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In JoVE (1)
- Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering
Other Publications (14)
- The Annals of Thoracic Surgery
- Journal of Biomedical Materials Research. Part A
- Annals of Biomedical Engineering
- Journal of Biomechanics
- Journal of Biomechanical Engineering
- Journal of Biomechanical Engineering
- Biomechanics and Modeling in Mechanobiology
- Tissue Engineering. Part C, Methods
- Journal of Orthopaedic Surgery and Research
- Journal of Biomedical Materials Research. Part A
- Cells, Tissues, Organs
- PloS One
- Journal of Biomechanics
Articles by Kristen L. Billiar in JoVE
Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering
Tracy A. Gwyther, Jason Z. Hu, Kristen L. Billiar, Marsha W. Rolle
Biomedical Engineering Department, Worcester Polytechnic Institute
This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.
Other articles by Kristen L. Billiar on PubMed
The Annals of Thoracic Surgery. Sep, 2005 | Pubmed ID: 16122464
The incidence of severe sternal wound complications in high-risk cardiac patients presents a significant need for more stabile sternal fixation techniques after median sternotomy procedures. Rigid metal plates, a potential alternative to wire fixation, are thought to promote faster sternal healing by reducing motion at the wound site. The goal of this study was to compare the stability provided by commercially available sternal plates with standard wires using an in vitro model.
Biaxial Mechanical Evaluation of Cholecyst-derived Extracellular Matrix: a Weakly Anisotropic Potential Tissue Engineered Biomaterial
Journal of Biomedical Materials Research. Part A. Apr, 2007 | Pubmed ID: 17269134
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.
Biomaterials. Apr, 2007 | Pubmed ID: 17280714
We report on a culture method for the rapid production of a strong and thick natural matrix by human cells for tissue engineering applications. Dermal fibroblasts were cultured for three weeks at high density on porous substrates in serum-containing or chemically defined media. The mechanical and biochemical properties of the resulting cell-derived matrix (CDM) were compared to those of standard fibroblast-populated collagen and fibrin gels and native human skin. We found that the ultimate tensile strength of CDM cultured in our chemically defined media (313+/-8.7 kPa) is significantly greater than for collagen gels (168+/-39.3 kPa), fibrin gels (133+/-8.0 kPa) and CDM cultured with serum (223+/-9.0 kPa), but less than native skin (713+/-55.2 kPa). In addition to the biomechanics, this *CDM is also biochemically more similar to native skin than the collagen and fibrin gels in terms of all parameters measured. As *CDM is produced by human cells in a chemically defined culture medium and is mechanically robust, it may be a viable living tissue equivalent for many connective tissue replacement applications requiring initial mechanical stability yet a high degree of biocompatibility.
Annals of Biomedical Engineering. May, 2007 | Pubmed ID: 17377844
Rigid metal plates are a promising alternative to wires for reapproximating the sternum after open-heart surgery due to their potential ability to reduce motion at the wound site and thereby reduce the likelihood of post-operative healing complications. Despite initial clinical success, the use of plates has been limited, in part, by insufficient knowledge about their most effective placement. This study compares the ability of five plate configurations to provide stable closure by limiting sternal separation. Commercially available x-shaped and box-shaped plates were used and combinations of parameters (plate type, location, and number of plates) were investigated in vitro. Lateral distraction tests using controlled, uniform loading were conducted on 15 synthetic sterna and the distractions between separated sternum halves were measured at seven locations. Distractions at the xiphoid, a critical region clinically, varied widely from 0.03 +/- 0.53 mm to 4.24 +/- 1.26 mm depending on all three plate parameters. Of the configurations tested, three x-shaped plates and one box-shaped plate resisted sternal separation most effectively. These results provide the first comparison of plate configurations for stabilizing a sternotomy. However, basic mechanical analyses indicate that sternal loading in vivo is non-uniform; future studies will need to accurately quantify in vivo loading to improve in vitro test methods.
Journal of Biomechanics. 2008 | Pubmed ID: 18384794
Murine models are commonly used to investigate bone healing and test new treatments before human trials. Our objective was to design an improved murine femur fracture device and determine optimal mass and velocity settings for maximal likelihood of transverse fracture. Fracture reproducibility was maximized using an adjustable kinetic energy level, a novel mouse positioning system and an electromagnet striker release assembly. Sixty wild-type mice of 8-12-week-old male and female with a weight of 26.4+/-6.1g were subjected to an experimental postmortem fracture in the left and right femur (n=120) using variable kinetic energy inputs. A best-fit prediction equation for transverse fracture was developed using multivariate linear regression. Transverse fracture was shown to correlate most highly with kinetic energy with a maximum likelihood at mv2=292 where m is mass (g) and v is velocity (m/s). Model validation with a group of 134 anesthetized C57BL/6 mice resulted in a favorable transverse fracture rate of 85.8%. Simple modifications to existing fracture devices can improve accuracy and reproducibility. The results may assist researchers studying the effects of genetic modifications and novel treatments on boney healing in murine femur fracture models. Maintaining kinetic energy parameters within suggested ranges may also aid in ensuring accuracy and reproducibility.
Journal of Biomechanical Engineering. Oct, 2008 | Pubmed ID: 19045511
The development of more effective fixation devices for reapproximating and immobilizing the sternum after open-heart surgery is limited by current methods for evaluating these devices. In particular, precise emulation of in vivo sternal loading has not been achieved in controlled model systems. The present study is an initial effort to determine the in vivo loading parameters needed to improve current in vitro and in silico (computational) models. Towards this goal, the direction, magnitude, and distribution of loading along a midline sternotomy were characterized in a porcine model. Two instrumented plating systems were used to measure the forces across the bisected sternum in four anaesthetized Yorkshire pigs during spontaneous breathing, ventilated breathing, and coughing for four treatments: live, cadaveric, embalmed, and refrigerated. Changes in forces incurred by death and embalming were also investigated to evaluate the potential applicability of cadavers as models for testing sternal fixation devices. The magnitudes of the respiratory forces in three orthogonal directions ranged from 0.4 N to 43.8 N, many fold smaller than previously estimated. Dynamic forces were highest in the lateral direction during coughing and low in all directions during normal breathing. No significant differences in force were found between the four treatments, most likely due to the unexpectedly low magnitude of forces in all groups. These results provide the first measurements of in vivo sternal forces and indicate that small cyclic fatigue loads rather than large quasistatic loads should be applied in future model systems to best evaluate the mechanical performance of fixation devices.
Journal of Biomechanical Engineering. May, 2009 | Pubmed ID: 19388775
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.
Biomechanics and Modeling in Mechanobiology. Jun, 2010 | Pubmed ID: 20169395
Cells within connective tissues routinely experience a wide range of non-uniform mechanical loads that regulate many cell behaviors. In this study, we developed an experimental system to produce complex strain patterns for the study of strain magnitude, anisotropy, and gradient effects on cells in culture. A standard equibiaxial cell stretching system was modified by affixing glass coverslips (5, 10, or 15 mm diameter) to the center of 35 mm diameter flexible-bottomed culture wells. Ring inserts were utilized to limit applied strain to different levels in each individual well at a given vacuum pressure thus enabling parallel experiments at different strain levels. Deformation fields were measured using high-density mapping for up to 6% applied strain. The addition of the rigid inclusion creates strong circumferential and radial strain gradients, with a continuous range of stretch anisotropy ranging from strip biaxial to equibiaxial strain and radial strains up to 24% near the inclusion. Dermal fibroblasts seeded within our 2D system (5 mm inclusions; 2% applied strain for 2 days at 0.2 Hz) demonstrated the characteristic orientation perpendicular to the direction of principal strain. Dermal fibroblasts seeded within fibrin gels (5 mm inclusions; 6% applied strain for 8 days at 0.2 Hz) oriented themselves similarly and compacted their surrounding matrix to an increasing extent with local strain magnitude. This study verifies how inhomogeneous strain fields can be produced in a tunable and simply constructed system and demonstrates the potential utility for studying gradients with a continuous spectrum of strain magnitudes and anisotropies.
Multichanneled Collagen Conduits for Peripheral Nerve Regeneration: Design, Fabrication, and Characterization
Tissue Engineering. Part C, Methods. Dec, 2010 | Pubmed ID: 20528663
In the absence of donor tissues, conduits are needed for axons to regenerate across nerve defects, yet single-channel conduits may result in axonal dispersion, and multichannel synthetic polymer conduits have failed due to dimensional instability. The goal of this study was to create a robust collagen-based nerve conduit with multiple submillimeter-diameter channels to facilitate nerve guidance. Toward this goal, we have developed a novel multistep molding technique to create single-, four-, and seven-channel conduits from collagen and examined the effects of crosslinking with 0-60 mM (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide [EDC] in N-hydroxysuccinimide) on geometric, enzymatic, and thermal stability, mechanical properties, and cellular behavior. Multichannel collagen conduits crosslinked with 30 mM EDC and 10 mM N-hydroxysuccinimide demonstrated low degradation rate (∼10% at 2 days), high shrinkage temperature (>75°C), and constant channel morphology out to 30 days in saline. Neurite outgrowth remained unaffected from cultured dorsal root ganglia explants seeded on collagen scaffolds with up to 30 mM EDC crosslinking. Compared with single-channel conduits, multichannel collagen conduits showed superior structural compressive, tensile, and bending stiffness. Taken together, these results suggest that the crosslinked multichannel collagen conduits possess favorable material and mechanical properties for nerve regeneration applications.
Journal of Orthopaedic Surgery and Research. 2010 | Pubmed ID: 20942976
Development of a Cell-derived Matrix: Effects of Epidermal Growth Factor in Chemically Defined Culture
Journal of Biomedical Materials Research. Part A. Feb, 2010 | Pubmed ID: 19235212
Extracellular matrices without animal components and with high mechanical strength are needed for the development of the next generation of viable skin replacements. The goal of this study was to determine the optimal concentration of epidermal growth factor (EGF) to maximize the strength and collagen content of cell-derived matrix (CDM) produced by fibroblasts in vitro in serum-free media. Scaffold-free CDM samples were produced by human dermal fibroblasts in the presence of 0-50 ng/mL EGF in chemically defined media. After 21 days of culture, a membrane inflation system was used to measure the biaxial tensile strength, failure stretch ratio, and thickness of each treatment group. The fibroblasts treated with 5 ng/mL EGF produced the thickest matrix (270 microm). A thinner (130 microm) matrix, produced when the fibroblasts were treated with 0.5 ng/mL, had an ultimate tensile strength (895 kPa), greater than two times that of the other treatment groups. The fibroblasts treated with 0.5 ng/mL also had the highest collagen density (23.5 mg/cm(3)). Fibroblasts stimulated with the lowest (0.05 ng/mL) and highest (50 ng/mL) concentrations of EGF produced significantly weaker matrices and lower collagen densities. There was no significant correlation between UTS and collagen density suggesting that mechanisms other than density contribute to the strength of the matrix. Taken together, these data indicate that the optimal EGF concentration depends upon the relative importance of matrix strength and volume in a given application and that 0.5-5.0 ng/mL EGF promotes production of a robust extracellular matrix in only 3 weeks.
Cells, Tissues, Organs. 2011 | Pubmed ID: 21252472
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.
PloS One. 2011 | Pubmed ID: 21858051
Cells have the ability to actively sense their mechanical environment and respond to both substrate stiffness and stretch by altering their adhesion, proliferation, locomotion, morphology, and synthetic profile. In order to elucidate the interrelated effects of different mechanical stimuli on cell phenotype in vitro, we have developed a method for culturing mammalian cells in a two-dimensional environment at a wide range of combined levels of substrate stiffness and dynamic stretch. Polyacrylamide gels were covalently bonded to flexible silicone culture plates and coated with monomeric collagen for cell adhesion. Substrate stiffness was adjusted from relatively soft (G' = 0.3 kPa) to stiff (G' = 50 kPa) by altering the ratio of acrylamide to bis-acrylamide, and the silicone membranes were stretched over circular loading posts by applying vacuum pressure to impart near-uniform stretch, as confirmed by strain field analysis. As a demonstration of the system, porcine aortic valve interstitial cells (VIC) and human mesenchymal stem cells (hMSC) were plated on soft and stiff substrates either statically cultured or exposed to 10% equibiaxial or pure uniaxial stretch at 1 Hz for 6 hours. In all cases, cell attachment and cell viability were high. On soft substrates, VICs cultured statically exhibit a small rounded morphology, significantly smaller than on stiff substrates (p<0.05). Following equibiaxial cyclic stretch, VICs spread to the extent of cells cultured on stiff substrates, but did not reorient in response to uniaxial stretch to the extent of cells stretched on stiff substrates. hMSCs exhibited a less pronounced response than VICs, likely due to a lower stiffness threshold for spreading on static gels. These preliminary data demonstrate that inhibition of spreading due to a lack of matrix stiffness surrounding a cell may be overcome by externally applied stretch suggesting similar mechanotransduction mechanisms for sensing stiffness and stretch.
Planar Biaxial Characterization of Diseased Human Coronary and Carotid Arteries for Computational Modeling
Journal of Biomechanics. Jan, 2012 | Pubmed ID: 22236530
Computational models have the potential to provide precise estimates of stresses and strains associated with sites of coronary plaque rupture. However, lack of adequate mathematical description of diseased human vessel wall mechanical properties is hindering computational accuracy. The goal of this study is to characterize the behavior of diseased human coronary and carotid arteries using planar biaxial testing. Diseased coronary specimens exhibit relatively high stiffness (50-210kPa) and low extensibility (1-10%) at maximum equibiaxial stress (250kPa) compared to human carotid specimens and values commonly reported for porcine coronary arteries. A thick neointimal layer observed histologically appears to be associated with heightened stiffness and the direction of anisotropy of the specimens. Fung, Choi-Vito and modified Mooney-Rivlin constitutive equations fit the multiaxial data from multiple stress protocols well, and parameters from representative coronary specimens were utilized in a finite element model with fluid-solid interactions. Computed locations of maximal stress and strain are substantially altered, and magnitudes of maximum principal stress (48-65kPa) and strain (6.5-8%) in the vessel wall are lower than previously predicted using parameters from uniaxial tests. Taken together, the results demonstrate the importance of utilizing disease-matched multiaxial constitutive relationships within patient-specific computational models to accurately predict stress and strain within diseased coronary arteries.