INTRODUCTION: Regeneration of thicker or larger tissues of clinically relevant size
remains a challenge due to poor oxygen diffusion into cells that are contained within
non-vascularized tissue-engineered constructs. In our previous work, we fabricated
vascularized tissue engineered constructs that remained viable for more than 28 days
in static culture. However, without exposing the vascular lining cells to flow, their
functionality and in vivo stability are suboptimal. Here, we “prime” the constructs
by dynamically perfusing them and determine how flow induced shear stress optimizes
the endoluminal surfaces of our tissue-engineered vessels.
MATERIALS AND METHODS: Pluronic F127 fibers, 1.5 mm in diameter, were sacrificed in
type I collagen, creating a central looped microchannel. 100μL polyculture cell suspension
mixture of 5 x 106 cells/mL of human foreskin fibroblasts and 5 x 106 cells/mL of
human aortic smooth muscle cells was seeded into the microchannel. The following day,
a 100μL cell suspension of 5 x 105 cells/mL of human placental pericytes and 5 x 106
cells/mL of human umbilical vein endothelial cells was seeded into the microchannel.
All constructs underwent daily media changes in static culture for 7 days, and then
half were perfused at 10 dynes/cm2 for an additional 7 days. After 14 days, scaffolds
were fixed and processed.
RESULTS: After 7 and 14 days of culture, constructs formed intact endoluminal linings
along the microchannel with increasing thickness over time. CD31 expressing endothelial
cells were noted along the luminal surface after 7 days and throughout the endoluminal
lining after 14 days, establishing a neointima. Constructs undergoing static and dynamic
culture had robust, vascular linings that spanned the entire microchannel. However,
dynamic constructs had a 59% thicker lining in the channel (p=0.0057). Ki67 staining
demonstrated statistically significant increased cell proliferation in constructs
that experienced dynamic perfusion suggesting stimulation by the shear stress (p=0.0429).
CONCLUSION: Shear stress through dynamic perfusion was used to optimize the development
of a layer of vascular lining cells to provide a non-thrombogenic surface to allow
continuous blood flow in these tissue engineered vessels. Exposing pre-vascularized
engineered tissues to controlled perfusion produces vessels with architecture that
more accurately recapitulates the in vivo phenotype and provides a surface for thrombosis-free
blood flow, allowing for surgical implantation via microanastomosis.
None of the authors has a financial interest in any of the products, devices, or drugs
mentioned in this manuscript.