Dual-core coaxial bioprinting of double-channel constructs with a potential for perfusion and interaction of cells.

Coaxial bioprinting of hydrogel tubes has tremendous potential in the fabrication of highly complex large-scale vascularized structures, however, constructs with bioinks of simultaneous weak printability and perfusable networks have not been reported. Here, we report a coaxial printing method in which double-channel filaments are three-dimensional (3D) extrusion-bioprinted using a customized dual-core coaxial nozzle. The filament in one channel can perform core/shell role and the other channel can play a role in perfusion. These parallel channels within filaments are separated by an interval wall of alginate, whose thickness (∼50µm) is beneficial to supplement nutrients via perfusion. Different cell-laden hydrogels of weak mechanics were used to test the adaptability and perfusability of our method, and the results showed that dynamic perfusion maintained higher viability and functions than static culture. By combining with a bioprinter, 8-layer perfusable double-channel constructs were fabricated, and the cell viabilities gradually decreased with the reduction in nutrients and oxygen in the downstream medium. Furthermore, the double-channel filaments were tested as a platform to mimic dynamic functions between cells through sequential perfusion by using Mouse insulinoma 6 (Min6) and Hepatocellular carcinoma (HepG2) as the model cells. These results demonstrated the insulin secreted by Min6 upstream simulated and increased the uptake of glucose by the downstream HepG2 cells. In conclusion, our study provided evidence for the probability of all-in-one fabrication of 3D double-channel perfusable constructs with high simplicity, expansibility, and versability. Our strategy has significant potential for building large-scale tissue constructs for applications in tissue engineering, possibly even in drug screening and regenerative medicine.

Capsaicin ameliorates intermittent high glucose-mediated endothelial senescence via the TRPV1/SIRT1 pathway.

BACKGROUND: Patients with diabetes have accelerated vascular aging when compared with healthy individuals. Hyperglycemia, especially intermittent high glucose (IHG), is the main cause of vascular endothelial senescence. Capsaicin, a major component of chili pepper is thought to contribute to cardiovascular protection by spicy food. OBJECTIVE: To investigate the pathway related with the effects of capsaicin on endothelial cell senescence induced by IHG. METHODS: HUVECs were exposed to IHG (5 mM or 33 mM glucose, alternating every 12 hours for 3 days) and treated with capsaicin at 0.3, 1 and 3 µM. To determine endothelial cell senescence, we examined the senescence-related ß-galactosidase staining, cell cycle arrest, cell viability, as well as production of reactive oxygen species (ROS). To evaluate the involvement of TRPV1/[Ca2+]i/CaMKII/AMPK/SIRT1 pathway in anti- senescence effects of capsaicin, HUVECs were treated with CAPZ (a TRPV1 antagonist), BAPTA-AM (an intracellular calcium chelator), KN62 (a CaMKII antagonist), compound C (an AMPK inhibitor), or EX527 (a SIRT1 inhibitor). To knockdown TRPV1, HUVECs were transfected with shRNA lentivirus targeting TRPV1. The levels of SIRT1, p21, TRPV1, AMPK and phospho-AMPK were evaluated by western blotting. RESULTS: IHG suppressed the levels of SIRT1 and enhanced endothelial senescence. Capsaicin upregulated SIRT1 expression and downregulated the senescence marker, p21, thereby protecting endothelial cells from IHG-induced senescence as indicated by relieved G0/G1 phase arrest, improved cell viabilities, and reduced counts of senescent cells and ROS production. Pre-treatment with CAPZ, BAPTA-AM, KN62 or compound C abrogated the anti-senescence effects of capsaicin. Capsaicin restored AMPK phosphorylation and IHG-inhibited TRPV1 expression. Moreover, TRPV1 silencing suppressed SIRT1 expression and abolished the anti-senescence effects of capsaicin. CONCLUSION: Capsaicin elevates SIRT1 levels through TRPV1/[Ca2+]i/CaMKII/AMPK pathway and suppresses IHG-mediated endothelial cell senescence. This study provides initial evidence that capsaicin is a potential candidate for the prevention of vascular aging in diabetes.