Engineered tissue grafts: opportunities and challenges in regenerative medicine.

<p>The human body has limited regenerative capacity in most of the major tissues and organs. This fact has spurred the field of regenerative medicine, promising to repair damage following traumatic injury or disease. Multiple therapeutic strategies are being explored including small molecules, gene delivery, and stem cells; however, tissue engineering remains a primary approach to achieving regeneration. Organ transplantation demonstrates that damaged tissues can be replaced, but technology to regenerate complex organs de novo is not yet available. Instead, tissue engineering can augment the body's own regenerative ability by replacing tissue sections and enhancing the regenerative cascade. As a consequence of these opportunities, it is timely to review the criteria and current status of engineered tissue grafts designed as patches to replace or regenerate damaged or diseased tissue and restore organ function. This topic will be explored starting from the biomaterials and cells incorporated into the engineered graft, the environment into which the graft is implanted and the integration of the engineered graft with the host. Common issues will be addressed that are relevant to regeneration in multiple tissue and organ systems. Specific examples will focus on engineered grafts for myocardial and corneal repair to illustrate the tissue-specific challenges and opportunities and highlight the innovation needed as the field moves forward.</p

Fabrication of freestanding alginate microfibers and microstructures for tissue engineering applications.

<p>Natural biopolymers such as alginate have become important materials for a variety of biotechnology applications including drug delivery, cell encapsulation and tissue engineering. This expanding use has spurred the development of new approaches to engineer these materials at the nano- and microscales to better control cell interactions. Here we describe a method to fabricate freestanding alginate-based microfibers and microstructures with tunable geometries down to approximately 3 µm. To do this, a polydimethylsiloxane stamp is used to micromold alginate or alginate-fibrin blends onto a sacrificial layer of thermally-sensitive poly(N-isopropylacrylamide) (PIPAAm). A warm calcium chloride solution is then used to crosslink the alginate and, upon cooling below the lower critical solution temperature (~32 °C), the PIPAAm layer dissolves and releases the alginate or alginate-fibrin as freestanding microfibers and microstructures. Proof-of-concept experiments demonstrate that C2C12 myoblasts seeded onto the alginate-fibrin microfibers polarize along the fiber length forming interconnected cell strands. Thus, we have developed the ability to engineer alginate-based microstructured materials that can selectively bind cells and direct cellular assembly.</p

Surface-initiated assembly of protein nanofabrics.

Cells and tissues are self-organized within an extracellular matrix (ECM) composed of multifunctional, nano- to micrometer scale protein fibrils. We have developed a cell-free, surface-initiated assembly technique to rebuild this ECM structure in vitro. The matrix proteins fibronectin, laminin, fibrinogen, collagen type I, and collagen type IV are micropatterned onto thermosensitive surfaces as 1 to 10 nm thick, micrometer to centimeter wide networks, and released as flexible, free-standing nanofabrics. Independent control of microstructure and protein composition enables us to engineer the mechanical and chemical anisotropy. Fibronectin nanofabrics are highly extensible (>4-fold) and serve as scaffolds for engineering synchronously contracting, cardiac muscle; demonstrating biofunctionality comparable to cell-generated ECM.</p

Engineered skeletal muscle tissue for soft robotics: fabrication strategies, current applications, and future challenges.

<p>Skeletal muscle is a scalable actuator system used throughout nature from the millimeter to meter length scales and over a wide range of frequencies and force regimes. This adaptability has spurred interest in using engineered skeletal muscle to power soft robotics devices and in biotechnology and medical applications. However, the challenges to doing this are similar to those facing the tissue engineering and regenerative medicine fields; specifically, how do we translate our understanding of myogenesis in vivo to the engineering of muscle constructs in vitro to achieve functional integration with devices. To do this researchers are developing a number of ways to engineer the cellular microenvironment to guide skeletal muscle tissue formation. This includes understanding the role of substrate stiffness and the mechanical environment, engineering the spatial organization of biochemical and physical cues to guide muscle alignment, and developing bioreactors for mechanical and electrical conditioning. Examples of engineered skeletal muscle that can potentially be used in soft robotics include 2D cantilever-based skeletal muscle actuators and 3D skeletal muscle tissues engineered using scaffolds or directed self-organization. Integration into devices has led to basic muscle-powered devices such as grippers and pumps as well as more sophisticated muscle-powered soft robots that walk and swim. Looking forward, current, and future challenges include identifying the best source of muscle precursor cells to expand and differentiate into myotubes, replacing cardiomyocytes with skeletal muscle tissue as the bio-actuator of choice for soft robots, and vascularization and innervation to enable control and nourishment of larger muscle tissue constructs.</p

ECM protein nanofibers and nanostructures engineered using surface-initiated assembly.

<p>The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.</p

Nano- and Microstructured ECM and Biomimetic Scaffolds for Cardiac Tissue Engineering

<p>This chapter provides an overview of the heart and the biomimetic approaches used to engineer cardiac muscle tissue. It first describes the unique structure and function of cardiac muscle (myocardium), discusses how this dictates scaffold design, and then defines the benchmarks used to evaluate the performance of engineered cardiac tissue. Next, the chapter presents the different techniques researchers have developed to fabricate tissue-engineered cardiac scaffolds that mimic extracellular matrix (ECM) cues observed in the heart. For each case, the relevant physical, mechanical and/or chemical properties and the particular advantages and limitations of the approach, are discussed. Finally, the chapter discusses the persistent challenges to engineering functional cardiac tissue and the future directions of the field. Nano- and microfabrication techniques for cardiac tissue engineering enable the formation of scaffolds with anisotropic structure on the same scale as fibers in the native cardiac ECM.</p

Development of polydimethylsiloxane substrates with tunable elastic modulus to study cell mechanobiology in muscle and nerve.

<p>Mechanics is an important component in the regulation of cell shape, proliferation, migration and differentiation during normal homeostasis and disease states. Biomaterials that match the elastic modulus of soft tissues have been effective for studying this cell mechanobiology, but improvements are needed in order to investigate a wider range of physicochemical properties in a controlled manner. We hypothesized that polydimethylsiloxane (PDMS) blends could be used as the basis of a tunable system where the elastic modulus could be adjusted to match most types of soft tissue. To test this we formulated blends of two commercially available PDMS types, Sylgard 527 and Sylgard 184, which enabled us to fabricate substrates with an elastic modulus anywhere from 5 kPa up to 1.72 MPa. This is a three order-of-magnitude range of tunability, exceeding what is possible with other hydrogel and PDMS systems. Uniquely, the elastic modulus can be controlled independently of other materials properties including surface roughness, surface energy and the ability to functionalize the surface by protein adsorption and microcontact printing. For biological validation, PC12 (neuronal inducible-pheochromocytoma cell line) and C2C12 (muscle cell line) were used to demonstrate that these PDMS formulations support cell attachment and growth and that these substrates can be used to probe the mechanosensitivity of various cellular processes including neurite extension and muscle differentiation.</p

Optimizing the structure and contractility of engineered skeletal muscle thin films.

<p>An experimental system was developed to tissue engineer skeletal muscle thin films with well-defined tissue architecture and to quantify the effect on contractility. Using the C2C12 cell line, the authors tested whether tailoring the width and spacing of micropatterned fibronectin lines can be used to increase myoblast differentiation into functional myotubes and maximize uniaxial alignment within a 2-D sheet. Using a combination of image analysis and the muscular thin film contractility assay, it was demonstrated that a fibronectin line width of 100μm and line spacing of 20μm is able to maximize the formation of anisotropic, engineered skeletal muscle with consistent contractile properties at the millimeter length scale. The engineered skeletal muscle exhibited a positive force-frequency relationship, could achieve tetanus and produced a normalized peak twitch stress of 9.4±4.6kPa at 1Hz stimulation. These results establish that micropatterning technologies can be used to control skeletal muscle differentiation and tissue architecture and, in combination with the muscular thin film contractility, assay can be used to probe structure-function relationships. More broadly, an experimental platform is provided with the potential to examine how a range of microenvironmental cues such as extracellular matrix protein composition, micropattern geometries and substrate mechanics affect skeletal muscle myogenesis and contractility.</p

Biohybrid thin films for measuring contractility in engineered cardiovascular muscle.

In vitro cardiovascular disease models need to recapitulate tissue-scale function in order to provide in vivo relevance. We have developed a new method for measuring the contractility of engineered cardiovascular smooth and striated muscle in vitro during electrical and pharmacological stimulation. We present a growth theory-based finite elasticity analysis for calculating the contractile stresses of a 2D anisotropic muscle tissue cultured on a flexible synthetic polymer thin film. Cardiac muscle engineered with neonatal rat ventricular myocytes and paced at 0.5 Hz generated stresses of 9.2 +/- 3.5 kPa at peak systole, similar to measurements of the contractility of papillary muscle from adult rats. Vascular tissue engineered with human umbilical arterial smooth muscle cells maintained a basal contractile tone of 13.1 +/- 2.1 kPa and generated another 5.1 +/- 0.8 kPa when stimulated with endothelin-1. These data suggest that this method may be useful in assessing the efficacy and safety of pharmacological agents on cardiovascular tissue.</p

Shrink Wrapping Cells in a Defined Extracellular Matrix to Modulate the Chemo-Mechanical Microenvironment

<p>Cell–matrix interactions are important for the physical integration of cells into tissues and the function of insoluble, mechanosensitive signaling networks. Studying these interactions <em>in vitro</em> can be difficult because the extracellular matrix (ECM) proteins that adsorb to <em>in vitro</em> cell culture surfaces do not fully recapitulate the ECM-dense basement membranes to which cells such as cardiomyocytes and endothelial cells adhere to <em>in vivo</em>. Towards addressing this limitation, we have developed a surface-initiated assembly process to engineer ECM proteins into nanostructured, microscale sheets that can be shrink wrapped around single cells and small cell ensembles to provide a functional and instructive matrix niche. Unlike current cell encapsulation technology using alginate, fibrin or other hydrogels, our engineered ECM is similar in density and thickness to native basal lamina and can be tailored in structure and composition using the proteins fibronectin, laminin, fibrinogen, and/or collagen type IV. A range of cells including C2C12 myoblasts, bovine corneal endothelial cells and cardiomyocytes survive the shrink wrapping process with high viability. Further, we demonstrate that, compared to non-encapsulated controls, the engineered ECM modulates cytoskeletal structure, stability of cell–matrix adhesions and cell behavior in 2D and 3D microenvironments.</p