Miles Montgomery

Biodegradable scaffold with built-in vasculature for organ-on-a-chip engineering and direct surgical anastomosis

We report the fabrication of a scaffold (hereafter referred to as AngioChip) that supports the assembly of parenchymal cells on a mechanically tunable matrix surrounding a perfusable, branched, three-dimensional microchannel network coated with endothelial cells. The design of AngioChip decouples the material choices for the engineered vessel network and for cell seeding in the parenchyma, enabling extensive remodelling while maintaining an open-vessel lumen. The incorporation of nanopores and micro-holes in the vessel walls enhances permeability, and permits intercellular crosstalk and extravasation of monocytes and endothelial cells on biomolecular stimulation. We also show that vascularized hepatic tissues and cardiac tissues engineered by using AngioChips process clinically relevant drugs delivered through the vasculature, and that millimeter-thick cardiac tissues can be engineered in a scalable manner. Moreover, we demonstrate that AngioChip cardiac tissues implanted via direct surgical anastomosis to the femoral vessels of rat hindlimbs establish immediate blood perfusion.

Perfusable branching microvessel bed for vascularization of engineered tissues

Vascularization is critical for the survival of engineered tissues in vitro and in vivo. In vivo, angiogenesis involves endothelial cell proliferation and sprouting followed by connection of extended cellular processes and subsequent lumen propagation through vacuole fusion. We mimicked this process in engineering an organized capillary network anchored by an artery and a vein. The network was generated by inducing directed capillary sprouting from vascular explants on micropatterned substrates containing thymosin β4-hydrogel. The capillary outgrowths connected between the parent explants by day 21, a process that was accelerated to 14 d by application of soluble VEGF and hepatocyte growth factor. Confocal microscopy and transmission electron microscopy indicated the presence of tubules with lumens formed by endothelial cells expressing CD31, VE-cadherin, and von Willebrand factor. Cardiac tissues engineered around the resulting vasculature exhibited improved functional properties, cell striations, and cell–cell junctions compared with tissues without prevascularization. This approach uniquely allows easy removal of the vasculature from the microfabricated substrate and easy seeding of the tissue specific cell types in the parenchymal space.

Biomaterial based cardiac tissue engineering and its applications.

Cardiovascular disease is a leading cause of death worldwide, necessitating the development of effective treatment strategies. A myocardial infarction involves the blockage of a coronary artery leading to depletion of nutrient and oxygen supply to cardiomyocytes and massive cell death in a region of the myocardium. Cardiac tissue engineering is the growth of functional cardiac tissue in vitro on biomaterial scaffolds for regenerative medicine application. This strategy relies on the optimization of the complex relationship between cell networks and biomaterial properties. In this review, we discuss important biomaterial properties for cardiac tissue engineering applications, such as elasticity, degradation, and induced host response, and their relationship to engineered cardiac cell environments. With these properties in mind, we also emphasize in vitro use of cardiac tissues for high-throughput drug screening and disease modelling.

Flexible shape-memory scaffold for minimally invasive delivery of functional tissues

Despite great progress in engineering functional tissues for organ repair, including the heart, an invasive surgical approach is still required for their implantation. Here, we designed an elastic and microfabricated scaffold using a biodegradable polymer (poly(octamethylene maleate (anhydride) citrate)) for functional tissue delivery via injection. The scaffolds shape memory was due to the microfabricated lattice design. Scaffolds and cardiac patches (1 cm × 1 cm) were delivered through an orifice as small as 1 mm, recovering their initial shape following injection without affecting cardiomyocyte viability and function. In a subcutaneous syngeneic rat model, injection of cardiac patches was equivalent to open surgery when comparing vascularization, macrophage recruitment and cell survival. The patches significantly improved cardiac function following myocardial infarction in a rat, compared with the untreated controls. Successful minimally invasive delivery of human cell-derived patches to the epicardium, aorta and liver in a large-animal (porcine) model was achieved.

Cardiac Tissue Vascularization From Angiogenesis to Microfluidic Blood Vessels

Myocardial infarction results from a blockage of a major coronary artery that shuts the delivery of oxygen and nutrients to a region of the myocardium, leading to massive cardiomyocytes death and regression of microvasculature. Growth factor and cell delivery methods have been attempted to revascularize the ischemic myocardium and prevent further cell death. Implantable cardiac tissue patches were engineered to directly revascularize as well as remuscularize the affected muscle. However, inadequate vascularization in vitro and in vivo limits the efficacy of these new treatment options. Breakthroughs in cardiac tissue vascularization will profoundly impact ischemic heart therapies. In this review, we discuss the full spectrum of vascularization approaches ranging from biological angiogenesis to microfluidic blood vessels as related to cardiac tissue engineering.

Platform technology for scalable assembly of instantaneously functional mosaic tissues

A biodegradable polymer-based approach enables vertical and horizontal assembly of living tissues in a single step using a hook and look mechanism. Engineering mature tissues requires a guided assembly of cells into organized three-dimensional (3D) structures with multiple cell types. Guidance is usually achieved by microtopographical scaffold cues or by cell-gel compaction. The assembly of individual units into functional 3D tissues is often time-consuming, relying on cell ingrowth and matrix remodeling, whereas disassembly requires an invasive method that includes either matrix dissolution or mechanical cutting. We invented Tissue-Velcro, a bio-scaffold with a microfabricated hook and loop system. The assembly of Tissue-Velcro preserved the guided cell alignment realized by the topographical features in the 2D scaffold mesh and allowed for the instant establishment of coculture conditions by spatially defined stacking of cardiac cell layers or through endothelial cell coating. The assembled cardiac 3D tissue constructs were immediately functional as measured by their ability to contract in response to electrical field stimulation. Facile, on-demand tissue disassembly was demonstrated while preserving the structure, physical integrity, and beating function of individual layers.

Spatial and Electrical Factors Regulating Cardiac Regeneration and Assembly

Functional human engineered cardiac tissue may one day be used to repair defects and disastrous results of myocardial infarction or used in high throughput drug screening and development applications. Recent advances in stem cell biology allow the production of immature autologous human cardiomyocytes. Tissue engineers aim to mature these cells to produce high fidelity human engineered cardiac tissue. Generating adult-like human cardiac tissue in vitro requires the application of cues that guide cellular self-assembly to resemble native tissue morphology and function. The myocardium is a highly organized tissue that allows anisotropic action potential propagation and contraction resulting in an efficient pumping action. Factors that guide cardiac assembly in vitro can be assigned into two main groups: spatial and electrical. Anisotropic topographical cues engineered into scaffolds can direct cardiomyocyte assembly. Static and cyclic stretch can be applied during the culture of cardiac tissue to promote alignment and stimulate hypertrophy. Application of an external electric field during culture can promote cellular alignment, tissue ultrastructure, and calcium handling. Biomaterials can be designed to facilitate action potential propagation throughout the cardiac tissue. Here we give a comprehensive review of spatial and electrical cues that direct the assembly of engineered cardiac tissue.

Microfabrication of AngioChip, a biodegradable polymer scaffold with microfluidic vasculature

Microengineered biomimetic systems for organ-on-a-chip or tissue engineering purposes often fail as a result of an inability to recapitulate the in vivo environment, specifically the presence of a well-defined vascular system. To address this limitation, we developed an alternative method to cultivate three-dimensional (3D) tissues by incorporating a microfabricated scaffold, termed AngioChip, with a built-in perfusable vascular network. Here, we provide a detailed protocol for fabricating the AngioChip scaffold, populating it with endothelial cells and parenchymal tissues, and applying it in organ-on-a-chip drug testing in vitro and surgical vascular anastomosis in vivo. The fabrication of the AngioChip scaffold is achieved by a 3D stamping technique, in which an intricate microchannel network can be embedded within a 3D scaffold. To develop a vascularized tissue, endothelial cells are cultured in the lumen of the AngioChip network, and parenchymal cells are encapsulated in hydrogels that are amenable to remodeling around the vascular network to form functional tissues. Together, these steps yield a functional, vascularized network in vitro over a 14-d period. Finally, we demonstrate the functionality of AngioChip-vascularized hepatic and cardiac tissues, and describe direct surgical anastomosis of the AngioChip vascular network on the hind limb of a Lewis rat model.This protocol describes the fabrication of AngioChip, a microfabricated scaffold for engineering vascularized tissues. In addition, the protocol contains procedures for immunostaining, drug screening and surgical anastomosis of the AngioChip.