Shulamit Levenberg

Engineering vascularized skeletal muscle tissue

One of the major obstacles in engineering thick, complex tissues such as muscle is the need to vascularize the tissue in vitro. Vascularization in vitro could maintain cell viability during tissue growth, induce structural organization and promote vascularization upon implantation. Here we describe the induction of endothelial vessel networks in engineered skeletal muscle tissue constructs using a three-dimensional multiculture system consisting of myoblasts, embryonic fibroblasts and endothelial cells coseeded on highly porous, biodegradable polymer scaffolds. Analysis of the conditions for induction and stabilization of the vessels in vitro showed that addition of embryonic fibroblasts increased the levels of vascular endothelial growth factor expression in the construct and promoted formation and stabilization of the endothelial vessels. We studied the survival and vascularization of the engineered muscle implants in vivo in three different models. Prevascularization improved the vascularization, blood perfusion and survival of the muscle tissue constructs after transplantation.

Nanoliter-scale synthesis of arrayed biomaterials and application to human embryonic stem cells

Identification of biomaterials that support appropriate cellular attachment, proliferation and gene expression patterns is critical for tissue engineering and cell therapy. Here we describe an approach for rapid, nanoliter-scale synthesis of biomaterials and characterization of their interactions with cells. We simultaneously characterize over 1,700 human embryonic stem cell–material interactions and identify a host of unexpected materials effects that offer new levels of control over human embryonic stem cell behavior.

Differentiation of human embryonic stem cells on three-dimensional polymer scaffolds

Human embryonic stem (hES) cells hold promise as an unlimited source of cells for transplantation therapies. However, control of their proliferation and differentiation into complex, viable 3D tissues is challenging. Here we examine the use of biodegradable polymer scaffolds for promoting hES cell growth and differentiation and formation of 3D structures. We show that complex structures with features of various committed embryonic tissues can be generated, in vitro, by using early differentiating hES cells and further inducing their differentiation in a supportive 3D environment such as poly(lactic-co-glycolic acid)/poly(l-lactic acid) polymer scaffolds. We found that hES cell differentiation and organization can be influenced by the scaffold and directed by growth factors such as retinoic acid, transforming growth factor β, activin-A, or insulin-like growth factor. These growth factors induced differentiation into 3D structures with characteristics of developing neural tissues, cartilage, or liver, respectively. In addition, formation of a 3D vessel-like network was observed. When transplanted into severe combined immunodeficient mice, the constructs continue to express specific human proteins in defined differentiated structures and appear to recruit and anastamose with the host vasculature. This approach provides a unique culture system for addressing questions in cell and developmental biology, and provides a potential mechanism for creating viable human tissue structures for therapeutic applications.

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.

Advances in Tissue Engineering

The clinical goals of tissue engineering are to restore, repair, or replace damaged or lost tissues in the body. Significant progress has been made in recent years, which includes the use of cells or polymer scaffolds as well as combinations of cells and polymers for engineering three-dimensional tissue constructs. However, major challenges still need to be addressed in order for these studies to progress into their clinical applications. The challenges include (1) developing functional polymers, (2) exploring more sources of human cells, and (3) finding ways to keep the engineered construct viable in vitro and in vivo. In addition to clinical applications, tissue engineering can provide new tools for studying cell and developmental biology by providing approaches for cell and tissue growth in three-dimensional environments. In this review we describe recent attempts in addressing some of the challenges of tissue engineering and discuss how such approaches may provide new insights into regulation of cell growth and differentiation.

p27 is involved in N-cadherin-mediated contact inhibition of cell growth and S-phase entry

In this study the direct involvement of cadherins in adhesion-mediated growth inhibition was investigated. It is shown here that overexpression of N-cadherin in CHO cells significantly suppresses their growth rate. Interaction of these cells and two additional fibroblastic lines with synthetic beads coated with N-cadherin ligands (recombinant N-cadherin ectodomain or specific antibodies) leads to growth arrest at the G1 phase of the cell cycle. The cadherin-reactive beads inhibit the entry into S phase and the reduction in the levels of cyclin-dependent kinase (cdk) inhibitors p21 and p27, following serum-stimulation of starved cells. In exponentially growing cells these beads induce G1 arrest accompanied by elevation in p27 only. We propose that cadherin-mediated signaling is involved in contact inhibition of growth by inducing cell cycle arrest at the G1 phase and elevation of p27 levels.

Cell–scaffold mechanical interplay within engineered tissue

Effective tissue engineering requires appropriate selection of cells and scaffold, where the latter serves as a mechanical and biological support for cell growth and functionality. The optimal combination of cell source and scaffold properties can vary for each desired application. Such preconditions necessitate enhanced understanding of the interactions between cells and scaffold within engineered tissue. Several studies have examined the deforming effects cells induce in scaffolds via exertion of contractile forces. In contrast, other studies focus on the scaffolds biochemical and mechanical properties and their effects on cell behavior. This review summarizes the mechanical interplay between cells and scaffold within engineered tissue. We present evidence for contractile forces exerted by cells on three-dimensional (3D) scaffolds and discuss existing methods for their quantification. In addition, we address some theories related to the effects of scaffold stiffness and mechanical stimulation on cell behavior. Further understanding of the reciprocal effects between cells and scaffold will provide both enhanced knowledge regarding the expected properties of engineered tissue and more competent tissue regeneration techniques.

Improved vascular organization enhances functional integration of engineered skeletal muscle grafts

Severe traumatic events such as burns, and cancer therapy, often involve a significant loss of tissue, requiring surgical reconstruction by means of autologous muscle flaps. The scant availability of quality vascularized flaps and donor site morbidity often limit their use. Engineered vascularized grafts provide an alternative for this need. This work describes a first-time analysis, of the degree of in vitro vascularization and tissue organization, required to enhance the pace and efficacy of vascularized muscle graft integration in vivo. While one-day in vitro was sufficient for graft integration, a three-week culturing period, yielding semiorganized vessel structures and muscle fibers, significantly improved grafting efficacy. Implanted vessel networks were gradually replaced by host vessels, coupled with enhanced perfusion and capillary density. Upregulation of key graft angiogenic factors suggest its active role in promoting the angiogenic response. Transition from satellite cells to mature fibers was indicated by increased gene expression, increased capillary to fiber ratio, and similar morphology to normal muscle. We suggest a “relay” approach in which extended in vitro incubation, enabling the formation of a more structured vascular bed, allows for graft-host angiogenic collaboration that promotes anastomosis and vascular integration. The enhanced angiogenic response supports enhanced muscle regeneration, maturation, and integration.

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.