Biomedical subjects

Marc E Levenston

Publications and source records attributed to Marc E Levenston.

15 recordsLinked to original sources

PubMed2006

Analysis of cartilage matrix fixed charge density and three-dimensional morphology via contrast-enhanced microcomputed tomography.

Small animal models of osteoarthritis are often used for evaluating the efficacy of pharmacologic treatments and cartilage repair strategies, but noninvasive techniques capable of monitoring matrix-level changes are limited by the joint size and the low radiopacity of soft tissues. Here we present a technique for the noninvasive imaging of cartilage at micrometer-level resolution based on detecting the equilibrium partitioning of an ionic contrast agent via microcomputed tomography. The approach exploits electrochemical interactions between the molecular charges present in the cartilage matrix and an ionic contrast agent, resulting in a nonuniform equilibrium partitioning of the ionic contrast agent reflecting the proteoglycan distribution. In an in vitro model of cartilage degeneration we observed changes in x-ray attenuation magnitude and distribution consistent with biochemical and histological analyses of sulfated glycosaminoglycans, and x-ray attenuation was found to be a strong predictor of sulfated glycosaminoglycan density. Equilibration with the contrast agent also permits direct in situ visualization and quantification of cartilage surface morphology. Equilibrium partitioning of an ionic contrast agent via microcomputed tomography thus provides a powerful approach to quantitatively assess 3D cartilage composition and morphology for studies of cartilage degradation and repair.

Animals↗

PubMed2006

Inhibition of in vitro chondrogenesis in RGD-modified three-dimensional alginate gels.

The goal of this study was to investigate the effects of adhesion to the arginine-glycine-aspartic acid (RGD) sequence on the chondrogenesis of bone marrow stromal cells (BMSCs). Synthetic RGE- and RGD-containing peptides were conjugated to sodium alginate, and bovine BMSCs were seeded onto 2D alginate surfaces or encapsulated in 3D gels. BMSCs spread specifically on RGD-modified surfaces, and spreading was inhibited by a soluble RGD peptide and by anti-beta1 and anti-alpha(v)beta3 integrin blocking antibodies. After 7 days in 3D gel culture, the chondrogenic supplements (TGF-beta1 and dexamethasone) significantly stimulated chondrocytic gene expression (collagen II, aggrecan, and Sox-9) and matrix accumulation (collagen II and sGAG) in RGE-modified gels, but this response was inhibited in the RGD-modified gels. Inhibition of sGAG synthesis increased with increasing RGD density, and synthesis was partially rescued by adding a soluble RGD peptide. Addition of an anti-alpha(v)beta3 integrin blocking antibody had no effect on chondrogenesis, while an anti-alpha5 antibody reduced sGAG accumulation. Overall, this study demonstrates that interaction with the RGD motif significantly inhibits the initial chondrogenesis of BMSCs within 3D alginate gels. These results provide new insights into the role of cell-matrix interactions in regulating chondrogenesis and highlight the importance of choosing appropriate biomaterials for tissue engineering therapies.

Adsorption↗

PubMed2006

Dynamic compression regulates the expression and synthesis of chondrocyte-specific matrix molecules in bone marrow stromal cells.

The overall objective of the present study was to investigate the mechanotransduction of bovine bone marrow stromal cells (BMSCs) through the interactions between transforming growth factor beta1 (TGF-beta1), dexamethasone, and dynamic compressive loading. Overall, the addition of TGF-beta1 increased cell viability, extracellular matrix (ECM) gene expression, matrix synthesis, and sulfated glycosaminoglycan content over basal construct medium. The addition of dexamethasone further enhanced extracellular matrix gene expression and protein synthesis. There was little stimulation of ECM gene expression or matrix synthesis in any medium group by mechanical loading introduced on day 8. In contrast, there was significant stimulation of ECM gene expression and matrix synthesis in chondrogenic media by dynamic loading introduced on day 16. The level of stimulation was also dependent on the medium supplements, with the samples treated with basal medium being the least responsive and the samples treated with TGF-beta1 and dexamethasone being the most responsive at day 16. Both collagen I and collagen II gene expressions were more responsive to dynamic loading than aggrecan gene expression. Dynamic compression upregulated Smad2/3 phosphorylation in samples treated with basal and TGF-beta1 media. These findings suggest that interactions between mechanical stimuli and TGF-beta signaling may be an important mechanotransduction pathway for BMSCs, and they indicate that mechanosensitivity may vary during the process of chondrogenesis.

Animals↗

PubMed2006

Selective and non-selective metalloproteinase inhibitors reduce IL-1-induced cartilage degradation and loss of mechanical properties.

Articular cartilage undergoes matrix degradation and loss of mechanical properties when stimulated with proinflammatory cytokines such as interleukin-1 (IL-1). Aggrecanases and matrix metalloproteinases (MMPs) are thought to be principal downstream effectors of cytokine-induced matrix catabolism, and aggrecanase- or MMP-selective inhibitors reduce or block matrix destruction in several model systems. The objective of this study was to use metalloproteinase inhibitors to perturb IL-1-induced matrix catabolism in bovine cartilage explants and examine their effects on changes in tissue compression and shear properties. Explanted tissue was stimulated with IL-1 for up to 24 days in the absence or presence of inhibitors that were aggrecanase-selective, MMP-selective, or non-selective. Analysis of conditioned media and explant digests revealed that aggrecanase-mediated aggrecanolysis was delayed to varying extents with all inhibitor treatments, but that aggrecan release persisted. Collagen degradation was abrogated by MMP- and non-selective inhibitors and reduced by the aggrecanase inhibitor. The inhibitors delayed but did not reduce loss of the equilibrium compression modulus, whereas the losses of dynamic compression and shear moduli were delayed and reduced. The data suggest that non-metalloproteinase mechanisms participate in IL-1-induced matrix degradation and loss of tissue material properties.

Aggrecans↗

PubMed2006

A constitutive model for protein-based materials.

Protein-based materials are critical to the construction of tissue substitutes that exhibit precisely defined mechanical properties. Under physiologically relevant conditions, materials derived from natural or synthetic structural proteins are characterized by nonlinear elastic responses at medium and large deformations, time-dependent or viscoelastic behavior, and display the effects of strain-induced structural changes. Although a constitutive model that accurately describes mechanical behavior is essential for the rational design of tissue constructs, few models account for all of these characteristics. In this report, we present a new constitutive model for protein based materials, in which nonlinear elasticity is captured by the Arruda-Boyce eight-chain model, time dependant viscoelasticity is described by a generalized Maxwell model, and the effect of strain-induced structural change is incorporated using a network alteration theory originally proposed by Tobolsky. The model was applied to a number of protein-based materials and cell containing constructs, including recombinant elastin-mimetic protein polymers and fibroblast populated collagen gel matrices. Significantly, numerical implementation of this model is straightforward and mechanical behavior accurately described under a variety of loading conditions. Moreover, when calibrated using stress relaxation data alone, the model accurately predicted cyclic loading responses. Although limitations exist, this model provides a convenient tool to correlate viscoelastic data obtained by different testing modes and may assist in reducing the number of experimental tests required to fully capture the range of viscoelastic responses of protein-based materials.

Animals↗

PubMed2005

Biomechanical analysis of silicon microelectrode-induced strain in the brain.

The ability to successfully interface the brain to external electrical systems is important both for fundamental understanding of our nervous system and for the development of neuroprosthetics. Silicon microelectrode arrays offer great promise in realizing this potential. However, when they are implanted into the brain, recording sensitivity is lost due to inflammation and astroglial scarring around the electrode. The inflammation and astroglial scar are thought to result from acute injury during electrode insertion as well as chronic injury caused by micromotion around the implanted electrode. To evaluate the validity of this assumption, the finite element method (FEM) was employed to analyze the strain fields around a single Michigan Si microelectrode due to simulated micromotion. Micromotion was mimicked by applying a force to the electrode, fixing the boundaries of the brain region and applying appropriate symmetry conditions to nodes lying on symmetry planes. Characteristics of the deformation fields around the electrode including maximum electrode displacement, strain fields and relative displacement between the electrode and the adjacent tissue were examined for varying degrees of physical coupling between the brain and the electrode. Our analysis demonstrates that when physical coupling between the electrode and the brain increases, the micromotion-induced strain of tissue around the electrode decreases as does the relative slip between the electrode and the brain. These results support the use of neuro-integrative coatings on electrode arrays as a means to reduce the micromotion-induced injury response.

Biomechanical Phenomena↗

PubMed2005

Indentation testing of human articular cartilage: effects of probe tip geometry and indentation depth on intra-tissue strain.

Experimental determination of intra-tissue deformation during clinically applicable rapid indentation testing would be useful for understanding indentation biomechanics and for designing safe indentation probes and protocols. The objectives of this study were to perform two-dimensional (2-D) indentation tests, using indenters and protocols that are analogous to those in clinically oriented probes, of normal adult-human articular cartilage in order to determine: (1) intra-tissue strain maps and regions of high strain magnitude, and (2) the effects on strain of indenter geometry (rectangular prismatic and cylindrical) and indentation depth (40-190 microm). Epifluorescence microscopy of samples undergoing indentation and subsequent video image correlation analysis allowed determination of strain maps. Regions of peak strain were near the "edges" of indenter contact with the cartilage surface, and the strain magnitude in these regions ranged from approximately 0.05 to approximately 0.30 in compression and shear, a range with known biological consequences. With increasing indentation displacement, strain magnitudes generally increased in all regions of the tissue. Compared to indentation using a rectangular prismatic tip, indentation with a cylindrical tip resulted in slightly higher peak strain magnitudes while influencing a smaller region of cartilage. These results may be used to refine clinical indenters and indentation protocols.

Adult↗

PubMed2004

Oscillatory tension differentially modulates matrix metabolism and cytoskeletal organization in chondrocytes and fibrochondrocytes.

Several modes of mechanical stimulation, including compression, shear, and hydrostatic pressure, have been shown to modulate chondrocyte matrix synthesis, but the effects of mechanical tension have not been widely explored. Since articular cartilage is primarily loaded in compression, tension is not generally viewed as a major contributor to the stress state of healthy tissue. However, injury or attempted repair may cause tension to become more significant. Additionally, fibrocartilaginous tissues experience significant tensile stresses in their normal mechanical environment. In this study we investigated mechanical tension as a means to modulate matrix synthesis and cytoskeletal organization in bovine articular chondrocytes and meniscal fibrochondrocytes (MFCs) in a three-dimensional fibrin construct culture system. Oscillatory tension was applied to constructs at 1.0 Hz and 0-10% displacement variation using a custom device. For nearly all conditions and both cell types, oscillatory tension inhibited matrix synthesis as indicated by 3H-proline and 35S-sulfate incorporation. Additionally, oscillatory tension significantly increased proliferation by chondrocytes but not MFCs. Confocal imaging revealed that all cells initially displayed a rounded morphology, but over time MFCs spontaneously developed a three-dimensional, stellate morphology with numerous projections containing organized cytoskeletal filaments. Interestingly, while unloaded chondrocytes remained rounded, chondrocytes subjected to oscillatory tension developed a similar stellate morphology. Both the biochemical and morphological results of this study have important implications for successfully developing cartilage and fibrocartilage tissue replacements and repair strategies.

Animals↗

PubMed2004

Dynamic compression of chondrocyte-seeded fibrin gels: effects on matrix accumulation and mechanical stiffness.

OBJECTIVE: Various strategies have been tested to direct and control matrix synthesis in tissue engineered cartilage, including mechanical stimulation of the construct both before and after implantation. This study examined the effects of oscillatory compression on chondrocytes in a fibrin-based tissue engineered cartilage. DESIGN: Chondrocyte-seeded fibrin gels were cultured under unconfined mechanical compression for 10 or 20 days (free-swelling, 10% static, or 10+/-4% at 0.1 or 1Hz). During the culture period, accumulation of nitrite, sGAG, and proteolytic enzymes in the culture media were monitored. Following culture, the mechanical stiffness and biochemical content of the gels (DNA, sGAG, and hydroxyproline content and GAG Delta-disaccharide composition) were assessed. RESULTS: Compared to free-swelling conditions, static compression had little effect on the mechanical stiffness or biochemical content of the gels. Compared to static compression, oscillatory compression produced softer gels, inhibited sGAG and hydroxyproline accumulation in the gels, and stimulated accumulation of nitrite and sGAG in the culture media. Minimal differences were observed in DNA content and Delta-disaccharide composition across treatment conditions. CONCLUSIONS: In this study, oscillatory compression inhibited formation of cartilage-like tissues by chondrocytes in fibrin gels. These results suggest that the effects of mechanical stimuli on tissue engineered cartilage may vary substantially between different scaffold systems.

Animals↗

PubMed2004

Combined effects of growth factors and static mechanical compression on meniscus explant biosynthesis.

OBJECTIVE: To compare the actions of fibroblast growth factor-basic (bFGF), insulin-like growth factor-I (IGF-I), platelet derived growth factor-AB (PDGF-AB), and transforming growth factor-beta 1 (TGF-beta1) on bovine meniscus tissue explants with and without static mechanical compression. DESIGN: Meniscus tissue explants were cultured in a serum-free environment supplemented with an individual growth factor (1) over a range of concentrations for 4 days, (2) at a single concentration for 2-14 days, and (3) at a single concentration for 4 days coupled with graded levels of static compression. Explants were analyzed for accumulation of newly synthesized proteoglycan and total protein as measured by 35S-sulfate and 3H-proline incorporation, respectively. RESULTS: Over the range of chosen concentrations, TGF-beta1 was the most potent stimulator of both protein and proteoglycan production, whereas bFGF was the least effective stimulator. Over a 2-week period for all four growth factors, the stimulation of proteoglycan production was sustained while there was no stimulation of protein production during this period. The superposition of static mechanical compression inhibited matrix production in the presence of each anabolic factor, with comparable inhibition relative to uncompressed controls for all factors. CONCLUSIONS: The growth factors chosen exhibited an anabolic effect on the meniscus tissue explants, encouraging matrix production and deposition. The addition of static mechanical compression produced comparable relative inhibition of matrix production for each growth factor, suggesting that static compression and growth factors may modulate meniscal fibrochondrocyte biosynthesis via distinct pathways.

Animals↗

PubMed2004

The influence of cyclic tension amplitude on chondrocyte matrix synthesis: experimental and finite element analyses.

While not generally viewed as physiologically significant in articular cartilage, substantial tension can develop in fibrocartilage structures and in articular cartilage injuries. This study examined how different amplitudes of cyclic tension influence chondrocyte matrix synthesis. Bovine articular chondrocytes seeded in fibrin gels were loaded continuously for 48 hours at 1.0 Hz with displacements of 5%, 10%, or 20%. Protein and proteoglycan synthesis were measured by (3)H-proline and (35)S-sulfate incorporation, respectively. A poroelastic finite element model of the fibrin gel was developed to determine the strain distributions, hydrostatic pressures, and fluid velocities within the constructs at the various levels of displacement. Compared to unloaded controls, 10% and 20% displacements inhibited proteoglycan synthesis to the same extent, while 5% displacement had no effect. Tensile loading did not significantly affect protein synthesis. The finite element model predicted a wide range of strains and fluid velocities within the region of the gel analyzed for matrix synthesis, and the ranges overlapped for the different levels of displacement. These results indicate that the cyclic tension amplitude influences chondrocyte proteoglycan synthesis and that there may be a threshold in the response.

Cartilage, Articular↗

PubMed2002

Mechanical compression alters gene expression and extracellular matrix synthesis by chondrocytes cultured in collagen I gels.

Articular cartilage responds to its mechanical environment through altered cell metabolism and matrix synthesis. In this study, isolated articular chondrocytes were cultured in collagen type I gels and exposed to uniaxial static compression of 0%, 25%, or 50% of original thickness for 0.5, 4, and 24 h, and to oscillatory (25 +/- 4%, 1 Hz) compression for 24 h. The cellular response was assessed through competitive and real-time RT-PCR to quantify expression of genes for collagen type I, collagen type II, and aggrecan core protein, and through radiolabelled proline and sulfate incorporation to quantify protein and proteoglycan synthesis rates. Static compression for 24 h inhibited expression of collagen I and II mRNAs and inhibited 3H-proline and 35S-sulfate incorporation. The mRNA expression exhibited transient fluctuations at intermediate time points. Oscillatory compression had no effect upon mRNA expression, and 24 h after release from static compression, there was no difference in collagen II or aggrecan mRNA, while there was an inhibition of collagen I. We conclude that the chondrocytes maintained some aspects of their ability to sense and respond to static compression, despite a biochemical and mechanical environment which is different from that in tissue. This suggests that mechanical stimuli may be useful in modulating chondrocyte metabolism in tissue engineering systems using fibrillar protein scaffolds.

Aggrecans↗

PubMed2002

The influence of repair tissue maturation on the response to oscillatory compression in a cartilage defect repair model.

This study examined the effects of mechanical compression on engineered cartilage in a novel hybrid culture system. Cylindrical holes were cut in discs of bovine articular cartilage and filled with agarose gels containing chondrocytes. These constructs were compressed in radiolabeled medium under static or oscillatory unconfined compression. Oscillatory compression at 1 Hz significantly stimulated synthesis above static control levels. Control experiments indicate that oscillatory compression does not stimulate freshly cast gels (without annuli), but does so after several weeks. This may be because physiologic fluid flow levels do not occur until sufficient extracellular matrix has accumulated. Finite element models predict minimal fluid flow in the gel core, and minimal differences in flow patterns between free and constrained gels. However, the models predict fluid pressures in constrained gels to be substantially higher than those in free gels. Our results suggest that pressure variations may influence synthesis of engineered cartilage matrices, with implications for construct development and post-implantation survival.

Analysis of Variance↗

PubMedDate not supplied

Alterations in physical cross-linking modulate mechanical properties of two-phase protein polymer networks.

Physically cross-linked protein-based materials possess a number of advantages over their chemically cross-linked counterparts, including ease of processing and the ability to avoid the addition or removal of chemical reagents or unreacted intermediates. The investigations reported herein sought to examine the nature of physical cross-links within two-phase elastin-mimetic protein triblock copolymer networks through an analysis of macroscopic viscoelastic properties. Given the capacity of solution processing conditions, including solvent type and temperature to modulate the microstructure of two-phase protein polymer networks, viscoelastic properties were examined under conditions in which interphase block mixing had been either accentuated or diminished during network formation. Protein networks exhibited strikingly different properties in terms of elastic modulus, hysteresis, residual deformability, and viscosity in response to interdomain mixing. Thus, two-phase protein polymer networks exhibit tunable responses that extend the range of application of these materials to a variety of tissue engineering applications.

Biocompatible Materials↗

PubMedDate not supplied

Maturation and integration of tissue-engineered cartilages within an in vitro defect repair model.

This study compared the behavior of four different engineered cartilages in a hybrid culture system. First, the growth and maturation of tissue-engineered cartilages in isolation were compared to those grown in an in vitro articular cartilage defect repair model. Tissue-engineered cartilages using fibrin, agarose, or poly(glycolic acid) scaffolds were implanted into annular explants of articular cartilage and cultured for 20 or 40 days. Native tissue had a substantial influence on the DNA, sulfated glycosaminoglycan, and hydroxyproline content of the engineered tissues, suggesting that the presence of living tissue in the culture significantly altered cell proliferation and matrix accumulation. Second, the adhesion strength of various engineered cartilages to native tissue was measured and compared with the biochemical content of the engineered tissues. All scaffold treatments adhered to the native cartilage, but there were statistically significant differences in adhesive strength between the different scaffolds. The adhesive strength of all engineered scaffolds was significantly lower than that of native tissue to itself. In the engineered tissues, neither failure stress nor energy to failure correlated with gross biochemical content, suggesting that adhesion between native and engineered tissues is not purely a function of gross matrix synthesis.

Animals↗