Background: As many as 6.8 in 1000 live births are affected by a life-threatening condition called persistent pulmonary hypertension of the newborn (PPHN). Structural defects of the blood vessels and extracellular space are major contributors to the dysfunctions in PPHN. Studying structural changes in 2D cell culture models is challenging: the stiff plastic surface is incapable of accurately replicating native tissue environments. Therefore, using 3D bioprinting technology, I fabricated a functional tissue construct that closely mimics the tunica media of blood vessels.

Methods: Pulmonary and coronary arterial smooth muscle cells were encapsulated in a stiffness-modifiable bioink comprised of alginate, collagen-type I, and fibrinogen. Smooth muscle tissues were bioprinted as standalone biorings, or biorings constrained within a stiffness-tunable acellular load bearing frame. Tissue maturation and contractile function were assessed by changes in lumen area, and cell organization was determined using filamentous actin staining. Vascular remodelling was assessed by gene expression for smooth muscle phenotypic markers. To assess stiffness dependent contractile response, biorings were treated with KCl and Cytochalasin D. To assess the effects of hypoxic stress, the biorings were exposed to 10% hypoxia.

Results: Stiff standalone biorings were structurally stable, but cells remained ‘balled up’ and were unable to spread and mature. Softer biorings showed signs of cell spreading but were structurally unstable. Addition of an acellular load bearing frame to the biorings reduced compaction while still allowing cells to mature. The degree of tissue compaction in biorings changed depending on the stiffness of the load bearing frame. In all cases, cells printed in soft biorings with stiff frames had well-organized bundles of actin filaments consistent with real vascular smooth muscle. Stiffness-dependent contractile responses were observed, with the strongest contractions observed in biorings with the stiffest acellular frame. No significant changes in phenotypic remodelling were observed upon hypoxia exposure.

Conclusion: The stiffness-tunable bioink and physical design produced a physiologically relevant structure which mimicked the structural defects seen in vascular diseases. This makes my model a novel and powerful tool that will enable us to understand how vessel stiffness affects the dysfunction in PPHN.