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Biomedical subjects

Lori A Setton

Publications and source records attributed to Lori A Setton.

At least 19 recordsLinked to original sources
PubMed2006

A thermally responsive biopolymer for intra-articular drug delivery.

Intra-articular drug delivery is the preferred standard for targeting pharmacologic treatment directly to joints to reduce undesirable side effects associated with systemic drug delivery. In this study, a biologically based drug delivery vehicle was designed for intra-articular drug delivery using elastin-like polypeptides (ELPs), a biopolymer composed of repeating pentapeptides that undergo a phase transition to form aggregates above their transition temperature. The ELP drug delivery vehicle was designed to aggregate upon intra-articular injection at 37 degrees C, and form a drug 'depot' that could slowly disaggregate and be cleared from the joint space over time. We evaluated the in vivo biodistribution and joint half-life of radiolabeled ELPs, with and without the ability to aggregate, at physiological temperatures encountered after intra-articular injection in a rat knee. Biodistribution studies revealed that the aggregating ELP had a 25-fold longer half-life in the injected joint than a similar molecular weight protein that remained soluble and did not aggregate. These results suggest that the intra-articular joint delivery of ELP-based fusion proteins may be a viable strategy for the prolonged release of disease-modifying protein drugs for osteoarthritis and other arthritides.

Animals↗
PubMed2006

Measurement of intracellular strain on deformable substrates with texture correlation.

Mechanical stimuli are important factors that regulate cell proliferation, survival, metabolism and motility in a variety of cell types. The relationship between mechanical deformation of the extracellular matrix and intracellular deformation of cellular sub-regions and organelles has not been fully elucidated, but may provide new insight into the mechanisms involved in transducing mechanical stimuli to biological responses. In this study, a novel fluorescence microscopy and image analysis method was applied to examine the hypothesis that mechanical strains are fully transferred from a planar, deformable substrate to cytoplasmic and intranuclear regions within attached cells. Intracellular strains were measured in cells derived from the anulus fibrosus of the intervertebral disc when attached to an elastic silicone membrane that was subjected to tensile stretch. Measurements indicated cytoplasmic strains were similar to those of the underlying substrate, with a strain transfer ratio (STR) of 0.79. In contrast, nuclear strains were much smaller than those of the substrate, with an STR of 0.17. These findings are consistent with previous studies indicating nuclear stiffness is significantly greater than cytoplasmic stiffness, as measured using other methods. This study provides a novel method for the study of cellular mechanics, including a new technique for measuring intranuclear deformations, with evidence of differential magnitudes and patterns of strain transferred from the substrate to cell cytoplasm and nucleus.

Animals↗
PubMed2006

Molecular phenotypes of notochordal cells purified from immature nucleus pulposus.

The immature nucleus pulposus (NP) is populated by cells of notochordal-origin that are larger and contain an extensive cytoskeletal network and numerous vacuoles. The disappearance of these cells with age is believed important in regulating metabolic shifts that may contribute to age-related disc degeneration. The precise biological function of these notochordal cells in the immature NP remains unclear, however, because of challenges in studying the mixed cell population in the NP. In this study, notochordal-like cells were purified from immature NP cells using a new fluorescence-activated cell sorting (FACS) protocol with auto-fluorescence and size analysis. The unique molecular phenotypes of sorted notochordal-like cells were characterized by the mRNA expression pattern for key matrix proteins and modulators, and by the expression of cell-matrix receptor integrin subunits. An FACS analysis showed that the immature NP contained a majority of cells that were larger than anulus fibrosus (AF) cells and with fluorescence higher than AF cells. In comparison with the small NP cells separated by the FACS protocol, sorted notochordal-like cells expressed lower mRNA levels of type I collagen, biglycan, TIMP1, HSP70 and c-fos, and did not express detectable mRNA levels of decorin, lumican, multiple MMPs or IL-1beta via real-time quantitative RT-PCR. A greater number of these notochordal-like cells also expressed the higher levels of alpha6, alpha1 and beta1 integrin subunits as compared to small NP cells. Together, our results point towards a unique molecular phenotype for these notochordal-like cells of NP, characterized by the absence of gene expression for specific small proteoglycans and higher protein expression of integrin subunits that regulate interactions with collagens and laminin. Future studies will be important for revealing if this unique molecular profile is coordinated with functional differences in pericellular matrix regions and/or integrin-mediated cell-matrix interactions for these notochordal-like cells within the NP.

Animals↗
PubMed2006

A mechano-chemical model for the passive swelling response of an isolated chondron under osmotic loading.

The chondron is a distinct structure in articular cartilage that consists of the chondrocyte and its pericellular matrix (PCM), a narrow tissue region surrounding the cell that is distinguished by type VI collagen and a high glycosaminoglycan concentration relative to the extracellular matrix. We present a theoretical mechano-chemical model for the passive volumetric response of an isolated chondron under osmotic loading in a simple salt solution at equilibrium. The chondrocyte is modeled as an ideal osmometer and the PCM model is formulated using triphasic mixture theory. A mechano-chemical chondron model is obtained assuming that the chondron boundary is permeable to both water and ions, while the chondrocyte membrane is selectively permeable to only water. For the case of a neo-Hookean PCM constitutive law, the model is used to conduct a parametric analysis of cell and chondron deformation under hyper- and hypo-osmotic loading. In combination with osmotic loading experiments on isolated chondrons, model predictions will aid in determination of pericellular fixed charge density and its relative contribution to PCM mechanical properties.

Biomechanical Phenomena↗
PubMed2006

Finite element modeling predictions of region-specific cell-matrix mechanics in the meniscus.

The knee meniscus exhibits significant spatial variations in biochemical composition and cell morphology that reflect distinct phenotypes of cells located in the radial inner and outer regions. Associated with these cell phenotypes is a spatially heterogeneous microstructure and mechanical environment with the innermost regions experiencing higher fluid pressures and lower tensile strains than the outer regions. It is presently unknown, however, how meniscus tissue mechanics correlate with the local micromechanical environment of cells. In this study, theoretical models were developed to study mechanics of inner and outer meniscus cells with varying geometries. The results for an applied biaxial strain predict significant regional differences in the cellular mechanical environment with evidence of tensile strains along the collagen fiber direction of approximately 0.07 for the rounded inner cells, as compared to levels of 0.02-0.04 for the elongated outer meniscus cells. The results demonstrate an important mechanical role of extracellular matrix anisotropy and cell morphology in regulating the region-specific micromechanics of meniscus cells, that may further play a role in modulating cellular responses to mechanical stimuli.

Animals↗
PubMed2006

Region-specific constitutive gene expression in the adult porcine meniscus.

The knee meniscus exhibits extensive spatial variations in native healing capacity, biochemical composition, and cell morphology that suggest the existence of distinct phenotypes for meniscus cells. Constitutive gene expression levels of appropriate extracellular matrix proteins may serve as useful molecular markers of cellular phenotypes; however, relatively little is known of variations in the gene expression for meniscus cells of different regions of the tissue. The objective of the present study was to evaluate constitutive differences between radial inner and outer regions in gene expression for extracellular matrix proteins relevant to the meniscus. A secondary objective was to determine if these region-specific differences in gene expression are maintained after periods of monolayer culture. The innermost regions of the meniscus were found to constitutively express higher mRNA levels for proteins highly expressed in articular cartilage, including aggrecan, type II collagen, and NOS2. In contrast, the outer meniscus was found to contain higher gene expression for proteins associated with fibrous tissues including type I collagen, and the proteases MMP2 and MMP3. Isolated inner and outer meniscus cells maintained these region-specific gene expression patterns for collagens and proteoglycans during short-term monolayer culture. The results provide new information that suggests the utility of constitutive gene expression levels as molecular markers to distinguish tissue and cells of the inner and outer meniscus.

Animals↗
PubMed2006

Chondrocytic differentiation of human adipose-derived adult stem cells in elastin-like polypeptide.

Human adipose derived adult stem (hADAS) cells have the ability to differentiate into a chondrogenic phenotype in three-dimensional culture and media containing dexamethasone and TGF-beta. The current study examined the potential of a genetically engineered elastin-like polypeptide (ELP) to promote the chondrocytic differentiation of hADAS cells without exogenous chondrogenic supplements. hADAS cells were cultured in ELP hydrogels in either chondrogenic or standard medium at 5% O2 for up to 2 weeks. By day 14, constructs cultured in either medium exhibited significant increases in sulfated glycosaminoglycan (up to 100%) and collagen contents (up to 420%). Immunolabeling confirmed that the matrix formed consisted mainly of type II and not type I collagen. The composition of the constructs cultured in either medium did not differ significantly. To assess the effect of oxygen tension on the differentiation of the above constructs, samples were cultured in standard medium at either 5% or 20% O2 for 7 days and their gene expression profile was evaluated using real time RT-PCR. In both cases, the hADAS-ELP constructs upregulated SOX9 and type II collagen gene expression, while type I collagen was downregulated. However, constructs cultured in 20% O2 highly upregulated type X collagen, which was not detected in the 5% O2 cultures. The study suggests that ELP can promote chondrogenesis for hADAS cells in the absence of exogenous TGF-beta1 and dexamethasone, especially under low oxygen tension conditions.

Adipose Tissue↗
PubMed2006

Biodendrimer-based hydrogel scaffolds for cartilage tissue repair.

Photo-crosslinkable dendritic macromolecules are attractive materials for the preparation of cartilage tissue engineering scaffolds that may be optimized for in situ formation of hydrated, mechanically stable, and well-integrated hydrogel scaffolds supporting chondrocytes and chondrogenesis. We designed and synthesized a novel hydrogel scaffold for cartilage repair, based on a multivalent and water-soluble tri-block copolymer consisting of a poly(ethylene glycol) core and methacrylated poly(glycerol succinic acid) dendrimer terminal blocks. The terminal methacrylates allow mild and biocompatible photo-crosslinking with a visible light, facilitating in vivo filling of irregularly shaped defects with the dendrimer-based scaffold. The multivalent dendrimer constituents allow high crosslink densities that inhibit swelling after crosslinking while simultaneously introducing biodegradation sites. The mechanical properties and water content of the hydrogel can easily be tuned by changing the biodendrimer concentration. In vitro chondrocyte encapsulation studies demonstrate significant synthesis of neocartilaginous material, containing proteoglycans and type II collagen.

Animals↗
PubMed2006

Biaxial strain effects on cells from the inner and outer regions of the meniscus.

During knee joint loading, the fibrocartilaginous menisci experience significant spatial variations in mechanical stimuli. Meniscus cells also exhibit significant variations in biosynthesis and gene expression depending on their location within the tissue. These metabolic patterns are consistent with a more chondrocytic phenotype for cells located within the avascular inner two-thirds compared with a more fibroblastic phenotype for cells within the vascularized outer periphery. The spatial distribution of cell biosynthesis and gene expression patterns within the meniscus suggest that cells may exhibit intrinsically different responses to mechanical stimuli. The objective of our study was to test for intrinsic differences in the responsiveness of these meniscus cell populations to an equivalent mechanical stimulus. Cellular biosynthesis and gene expression for extracellular matrix proteins in isolated inner and outer meniscus cells in monolayer were quantified following cyclic biaxial stretch. The results demonstrate that inner and outer meniscus cells exhibit significant differences in matrix biosynthesis and gene expression regardless of stretching condition. Both inner and outer meniscus cells responded to stretch with increased nitric oxide production and total protein synthesis. The results suggest that inner and outer meniscus cells may respond similarly to biaxial stretch in vitro with measures of biosynthesis and gene expression.

Aggrecans↗
PubMed2006

Compressive properties of mouse articular cartilage determined in a novel micro-indentation test method and biphasic finite element model.

The mechanical properties of articular cartilage serve as important measures of tissue function or degeneration, and are known to change significantly with osteoarthritis. Interest in small animal and mouse models of osteoarthritis has increased as studies reveal the importance of genetic background in determining predisposition to osteoarthritis. While indentation testing provides a method of determining cartilage mechanical properties in situ, it has been of limited value in studying mouse joints due to the relatively small size of the joint and thickness of the cartilage layer. In this study, we developed a micro-indentation testing system to determine the compressive and biphasic mechanical properties of cartilage in the small joints of the mouse. A nonlinear optimization program employing a genetic algorithm for parameter estimation, combined with a biphasic finite element model of the micro-indentation test, was developed to obtain the biphasic, compressive material properties of articular cartilage. The creep response and material properties of lateral tibial plateau cartilage were obtained for wild-type mouse knee joints, by the micro-indentation testing and optimization algorithm. The newly developed genetic algorithm was found to be efficient and accurate when used with the finite element simulations for nonlinear optimization to the experimental creep data. The biphasic mechanical properties of mouse cartilage in compression (average values: Young's modulus, 2.0 MPa; Poisson's ratio, 0.20; and hydraulic permeability, 1.1 x 10(-16) m4/N-s) were found to be of similar orders of magnitude as previous findings for other animal cartilages, including human, bovine, rat, and rabbit and demonstrate the utility of the new test methods. This study provides the first available data for biphasic compressive properties in mouse cartilage and suggests a promising method for detecting altered cartilage mechanics in small animal models of osteoarthritis.

Animals↗
PubMed2006

The pericellular matrix as a transducer of biomechanical and biochemical signals in articular cartilage.

The pericellular matrix (PCM) is a narrow tissue region surrounding chondrocytes in articular cartilage, which together with the enclosed cell(s) has been termed the "chondron." While the function of this region is not fully understood, it is hypothesized to have important biological and biomechanical functions. In this article, we review a number of studies that have investigated the structure, composition, mechanical properties, and biomechanical role of the chondrocyte PCM. This region has been shown to be rich in proteoglycans (e.g., aggrecan, hyaluronan, and decorin), collagen (types II, VI, and IX), and fibronectin, but is defined primarily by the presence of type VI collagen as compared to the extracellular matrix (ECM). Direct measures of PCM properties via micropipette aspiration of isolated chondrons have shown that the PCM has distinct mechanical properties as compared to the cell or ECM. A number of theoretical and experimental studies suggest that the PCM plays an important role in regulating the microenvironment of the chondrocyte. Parametric studies of cell-matrix interactions suggest that the presence of the PCM significantly affects the micromechanical environment of the chondrocyte in a zone-dependent manner. These findings provide support for a potential biomechanical function of the chondrocyte PCM, and furthermore, suggest that changes in the PCM and ECM properties that occur with osteoarthritis may significantly alter the stress-strain and fluid environments of the chondrocytes. An improved understanding of the structure and function of the PCM may provide new insights into the mechanisms that regulate chondrocyte physiology in health and disease.

Animals↗
PubMed2006

Mechanobiology of the intervertebral disc and relevance to disc degeneration.

Mechanical loading of the intervertebral disc may contribute to disc degeneration by initiating degeneration or by regulating cell-mediated remodeling events that occur in response to the mechanical stimuli of daily activity. This article is a review of the current knowledge of the role of mechanical stimuli in regulating intervertebral disc cellular responses to loading and the cellular changes that occur with degeneration. Intervertebral disc cells exhibit diverse biologic responses to mechanical stimuli, depending on the loading type, magnitude, duration, and anatomic zone of cell origin. The innermost cells respond to low-to-moderate magnitudes of static compression, osmotic pressure, or hydrostatic pressure with increases in anabolic cell responses. Higher magnitudes of loading may give rise to catabolic responses marked by elevated protease gene or protein expression or activity. The key regulators of these mechanobiologic responses for intervertebral disc cells will be the micromechanical stimuli experienced at the cellular level, which are predicted to differ from that measured for the extracellular matrix. Large hydrostatic pressures, but little volume change, are predicted to occur for cells of the nucleus pulposus during compression, while the highly oriented cells of the anulus fibrosus may experience deformations in tension or compression during matrix deformations. In general, the pattern of biologic response to applied loads suggests that the cells of the nucleus pulposus and inner portion of the anulus fibrosus experience comparable micromechanical stimuli in situ and may respond more similarly than cells of the outer portion of the anulus fibrosus. Changes in these features with degeneration are critically understudied, particularly degeneration-associated changes in cell-level mechanical stimuli and the associated mechanobiology. Little is known of the mechanisms that regulate cellular responses to intervertebral mechanobiology, nor is much known with regard to the precise mechanical stimuli experienced by cells during loading. Mechanical factors appear to regulate responses of the intervertebral disc cells through mechanisms involving intracellular Ca(2+) transients and cytoskeletal remodeling that may regulate downstream effects such as gene expression and posttranslational biosynthesis. Future studies should address the broader biologic responses to mechanical stimuli in intervertebral disc mechanobiology, the involved signaling mechanisms, and the apparently important interactions among mechanical factors, genetic factors, cytokines, and inflammatory mediators that may be critical in the regulation of intervertebral disc degeneration.

Animals↗
PubMed2005

Epitope tagging for tracking elastin-like polypeptides.

Elastin-like polypeptides (ELPs) are a class of biocompatible, non-immunogenic and crosslinkable biomaterials that offer promise for use as an injectable scaffold for cartilage repair. In this study, an oligohistidine (His(6)) epitope tag was incorporated at the N-terminus of an ELP using recombinant DNA techniques to permit tracking without compromising on material biocompatibility. His(6)-tagged ELPs were successfully detected by Western blot analysis and quantified by ELISAs following digestion with trypsin. The mass of His(6) tagged ELP fragments freed from a crosslinked ELP hydrogel after digestion with trypsin correlated highly with hydrogel weight loss, providing evidence of the tag's capability to enable tracking of enzymatic degradation of the ELP hydrogel. The His(6) tag also facilitated recognition of crosslinked ELPs from background staining of articular cartilage. These results suggest that the His(6) epitope tag has the potential to track ELP scaffold loss independently of newly formed tissue mass for evaluating matrix remodeling in vivo.

Amino Acid Sequence↗
PubMed2005

Patterning cells in highly deformable microstructures: effect of plastic deformation of substrate on cellular phenotype and gene expression.

We describe the fabrication of deformable microstructures by low-pressure-soft-microembossing (muSEmb) that provides in vitro experimental "test-beds" to investigate the interplay of mechanical and chemical stimuli on cell behavior in a highly controlled environment. Soft microembossing exploits the softness and plasticity of parafilm to fabricate microstructures by pressing a silicon master or an elastomeric poly(dimethylsiloxane) stamp into the parafilm. We demonstrate that a protein-resistant comb polymer can be printed into the raised features of the embossed microstructures, which imparts protein, and hence cell resistance to those regions of the microstructures. These two features of our fabrication methodology-microembossing followed by spatially selective transfer of a nonfouling polymer-forms the core of our strategy to pattern cells within the parafilm microstructures, such that the cells are confined within bottoms of the microstructures. Cell culture experiments demonstrated the preferential cell attachment of NIH 3T3 fibroblasts to the fibronectin (FN) micropatterns by immunofluorescence microscopy. The actin cytoskeleton realigned along the axis of applied mechanical stress, and stretched cells showed altered gene expression of cytoskeletal and matrix proteins in response to mechanical deformation. The use of parafilm as a substrate and muSEmb as a fabrication method provides a simple and widely accessible methodology to investigate cellular behavior under well-defined conditions of plastic deformation and surface ligand density.

Animals↗
PubMed2005

The biomechanical role of the chondrocyte pericellular matrix in articular cartilage.

The pericellular matrix (PCM) is a narrow tissue region that surrounds chondrocytes in articular cartilage. Previous parametric studies of cell-matrix interactions suggest that the mechanical properties of the PCM relative to those of the extracellular matrix (ECM) can significantly affect the micromechanical environment of the chondrocyte. The goal of this study was to use recently quantified mechanical properties of the PCM in a biphasic finite element model of the cell-PCM-ECM structure to determine the potential influence of the PCM on the mechanical environment of the chondrocyte under normal and osteoarthritic conditions. Our findings suggest that the mismatch between the Young's moduli of PCM and ECM amplifies chondrocyte compressive strains and exhibits a significant stress shielding effect in a zone-dependent manner. Furthermore, the lower permeability of PCM relative to the ECM inhibits fluid flux near the cell by a factor of 30, and thus may have a significant effect on convective transport to and from the chondrocyte. Osteoarthritic changes in the PCM and ECM properties significantly altered the mechanical environment of the chondrocyte, leading to approximately 66% higher compressive strains and higher fluid flux near the cell. These findings provide further support for a potential biomechanical role for the chondrocyte PCM, and suggest that changes in the properties of the PCM with osteoarthritis may alter the stress-strain and fluid flow environment of the chondrocytes.

Biomechanical Phenomena↗
PubMed2005

Zonal uniformity in mechanical properties of the chondrocyte pericellular matrix: micropipette aspiration of canine chondrons isolated by cartilage homogenization.

The pericellular matrix (PCM) is a region of tissue that surrounds chondrocytes in articular cartilage and together with the enclosed cells is termed the chondron. Previous studies suggest that the mechanical properties of the PCM, relative to those of the chondrocyte and the extracellular matrix (ECM), may significantly influence the stress-strain, physicochemical, and fluid-flow environments of the cell. The aim of this study was to measure the biomechanical properties of the PCM of mechanically isolated chondrons and to test the hypothesis that the Young's modulus of the PCM varies with zone of origin in articular cartilage (surface vs. middle/deep). Chondrons were extracted from articular cartilage of the canine knee using mechanical homogenization, and the elastic properties of the PCM were determined using micropipette aspiration in combination with theoretical models of the chondron as an elastic incompressible half-space, an elastic compressible bilayer, or an elastic compressible shell. The Young's modulus of the PCM was significantly higher than that reported for isolated chondrocytes but over an order of magnitude lower than that of the cartilage ECM. No significant differences were observed in the Young's modulus of the PCM between surface zone (24.0 +/- 8.9 kPa) and middle/deep zone cartilage (23.2 +/- 7.1 kPa). In combination with previous theoretical biomechanical models of the chondron, these findings suggest that the PCM significantly influences the mechanical environment of the chondrocyte in articular cartilage and therefore may play a role in modulating cellular responses to micromechanical factors.

Animals↗