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A Three-Dimensional Tissue Engineered Platform to Analyze the Effect of Various Microvascular Shear Stress Conditions
Julia Jin, BS1, Daniel Cheung, MS2, Kate Schole, BS2, Jonathan T. Butcher, PhD2, Jason A. Spector, MD1,2.
1Weill Cornell Medical College, New York, NY, USA, 2Cornell University, Ithaca, NY, USA.

PURPOSE: Open deep tissue wounds commonly result from severe burns, trauma, chronic diseases, irradiation, and infection. Autologous tissue transfer remains the gold standard for surgical reconstruction, but unfortunately can come with significant complications. The development of an on-demand, geometrically tunable tissue engineered substitute with an inherent vascular supply would transform reconstructive surgical practice, but regeneration of thicker or larger tissues of clinically relevant size remains a challenge due to poor oxygen diffusion into cells within these constructs. Hemodynamic shear stress alters cellular morphology and biological activity, especially luminal endothelial cells within blood vessels. However, these mechanisms have only been studied in isolation in vitro, and work within complex vascular networks has remained elusive. We have thus fabricated a unique three-dimensional vascular anatomy regime with various geometric angles to study the effects of differential shear stress on endothelial cell behavior.
METHODS: A multi-hairpin channel geometry with a diameter of 1.5 mm was designed in SolidWorks and created using a 3D-printer. Flow parameters were calculated using fluid simulations performed by Ansys Fluent to determine expected shear stresses throughout the channel. A Pluronic-F127 multi-hairpin channel was sacrificed in type-I collagen creating a central microchannel with encapsulated human placental pericytes in the collagen bulk. Subsequently, human aortic smooth muscle cells were intraluminally seeded into the macrochannel, followed by human umbilical vein endothelial cells 24 hours later. Constructs were statically cultured for 72 hours followed by dynamic perfusion at 10 dynes/cm2 for 10 days. After 14 days of culture, constructs were analyzed for changes in endothelial phenotype. Images were analyzed in ImageJ (NIH).
RESULTS: Fluid simulation verified the presence of different shear stresses at various regions in the channel. Acute angles provided regions of higher shear stress, and the plenum facilitated higher vorticity. The geometry allowed for multiple shear regimes within the construct for high throughput analysis. MicroCT analysis revealed the development of appropriately shaped microchannels that recapitulated the 3D printed geometry. Hemotoxylin and Eosin staining revealed the adherence of vascular cells along the microchannel. Regions of interest were stained with DAPI and confocal microscopy was used to take scans of the whole section. Images were converted to binary and the distribution of bulk cells around the vascularized microchannel was determined. Distribution of bulk cells was found to primarily localize around the microchannel, with 39% of cells being within 500μm of the microchannel. Although bulk cells were originally randomly mixed into a homogenous distribution, the localization of bulk cells suggested that the presence of shear stress promoted bulk cellular migration.
CONCLUSION: Using our novel platform, we have fabricated an anatomically appropriate vascularized model with different geometric angles to study flow dynamics and the effects of differential shear stress on recruitment of bulk cells to stabilize the neovessel. This physiological model overcomes the limitations of previous 2D and 3D flow studies with the potential to recapitulate in-vivo cell organization. We have integrated the capabilities of 3D printing and tissue engineering to develop printable biologically derived, cell-friendly hydrogels.


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