oxygen and nutrient diffusion in dense tissues. 16,42 The decrease in ring MTM was unexpected, as microsphere incorporation has been shown to increase tissue stiffness.…”

“…13 Briefly, a water-in-oil emulsion was created with 11.1 w/v% type A gelatin (Sigma-Aldrich) and olive oil (GiaRussa). Gelatin microspheres were crosslinked with 1% w/v genipin (Wako) for 3 h at room temperature.…”

“…10,12 In addition to SMC rings and tubes, this versatile cellular self-assembly system may enable fabrication of rings and tubes of other tissue types, including human cartilage. 13 Cellular self-assembly may have advantages over scaffold-based approaches for vascular tissue engineering. Compared to cells seeded on scaffold materials, selfassembled cellular constructs may have greater cell density, enhanced extracellular matrix (ECM) production and tissue strength, improved biological function, and lower susceptibility to degradation and infection, 11,[14][15][16] and thus may be more similar in structure and function to native tissue.…”