Design and Characterization of Thermal Inkjet Bioprinted Constructs for the Treatment of Diabetic Foot Ulcers and Other Wound Healing Impaired Conditions

Tissue engineering (TE) is a multidisciplinary practice focused on developing patient-specific constructs to repair, regenerate, or replace injured tissues and organs. One biofabrication process that has gained tremendous momentum in this field is bioprinting. Most, if not all bioprinting modalities embody the same principles of heat and shear due to mechanical forces or friction as cells are forced through narrow orifices. While it is important to examine the interaction between host and implanted constructs, it is also crucial to understand the direct effects that these bioprinting technologies have on cells before embedding them into TE applications. The purpose of this work is to determine the effects of thermal inkjet bioprinting (TIB) on cellular angiogenesis in vitro. Our in vivo histological analysis of implanted constructs containing TIB endothelial cells (ECs) in B-17SCID mice demonstrated significantly higher number of capillary-like structures as compared to constructs with manually pipetted (MP) ECs. Additionally, in vitro cellular morphological differences between TIB- and MP-ECs were noted. TIB cells had elongated protrusions at 5–6 times the size of MP cells. Moreover, an Annexin V-FITC and PI apoptosis assay showed a 75% apoptosis among TIB cells as compared to MP cells via flow cytometry analysis. After a 3-day incubation period, however, TIB cells demonstrated significantly higher viability as compared to the control group. Also, cytokine expression was assessed with the use of Milliplex magnetic bead panels, which confirmed significant overexpression of HSP70, IL-1α, VEGF-A, IL-8, and FGF-1 among TIB cells as compared to the control. Furthermore, a Human phospho-kinase array to determine intracellular kinase and protein activation showed a significant over activation of HSP27 and HSP60 in TIB cells as compared to MP cells as well as a decreased proliferative state among TIB-ECs. In all, we have demonstrated that constructs containing TIB-ECs offers an alternative method to inducing microvascular networks in vivo. Our in vitro data also supports our findings as they translate in vivo. The ability to create vast capillary networks, coupled with the fixed printing parameters of our TIB technology (i.e. heat, pressure, nozzle size, droplet size, and velocity) allows for versatile and repeatable means to create these constructs. Future in vitro work with appropriate controls for mechanical stress or possibly printing with the cartridge submerged within media to control for cellular stretching are recommended. Also, in vivo work with impaired wound healing such as a diabetic foot ulcer model are also suggested. Finally, a comparative study among different TIB modalities are also recommended. Looking ahead, bioengineered constructs using our TIB technology may find potential applications in organ on a chip, drug testing, and autonomic healing applications