Ayelet Lesman

Tissue Engineering of Vascularized Cardiac Muscle From Human Embryonic Stem Cells

Transplantation of a tissue-engineered heart muscle represents a novel experimental therapeutic paradigm for myocardial diseases. However, this strategy has been hampered by the lack of sources for human cardiomyocytes and by the scarce vasculature in the ischemic area limiting the engraftment and survival of the transplanted muscle. Beyond the necessity of endothelial capillaries for the delivery of oxygen and nutrients to the grafted muscle tissue, interactions between endothelial and cardiomyocyte cells may also play a key role in promoting cell survival and proliferation. In the present study, we describe the formation of synchronously contracting engineered human cardiac tissue derived from human embryonic stem cells containing endothelial vessel networks. The 3D muscle consisted of cardiomyocytes, endothelial cells (ECs), and embryonic fibroblasts (EmFs). The formed vessels were further stabilized by the presence of mural cells originating from the EmFs. The presence of EmFs decreased EC death and increased EC proliferation. Moreover, the presence of endothelial capillaries augmented cardiomyocyte proliferation and did not hamper cardiomyocyte orientation and alignment. Immunostaining, ultrastructural analysis (using transmission electron microscopy), RT-PCR, pharmacological, and confocal laser calcium imaging studies demonstrated the presence of cardiac-specific molecular, ultrastructural, and functional properties of the generated tissue constructs with synchronous activity mediated by action potential propagation through gap junctions. In summary, this is the first report of the construction of 3D vascularized human cardiac tissue that may have unique applications for studies of cardiac development, function, and tissue replacement therapy.

Vascularization—The Conduit to Viable Engineered Tissues

Long-term viability of thick three-dimensional engineered tissue constructs is a major challenge. Addressing it requires development of vessel-like network that will allow the survival of the construct in vitro and its integration in vivo owing to improved vascularization after implantation. Resulting from work of various research groups, several approaches were developed aiming engineered tissue vascularization: (1) embodiment of angiogenesis growth factors in the polymeric scaffolds for prolonged release, (2) coculture of endothelial cells with target tissue cells and angiogenesis signaling cells, (3) use of microfabrication methods for creating designed channels for allowing nutrients to flow and/or for directing endothelial cells attachment, and (4) decellularization of organs and blood vessels for creating extracellular matrix. A synergistic effect is expected by combining several of these approaches as already demonstrated in some of the latest studies. Current paper reviews the progress in each approach and recent achievements toward vascularization of engineered tissues.

Engineering vessel-like networks within multicellular fibrin-based constructs

Sufficient vascularization in engineered tissues can be achieved through coordinated application of improved biomaterial systems with proper cell types. In this study, we employed 3D fibrin gels alone or in combination with the synthetic poly(l-lactic acid) (PLLA)/polylactic-glycolic acid (PLGA) sponges to support in-vitro construct vascularization and to enhance neovascularization upon implantation. Two multicellular assays were embedded in these constructs: (a) co-culture of endothelial (EC) and fibroblast cells, and (b) a tri-culture combination of ECs, fibroblasts and tissue specific skeletal myoblast cells. In-vitro vessel network formation was examined under advanced confocal microscopy in various time points from cell seeding. Vessel network maturity levels and morphology were found to be highly regulated by fibrinogen concentrations in-vitro. Combination of PLLA/PLGA sponges with fibrin matrices provided added mechanical strength and featured highly mature vessels-like networks. Implantation studies revealed that the implanted ECs developed into 3D interconnected vessel-like networks in-vivo. The PLLA/PLGA scaffold proved to be a key stimulator of neovascularization and perfusion of implanted grafts. Our findings demonstrate that complex biomaterial platform involving fibrin and PLLA/PLGA synthetic scaffold provide a way to enhancing vascularization in-vitro and in-vivo.

Modeling of flow-induced shear stress applied on 3D cellular scaffolds: Implications for vascular tissue engineering.

Novel tissue‐culture bioreactors employ flow‐induced shear stress as a means of mechanical stimulation of cells. We developed a computational fluid dynamics model of the complex three‐dimensional (3D) microstructure of a porous scaffold incubated in a direct perfusion bioreactor. Our model was designed to predict high shear‐stress values within the physiological range of those naturally sensed by vascular cells (1–10 dyne/cm2), and will thereby provide suitable conditions for vascular tissue‐engineering experiments. The model also accounts for cellular growth, which was designed as an added cell layer grown on all scaffold walls. Five model variants were designed, with geometric differences corresponding to cell‐layer thicknesses of 0, 50, 75, 100, and 125 µm. Four inlet velocities (0.5, 1, 1.5, and 2 cm/s) were applied to each model. Wall shear‐stress distribution and overall pressure drop calculations were then used to characterize the relation between flow rate, shear stress, cell‐layer thickness, and pressure drop. The simulations showed that cellular growth within 3D scaffolds exposes cells to elevated shear stress, with considerably increasing average values in correlation to cell growth and inflow velocity. Our results provide in‐depth analysis of the microdynamic environment of cells cultured within 3D environments, and thus provide advanced control over tissue development in vitro. Biotechnol. Bioeng. 2010; 105: 645–654.

Contractile forces regulate cell division in three-dimensional environments

Live-cell imaging, combined with mapping of 3D matrix displacements, identifies sites at which cells apply contractile forces to the matrix and reveals roles for physical forces in cell division.

Vascularization shaping the heart

Myocardial infarction can lead to irreversible heart failure. In an attempt to restore function in the failing heart, tissue‐engineered cardiac constructs can be applied to repopulate scar tissue with a new pool of contractile cells. Effective engineering of viable thick complex tissue‐constructs requires intense vascularization. Furthermore, endothelial–cardiomyocyte crosstalk plays a key role in mutually enhancing tissue functionality, which can further improve construct survival. The ability to generate an engineered, vascularized muscle tissue was demonstrated by us using the skeletal and the cardiac muscle models. In the skeletal model, we showed that prevascularization of the construct promoted perfusion of the graft. More recently, we successfully generated a beating human cardiac muscle‐construct, containing an endothelial network, by co‐culturing human embryonic stem cell–derived‐cardiomyocytes, fibroblasts, and endothelial cells within biodegradable scaffolds. Such muscle‐constructs could contribute significantly to the emerging discipline of cardiovascular regenerative medicine as well as to the study of the important role of tissue vascularization.

Microbuckling of fibrin provides a mechanism for cell mechanosensing

Biological cells sense and respond to mechanical forces, but how such a mechanosensing process takes place in a nonlinear inhomogeneous fibrous matrix remains unknown. We show that cells in a fibrous matrix induce deformation fields that propagate over a longer range than predicted by linear elasticity. Synthetic, linear elastic hydrogels used in many mechanotransduction studies fail to capture this effect. We develop a nonlinear microstructural finite-element model for a fibre network to simulate localized deformations induced by cells. The model captures measured cell-induced matrix displacements from experiments and identifies an important mechanism for long-range cell mechanosensing: loss of compression stiffness owing to microbuckling of individual fibres. We show evidence that cells sense each other through the formation of localized intercellular bands of tensile deformations caused by this mechanism.

Cell Tri-Culture for Cardiac Vascularization

Poor graft survival is a critical obstacle toward production of clinically relevant engineered tissues. Here we utilize a multicellular culturing approach for induction of vascular networks embedded within cardiac tissue constructs. The construct is composed of human cardiomyocytes, endothelial cells (ECs), and embryonic fibroblast cells co-seeded onto highly porous three-dimensional (3D) scaffolds. The resulting vascularized cardiac constructs showed microstructural details characteristic of cardiomyocytes and nascent vessels and exhibited synchronous beating activity in vitro. Upon implantation, stable grafts were formed presenting intense vascularization, with evidence of anastomosis between the pre-formed endothelial capillaries and host neovessels.

Mechanical regulation of vascular network formation in engineered matrices

Generation of vessel networks within engineered tissues is critical for integration and perfusion of the implanted tissue in vivo. The effect of mechanical cues in guiding and stabilizing the vessels has begun to attract marked interest. This review surveys the impact of mechanical cues on formation of vascular networks in 2D and 3D gel matrices. We give less emphasis to regulation of endothelial monolayers and single endothelial cells. Several vascularization models have consistently found that the stress generated in the gel, and encountered by embedded cells, control various aspects of vascular network formation, including sprouting, branching, alignment, and vessel maturation. This internal stress is generated by cell contractile forces, and is balanced by gel stiffness and boundary constrains imposed on the gel. Actin and myosin II are key molecular players in controlling initiation of vessel sprouting and branching morphogenesis. Additionally, the impact of external mechanical cues on tissue vascularization, and studies supporting the notion that mechanical forces regulate vascularization in the live animal are reviewed.

Nonlinear Elasticity of the ECM Fibers Facilitates Efficient Intercellular Communication

Biological cells embedded in fibrous matrices have been observed to form intercellular bands of dense and aligned fibers through which they mechanically interact over long distances. Such matrix-mediated cellular interactions have been shown to regulate various biological processes. This study aimed to explore the effects of elastic nonlinearity of the fibers contained in the extracellular matrix (ECM) on the transmission of mechanical loads between contracting cells. Based on our biological experiments, we developed a finite-element model of two contracting cells embedded within a fibrous network. The individual fibers were modeled as showing linear elasticity, compression microbuckling, tension stiffening, or both of the latter two. Fiber compression buckling resulted in smaller loads in the ECM, which were primarily directed toward the neighboring cell. These loads decreased with increasing cell-to-cell distance; when cells were >9 cell diameters apart, no such intercellular interaction was observed. Tension stiffening further contributed to directing the loads toward the neighboring cell, though to a smaller extent. The contraction of two neighboring cells resulted in mutual attraction forces, which were considerably increased by tension stiffening and decayed with increasing cell-to-cell distances. Nonlinear elasticity contributed also to the onset of force polarity on the cell boundaries, manifested by larger contractile forces pointing toward the neighboring cell. The density and alignment of the fibers within the intercellular band were greater when fibers buckled under compression, with tension stiffening further contributing to this structural remodeling. Although previous studies have established the role of the ECM nonlinear mechanical behavior in increasing the range of force transmission, our model demonstrates the contribution of nonlinear elasticity of biological gels to directional and efficient mechanical signal transfer between distant cells, and rehighlights the importance of using fibrous gels in experimental settings for facilitating intercellular communication. VIDEO ABSTRACT.