Search

In this study, we created self-assembled smooth muscle cell (SMC) tissue rings (comprised entirely of cells and cell-derived matrix; CDM) and compared their structure and material properties with tissue rings created from SMC-seeded fibrin or collagen gels. All tissue rings were cultured statically for 7 days in supplemented growth medium (with ε-amino caproic acid, ascorbic acid, and insulin-transferrin-selenium), prior to uniaxial tensile testing and histology. Self-assembled CDM rings exhibited ultimate tensile strength and stiffness values that were two-fold higher than fibrin gel and collagen gel rings. Tensile testing of CDM, fibrin gel and collagen gel rings treated with deionized water to lyse cells showed little to no change in mechanical properties relative to untreated ring samples, indicating that the ECM dominates the measured ring mechanics. In addition, CDM rings cultured in supplemented growth medium were significantly stronger than CDM rings cultured in standard, unsupplemented growth medium. These results illustrate the potential utility of self-assembled cell rings as model CDM constructs for tissue engineering and biomechanical analysis of ECM material properties., (Copyright © 2012 Wiley Periodicals, Inc.)

During heart valve remodeling and in many disease states, valvular interstitial cells (VICs) shift to an activated myofibroblast phenotype characterized by enhanced synthetic and contractile activity. Pronounced alpha smooth muscle actin (αSMA)-positive stress fibers, the hallmark of activated myofibroblasts, are also observed in VICs cultured on stiff substrates especially in the presence of transforming growth factor-beta1 (TGF-β1), however, the detailed relationship between stiffness and VIC phenotype has not been explored. The goal of this study was to characterize VIC activation as a function of substrate stiffness over a wide range of stiffness levels including that of diseased valves (stiff), normal valves (compliant), and hydrogels for heart valve tissue engineering (very soft). VICs obtained from porcine aortic valves were cultured on stiff tissue culture plastic to activate them, then, cultured on collagen-coated polyacrylamide substrates of predefined stiffness in a high-throughput culture system to assess the persistence of activation. Metrics extracted from regression analysis demonstrate that relative to a compliant substrate, stiff substrates result in higher cell numbers, more pronounced expression of αSMA-positive stress fibers, and larger spread area which is in qualitative agreement with previous studies. Our data also indicate that VICs require a much lower substrate stiffness level to "deactivate" them than previously thought. The high sensitivity of VICs to substrate stiffness demonstrates the importance of the mechanical properties of materials used for valve repair or for engineering valve tissue., (Copyright © 2012 Wiley Periodicals, Inc.)

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.(1) Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.(2-5) Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues. To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.(6,7) These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength(8) which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (<30 kPa).(9,10) Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

Transforming growth factor-beta1 (TGF-beta1) is commonly used to promote matrix production for engineered tissues in vitro, yet it also enhances fibroblast contractility. For applications where contraction is undesirable, we hypothesized that epidermal growth factor (EGF) would yield equivalent mechanical properties without enhancing contractility. In this study, the response of human dermal fibroblasts to EGF (5 ng/mL) and TGF-beta1 (5 ng/mL) was determined within hemispheric fibrin-based gels by assessing matrix compaction and strength, cell number, collagen production, and contractility. After 3 weeks, both cytokines enhanced compaction relative to controls, and EGF roughly doubled matrix strength over controls and TGF-beta1-treated samples. TGF-beta1 induced alpha-smooth muscle actin (alphaSMA) expression whereas EGF did not. TGF-beta1 also increased retraction following substrate release while EGF reduced retraction. Treatment with cytochalasin D revealed that, regardless of growth factor, approximately 10% of the total retraction was due to residual matrix stress accumulated during cell-mediated remodeling. EGF increased the cell number by 17%, whereas TGF-beta1 decreased the cell number by 63% relative to controls. EGF and TGF-beta1 stimulated greater collagen content than controls by 49% and 33%, respectively. These data suggest that EGF may be an attractive alternative to TGF-beta1 for engineering fibrin-based connective tissue substitutes with adequate strength and minimal tissue contractility.

A new acellular, natural, biodegradable matrix has been discovered in the cholecyst-derived extracellular matrix (CEM). This matrix is rich in collagen and contains several other macromolecules useful in tissue remodeling. In this study, the principal material axes, collagen fiber orientations, and biaxial mechanical properties in a physiological loading regime were characterized. Fiber direction was determined by polarized light microscopy, and the principal axes and degree of anisotropy were determined mechanically. Macroscopic equibiaxial strain tests were then conducted on preconditioned specimens. While 13% of the area of CEM contains collagen fibers oriented between 50 degrees and 60 degrees from the neck-fundus axis, the principal material axis was oriented 63 degrees +/- 13.7 degrees , with an aspect ratio of 0.11 +/- 0.06, indicating a weak anisotropy in that direction. Under biaxial loading, CEM exhibited a large toe region followed by an exponential rise in stress in both principal and perpendicular axis directions, similar to other materials currently under research. There was no significant difference between the biaxial stress-strain profile and the burst stress-strain profile. The results demonstrate that CEM is weakly anisotropic and it has the ability to support large strains across a physiological loading regime., ((c) 2006 Wiley Periodicals, Inc.)

Understanding the effects of the mechanical environment on wound healing is critical for developing more effective treatments to reduce scar formation and contracture. The aim of this study was to investigate the effects of dynamic mechanical stretch on cell-mediated early wound remodeling independent of matrix alignment which obscures more subtle remodeling mechanisms. Cyclic equibiaxial stretch (16% stretch at 0.2 Hz) was applied to fibroblast-populated fibrin gel in vitro wound models for eight days. Compaction, density, tensile strength, and collagen content were quantified as functional measures of remodeling. Stretched samples were approximately ten times stronger, eight-fold more dense, and eight times thinner than statically cultured samples. These changes were accompanied by a 15% increase in net collagen but no significant differences in cell number or viability. When collagen crosslinking was inhibited in stretched samples, the extensibility increased and the strength decreased. The apparent weakening was due to a reduction in compaction rather than a decrease in ability of the tissue to withstand tensile forces. Interestingly, inhibiting collagen crosslinking had no measurable effects on the statically cultured samples. These results indicate that amplified cell-mediated compaction and even a slight addition in collagen content play substantial roles in mechanically induced wound strengthening. These findings increase our understanding of how mechanical forces guide the healing response in skin, and the methods employed in this study may also prove valuable tools for investigating stretch-induced remodeling of other planar connective tissues and for creating mechanically robust engineered tissues.

The propagation of mechanical signals through nonlinear fibrous tissues is much more extensive than through continuous synthetic hydrogels. Results from recent studies indicate that increased mechanical propagation arises from the fibrous nature of the material rather than the strain-stiffening property. The relative importance of different parameters of the fibrous network structure to this propagation, however, remains unclear. In this work, we directly compared the mechanical response of substrates of varying thickness subjected to a constant cell traction force using either a nonfibrous strain-stiffening continuum-based model or a volume-averaged fiber network model consisting of two different types of fiber network structures: one with low fiber connectivity (growth networks) and one with high fiber connectivity (Delaunay networks). The growth network fiber models predicted a greater propagation of substrate displacements through the model and a greater sensitivity to gel thickness compared to the more connected Delaunay networks and the nonlinear continuum model. Detailed analysis of the results indicates that rotational freedom of the fibers in a network with low fiber connectivity is critically important for enhanced, long-range mechanosensing. Our findings demonstrate the utility of multiscale models in predicting cells mechanosensing on fibrous gels, and they provide a more complete understanding of how cell traction forces propagate through fibrous tissues, which has implications for the design of engineered tissues and the stem cell niche. [ABSTRACT FROM AUTHOR]

The goal of this study was to develop a system to rapidly generate engineered tissue constructs from aggregated cells and cell-derived extracellular matrix (ECM) to enable evaluation of cell-derived tissue structure and function. Rat aortic smooth muscle cells seeded into annular agarose wells (2, 4 or 6 mm inside diameter) aggregated and formed thick tissue rings within 2 weeks of static culture (0.76 mm at 8 days; 0.94 mm at 14 days). Overall, cells appeared healthy and surrounded by ECM comprised of glycosoaminoglycans and collagen, although signs of necrosis were observed near the centers of the thickest rings. Tissue ring strength and stiffness values were superior to those reported for engineered tissue constructs cultured for comparable times. The strength (100-500 kPa) and modulus (0.5-2 MPa) of tissue rings increased with ring size and decreased with culture duration. Finally, tissue rings cultured for 7 days on silicone mandrels fused to form tubular constructs. Ring margins were visible after 7 days, but tubes were cohesive and mechanically stable, and histological examination confirmed fusion between ring subunits. This unique system provides a versatile new tool for optimization and functional assessment of cell-derived tissue, and a new approach to creating tissue-engineered vascular grafts. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Mechanical cues modulate fibroblast tractional forces and remodeling of extracellular matrix in healthy tissue, healing wounds, and engineered matrices. The goal of the present study is to establish dose-response relationships between stretch parameters (magnitude and duration per day) and matrix remodeling metrics (compaction, strength, extensibility, collagen content, contraction, and cellularity). Cyclic equibiaxial stretch of 2-16% was applied to fibroblast-populated fibrin gels for either 6 h or 24 h/day for 8 days. Trends in matrix remodeling metrics as a function of stretch magnitude and duration were analyzed using regression analysis. The compaction and ultimate tensile strength of the tissues increased in a dose-dependent manner with increasing stretch magnitude, yet remained unaffected by the duration in which they were cycled (6 h/day versus 24 h/day). Collagen density increased exponentially as a function of both the magnitude and duration of stretch, with samples stretched for the reduced duration per day having the highest levels of collagen accumulation. Cell number and failure tension were also dependent on both the magnitude and duration of stretch, although stretch-induced increases in these metrics were only present in the samples loaded for 6 h/day. Our results indicate that both the magnitude and the duration per day of stretch are critical parameters in modulating fibroblast remodeling of the extracellular matrix, and that these two factors regulate different aspects of this remodeling. These findings move us one step closer to fully characterizing culture conditions for tissue equivalents, developing improved wound healing treatments and understanding tissue responses to changes in mechanical environments during growth, repair, and disease states. [ABSTRACT FROM AUTHOR]

Abstract: Patients with repaired tetralogy of Fallot account for the majority of cases with late onset right ventricle (RV) failure. A new surgical procedure placing an elastic band in the right ventricle is proposed to improve RV function measured by ejection fraction. A multiphysics modeling approach is developed to combine cardiac magnetic resonance imaging, modeling, tissue engineering and mechanical testing to demonstrate feasibility of the new surgical procedure. Our modeling results indicated that the new surgical procedure has the potential to improve right ventricle ejection fraction by 2–7% which compared favorably with recently published drug trials to treat LV heart failure. [Copyright &y& Elsevier]