Introduction: The rise of spheroid-based tissue engineering has resulted in efficient strategies for skeletal defect regeneration through the formation of cartilage intermediate templates (1). As previously demonstrated, spheroid-based implants composed of cartilaginous microtissues can form ossicles upon implantation and regenerate critical size tibial defects (2). However, these implants were formed through microtissue self-assembly, and the formation of dense spheroid-based implants is a challenge through conventional extrusion based bioprinting. Novel, nozzle-free & rapid biofabrication method for engineering large microtissue-based implants need to be developed. In this work, we explore the use of two types of iron oxide nanoparticles (IONPs), their loading on cartilaginous microtissues and their potential as magnetically active building blocks for the biofabrication of callus-like mesotissues.
Methods: Human periosteum-derived cells (hPDCs), after 7 days of 2D expansion in DMEM complete medium were seeded in non-adherent microwells (AggreWell™400, STEMCELL Technologies) to form 3D spheroids. The spheroids were then cultured with either a chemically defined differentiation chondrogenic medium (CM) or basal medium for 7 days and then were incubated overnight with 0.5 μgr/mL dextran or citric acid coated IONPs. The treated spheroids were eventually placed in non-adherent 24 well plates and upon applying magnetic field, they were guided into forming a meso tissue (d=2 mm) which exhibited growth in size during culture (in chondrogenic medium) after 8 days. Microscope images were obtained at different timepoints, and non-treated spheroids were used as a control. Finally, image analysis was used for the quantification of implant’s growth.
Results: Following the characterization of the IONPs by TEM, XRD and FT-IR, spheroids cultured in CM had approximately 200 μm in diameter where the spheroids cultured in basal medium had an approximate diameter of 100 μm. Dextrose and citric acid coated IONPs after been incubated with the spheroids, they offered their magnetic actuation properties to the cartilaginous microtissues and thus they were able to respond to an external magnetic stimulus and be guided. The spheroids cultured with CM formed a granular mesotissue after the magnetic biofabrication, while spheroids cultured in BM formed a mesospheric tissue. In both samples, CM was used after the bioassembly and in both cases they doubled their size after 8 days in culture indicating that cells survive the process of biofabrication.
Conclusions: This biofabrication approach paves the way for a rapid and low shear method to produce scaffold-free implants formed from magnetically loaded cartilaginous microtissues under the guidance of a magnetic field. This approach overcomes limitations such as loss of microtissues during their handling in cell culture and moreover minimizes the time required for the biofabrication process in contrast to extrusion-based bioprinting.
 Laschke, M. W., & Menger, M. D. (2017). Life is 3D: boosting spheroid function for tissue engineering. Trends in biotechnology, 35(2), 133-144.
 G. Nilsson Hall, L.F. Mendes, C. Gklava, L. Geris, F.P. Luyten, I. Papantoniou, Developmentally Engineered Callus Organoid Bioassemblies Exhibit Predictive In Vivo Long Bone Healing, Adv. Sci. 7 (2020) 1–16.