Endothelial cells (ECs) have potential in bone tissue engineering due to their important role on vascularization. For repairing bone defects, co-culturing of ECs and bone marrow stem cells (BMSCs) is suggested to induce both the capillary network formation and bone regeneration. The communication between the two cell types has been experimented on settings based on cell number ratio, culture medium [1-2] and culture distance . With regards to 3D printing technology, EC suspensions have been previously flushed into engineered lumens or canals to initiate vessel formation in existing structures [4-5]. However, bioprinting is yet a rarely used form of technology for delivering ECs. Vascu-ink, containing fibrinogen and gelatin, aimed for soft, elastic and dynamic 3D environment, enabling the rapid maturation of bioprinted ECs. The aim of this study was to investigate the influence of a multimaterial bioink, vascu-ink, in a co-culture setting on angiogenic tissue maturation.
The performance of the vascu-ink with ECs was characterized as single bioink but including the co-culture effect by seeding BMSCs outside the bioink structures. To investigate the potential of vascu-ink as an EC carrier, the bioink was mixed with the ECs (1x107/ml) and bioprinted in a four-layered structure using a 3D-Bioplotter (EnvisionTEC) with a 250 μm metal needle. The structures were crosslinked externally with thrombin (2.5U/ml) and CaCl2 (100mM). BMSCs were seeded on the bottom of the well plate and the crosslinked vascu-ink samples were lifted on top. The co-cultures were then followed for structural integrity, cell viability (Live/Dead staining), metabolic activity (Cell Counting Kit -8) and tubular formation, via immunostaining, for up to 21 days. Endothelial growth media was used for the first three days and then switched into osteogenic media. After that, the media was changed three times a week.
The developed vascu-ink was very compliant as desired. The viability of the ECs was high throughout the culture period, cells were spreading and migrating, and the metabolic activity of ECs was maintained with the BMSCs. The printed structures survived the culture period but had gradually lost their fidelity as the material was preferred by both of the cell types. The desired tubular formations and organization of the ECs was recorded by both Live/Dead staining and immunostaining.
The promising results indicate that the developed bioink serves as a tissue specific cell carrier and culture environment. The co-culture of the two cell types was also beneficial in tubular formation.
1. Ma, J., et al., Tissue Eng Part C Methods. 17(3), 349-57 (2011).
2. Bidarra, S.J., et al., Stem Cell Res. 7(3), 186-97 (2011).
3. Piard, C., et al., Biomaterials 222, 119423 (2019).
4. Kolesky, D.B., et al., PNAS 113(12), 3179-84 (2016).
5. Miller, J.S., et al., Nat. Mater. 11(9), 768-74 (2012).