Tendon tissues have highly-anisotropic physical properties that are responsible for its biomechanical performance and biological organization. The recreation of its 3D extracellular matrix (ECM) and cellular patterns in bioengineered constructs remains challenging. The concept of magnetically-assisted 3D bioprinting with magnetic hydrogel bioinks can be exploited to fabricate anisotropic scaffolding materials with 3D architectures that resemble the organization of tendinous ECM and to modulate biophysical/biochemical cues that influence the fate of encapsulated cells. Moreover, magnetic nanoparticles (MNPs) remote response enables their use as magnetomechanical actuators to control cellular/tissue behavior. However, a main challenge hindering the implementation of this concept is how to control the 3D organization of magnetic elements during layer-by-layer printing without compromising the fidelity and resolution of printed constructs. To overcome this dichotomy, here we combine the concepts of magnetically and matrix-assisted 3D bioprinting technologies. This strategy enables to fabricate high-resolution constructs with magnetic bioinks that remain liquid for long enough before gelation to allow the orientation of magnetic elements, thus building 3D fibrillar patterns resembling the microstructure of tendon tissues.
Monodisperse iron oxide-based MNPs displaying extremely-high magnetization values were synthesized through thermal decomposition. These MNPs were then incorporated into electrospun polycaprolactone meshes, which were subsequently cryo-sectioned at different lengths to produce dispersed magnetic microfibers. Magnetically-responsive bioinks were prepared by mixing the magnetic short fibers with gelatin solutions and human adipose-derived stem cells (hASCs). The 3D extrusion bioprinting steps were performed under the presence of fairly uniform external magnetostatic fields produced by a two parallel magnets setup. Agarose and cellulose nanocrystals (CNCs)-based fluid gels (supplemented with transglutaminase for gelatin crosslinking) were tested as support baths.
Zinc-doping demonstrated to be the most efficient approach to increase the magnetic power of superparamagnetic iron oxide-based MNPs. Zn-Fe3O4 MNPs were used to prepare magnetically-responsive electrospun polycaprolactone microfibers with 20-100 µm of length. The incorporation of these microfibers and hASCs in gelatin solutions resulted in bioinks that enabled the fabrication of high-resolution 3D-printed constructs when using CNCs as suspension baths, but not when with the respective granular agarose gels. Exploiting the high magnetic power of the MNPs, very low particle concentrations and weak magnetic field strengths were enough to align the fibers during the layer-by-layer extrusion printing steps. The anisotropic microstructure of the biomimetic constructs induced elongated growth and phenotypic commitment of the encapsulated cells.
Our strategy allows the 3D manufacturing of biomimetic magnetic constructs that replicate the architecture of native tendons ECM. We established the design of MNPs with extremely-high magnetic power as a key factor to fabricate bioink hydrogels that can be manipulated using low contents of magnetic material and weak magnetic fields, minimizing the toxic/safety risks associated with these factors. The combination of magnetically-assisted 3D bioprinting strategies with the use of CNCs support baths has demonstrated to be essential for enabling the proposed concept. The resulting anisotropic 3D fibrillar microstructure of the printed constructs revealed effective on directing encapsulated cell fate. The effects of remote magnetomechanical actuation on cellular constructs is currently under investigation.