1,721,022 research outputs found
Intranuclear strain measured by iterative warping in cells under mechanical and osmotic stress
Full text linkThe nucleus is a membrane bound organelle and regulation center for gene expression in the cell. Mechanical forces transfer to the nucleus directly and indirectly through specific cellular cytoskeletal structures and pathways. There is increasing evidence that the transferred forces to the nucleus orchestrate gene expression activity. Methods to characterize nuclear mechanics typically study isolated cells or cells embedded in 3D gel matrices. Often report only aspect ratio and volume changes, measures that oversimplify the inherent complexity of internal strain patterns. This presents technical challenges to simultaneously observe small scale nuclear mechanics and gene expression levels inside the nuclei of cells embedded in their native extracellular environment. Therefore, a hybrid imaging and model based image registration technique has been developed to enabled us to explore links between biomechanical and biochemical signaling within individual cells. The hybrid technique uses an iterative warping deformable image registration to measure intranuclear strain fields that are correlated to nuclear structures. Three cell mechanics methods were developed to examine the mechanical response of the nucleus under different mechanical conditions. 1) Strain transfer from tissue to nuclei in a cartilage tissue deformation model paired with nascent RNA expression, 2) strain transfer to the nucleus with different cell types on a stretchable membrane, and 3) force traction microscopy of cells during osmotic stress. Intranuclear strain fields provide spatial details of the nucleus that when paired with single cell biochemical assays will provide insight into how mechanical forces transferred to the nucleus influence gene expression
Collagen densification as a model for cardiac fibrosis
No full textCardiac fibrosis is a disease state characterized by excessive collagenous matrix accumulation within the myocardium that can lead to ventricular dilation and systolic failure. Current treatment options are severely lacking due in part to the poor understanding of the complexity of molecular pathways involved in cardiac fibrosis. Therefore, a need exists for an in-vitro model system that recapitulates the defining features of a fibrotic cellular environment, namely extracellular mechanics and composition. Type I collagen, as the major matrix component of fibrotic tissue, is an attractive matrix choice for a fibrosis model, but demonstrates poor mechanical strength due to solubility limits. However, plastic compression of collagen matrices has been shown to significantly increase their mechanical properties. Here, we utilized confined compression of collagen oligomer matrices to achieve constructs with increased surface fibril concentration (3.07 fold increase to 13.4 mg/mL) and mean thickness (1.4 fold increase to 1.39 µm) that were subsequently used to study the spontaneous cardiomyocyte beating response on compressed gels. Beating intensity was shown to be significantly decreased in compressed collagen matrices, with a 2.4 fold decrease seen in calcium stain intensity. These results were consistent with the expected beating response on a pathologically stiff substrate. Plastic compression of collagen matrices therefore shows potentially as a mechanically and physiochemically relevant platform for in-vitro study of cardiac fibrosis
Controlled Magnetic Alignment of Oligomeric Collagen
No full textTissue engineering offers many potential solutions for replacement and repair of diseased or damage tissues through the use of engineered constructs. It has become clear that emulation of structural morphology is one important component for successful tissue scaffold development. As the main component of the extra-cellular matrix, collagen is a popular choice as a biopolymer for engineering biomimetic constructs. Magnetic fields have been shown to nondestructively align collagen gels during its sol-gel transition (i.e. polymerization). This technique is limited to the alignment of thin collagen gels and lacks control necessary to achieve targeted alignment profiles. The specific aims of this work were to 1) determine the efficacy of magnetic fields to align oligomeric collagen formulations, 2) evaluate the effect of fibrillogenesis temperature on the resultant fiber anisotropy of magnetic alignment, 3) quantify collagen fiber anisotropy on a bulk- and mico-scale, and 4) investigate the use of magnetic field, temperature, and concentration as tunable parameters for achieving targeted anisotropy levels and alignment depth. The results of this study indicate that magnetic alignment can be extended to oligomeric collagen and can be controlled through temperature and concentration manipulation
Synthesis and characterization of a lubricin mimic (mLub) to reduce friction on the articular cartilage surface
Full text linkThe lubricating proteoglycan, lubricin, facilitates the remarkable low friction and wear properties of articular cartilage in the synovial joints of the body. Lubricin lines the joint surfaces and plays a protective role as a boundary lubricant in sliding contact; down-regulation of lubricin is associated with cartilage degradation and the pathogenesis of osteoarthritis. An unmet need for early osteoarthritis treatment is the development of therapeutic molecules that mimic lubricin function and yet are also resistant to enzymatic degradation common in the damaged joint. Here, we engineered a lubricin mimic (mLub) that resists enzymatic degradation and binds to the articular surface to reduce friction. mLub was synthesized using a mucin-like chondroitin sulfate backbone with collagen II and hyaluronic acid (HA) binding peptides to promote interaction with the articular surface and synovial fluid constituents. In vitro and in vivo characterization confirmed the binding ability of mLub to isolated collagen II and HA, and to the cartilage surface. Following trypsin treatment to the cartilage surface, application of mLub, in combination with purified or commercially available hyaluronan, reduced the coefficient of friction to control levels as assessed over macro- to micro-scales by rheometry and atomic force microscopy. In vivo studies demonstrate an mLub residency time of less than 1 week. Enhanced lubrication by mLub reduces surface friction, ideal to help suppress the progression of degradation and cartilage loss in the joint. mLub therefore shows potential for viscosupplementation treatment in early osteoarthritis following injury
Biomechanics and relaxivity for functional imaging of articular cartilage injury and degradation
Full text linkOsteoarthritis (OA) is a major debilitating health concern and economic burden worldwide, affecting 27 million people in the United States alone. OA often follows tissue injury, and is marked by changes in the structure and biomechanical function of cartilage, including breakdown of extracellular matrix molecules, loss of bulk tissue stiffness, and increase in articular surface friction and wear. Unlike bone and many other tissues, cartilage lacks an intrinsic capacity for regeneration. Advanced OA is typically diagnosed by patient symptoms (e.g. joint pain) and confirmed by radiographic evaluation of joint space narrowing. However, the application of functional imaging to assess cartilage physiology may provide an early diagnosis of joint changes prior to patient symptoms. One such functional imaging modality, magnetic resonance imaging (MRI), may be used to characterize the mechanics of joint cartilage in vitro and in vivo, but it has not yet been applied to evaluate cartilage injury in defined damage models. Here, we studied the changes in MRI-assessed intratissue strain following cartilage injury, and correlate those changes with traditional assessment metrics such as relaxivity, biochemical composition, and microstructure. Osteochondral samples were harvested from the load-bearing region of juvenile bovine knees. Samples were exposed to injurious compressive loading at 100% strain/second and incubated over four weeks. Tissue strain throughout the cartilage interior was measured by displacements under applied loading by MRI (dualMRI) and coregistered to relaxivity measures of T1 and T2. Proteoglycan and collagen content, cartilage microstructure, and cell viability were also assessed by biochemical, histochemical, and microscopy assays. Injurious compressive strain magnitudes of 50% resulted in decreased chondrocyte viability. By three weeks post-injury, dualMRI strains in the compressive loading direction of injured cartilage increased compared to controls, suggesting a regional loss of tissue stiffness. T2 and sample height increased with incubation time. Changes in proteoglycan and collagen content, and microstructure, were also observed to change with incubation time. These finding indicate that dualMRI may be a promising technology to detect and diagnose the early onset of injury-related degeneration compared to conventional techniques like MR relaxivity. The results also indicate the utility and potential for functional imaging to assess disease progression and treatment
Magnetic manipulation and multimodal imaging for single cell direct mechanosensing
Full text linkThe study of internal mechanics of single cells is paramount to understand mechanisms of mechanoregulation. External loading and cell-mediated force generation result in changes in cell shape, rheology, and the deformation of subcellular structures such as the nucleus. Moreover, alterations in the processes that regulate these responses have been further correlated to specific pathologies. Cellular deformation is often studied through application of forces in the environment of the cell, relying on strain and stress transfer through focal adhesions and the cytoskeletal system. However, the transfer of these external forces to internal mechanics can introduce uncertainties in the interpretation of subcellular responses. Our group has focused on minimally-invasive techniques for the study of internal mechanical perturbation and mechanobiology measures. We have been particularly interested in multimodal imaging methods that combine and leverage nano-scale spatial localization, visualization, biophysical and physico-chemical analysis features to reveal information that cannot be attained by any single method alone. We recently fabricated novel atomic force microscopy (AFM) cantilevers, functionalized to generate small, highly-localized magnetic fields, for the controlled force application and sensing of single cells. In combination with AFM and fluorescence microscopy detection capabilities, this technique enables the selective stimulation and monitoring of cells injected with superparamagnetic microbeads. Though the targeted magnetic force application, we are able to apply various waveforms to direct the microdisplacements of the injected beads to allow insight into the structural architecture of the cell. Coupling this with AFM techniques further yields insight into internal and external mechanics over time. This technique can be extended to include studies of intranuclear strain dynamics through fluorescent labeling of specific cellular targets and image post-processing algorithms such as hyperelastic warping. Furthermore, the ability to alter the culture environment (e.g. to manipulate osmotic pressure or enable drug delivery) allows this technique to be a powerful single cell analysis tool for a diverse set of applications. We demonstrate the feasibility of this technique through the localized application of low magnetic fields that produce bead displacements in the micrometer scale. The effects of larger induced magnetic fields in the displacement field are also presented, along with validation and viability studies, and a range of practical applications for the study of single cells
An inside-out approach to nuclear mechanics: Genetic engineering of an in vitro laminopathy model
No full textLaminopathies are a group of genetic diseases affecting the nuclear lamina of metazoan cells with mutations in the genes LMNA, LMNB1, and LMNB2, which code for intermediate filament proteins called lamins. In LMNA alone, more than 180 mutations causing at least 14 diseases exist displaying a variety of phenotypes. Classical Hutchinson-Gilford Progeria Syndrome (HGPS) is a progeroid, or accelerated aging, laminopathy caused by a de novo heterozygous point mutation in LMNA (c. 1824 C\u3eT). The disease affects multiple organ systems resulting in apparent accelerated aging and early death due to atherosclerotic complications such as myocardial infarction or stroke. At the subcellular level, HGPS causes stiffening of the nuclear envelope, an increase in mechanosensitivity, inhibited DNA repair, aberrant chromatin organization, and other complications. Here, the CRISPR-Cas9 genome editing system has been used to successfully incorporate the HGPS mutation into the human HT1080 fibroblast cell line using paired D10A mutant Cas9 nickases guided by truncated guide RNAs (tru-gRNAs) with low levels of off-target mutagenesis. This in vitro genetic disease model may be used in nuclear mechanics studies of HGPS to further uncover disease mechanisms and potential treatments or in basic science research aimed at learning the roles played by nuclear lamins and their mutant forms
Displacement-encoded and quantitative MRI in human osteoarthritis
No full textOsteoarthritis (OA) is a debilitating, degenerative disease of articular cartilage in synovial joints afflicting an estimated 27 million adults in the United States alone with an annual cost of $80 billion. The mechanical function of cartilage declines during OA progression, resulting from softening of the tissue coupled with increased friction and wear. Magnetic resonance imaging (MRI) is currently a gold standard for diagnosing late-stage OA, but there remains a need to detect early- to mid-stage OA in vivo to possibly prevent the need for invasive and costly total joint replacement surgery. Moderate but limited success has been achieved with quantitative MRI (qMRI), including T1ρ and T 2 mapping. qMRI parameters are thought to be sensitive to biochemical changes in diseased cartilage, but ignore associated mechanical changes. Displacement-encoded MRI (deMRI) allows for the measurement of deformation in articular cartilage during cyclic loading, and may be sensitive to changing mechanical function. The specific aims of this work were 1) to understand the distribution of strains and related parameters in explanted cartilage from human volunteers with OA, 2) determine the ability of deMRI to assay and detect OA as defined by a quantitative metric, 3) compare the potential of deMRI to that of qMRI for OA detection, and 4) determine if these measures can be used together to best predict OA in the earliest stages
Top-Down and Bottom-Up Engineered Microenvironments for Cartilage Tissue Regeneration
No full textTissue engineering has the potential to mitigate and remedy many degenerative diseases and traumatic injuries. Osteoarthritis, characterized by the structural and biochemical degradation of articular cartilage, affects over 27 million individuals nationwide. The current clinical standard for cartilage repair includes methods such as mosaicplasty and autograft transplantation, where cartilage from non-load bearing regions is used to fill in defects in the cartilage structure. These methods suggest that the complex structure and biochemistry of native cartilage extracellular matrix positively contributes to cartilage healing and regeneration. We investigated this idea through a series of top-down and bottom-up tissue engineering approaches. Using a top-down approach, we studied the healing capability of decellularized cartilage allografts in an in vivo ovine osteoarthritis model. The results of this in vivo study showed positive cartilage healing and supported the model of tissue physicochemical microenvironment as an ideal environment for tissue regeneration. As a result, we investigated a bottom-up approach, attempting to recapitulate various aspects of the native cartilage tissue. In the first study, we investigated the ability of polymerizable collagen fibrils to be aligned in the presence of high magnetic fields, emulating the fibril alignment aspect of the cartilage superficial zone. Further, using a plastic compression approach, we developed gradient collagen structures that emulated the depth dependent alignment properties of articular cartilage while greatly increasing the density and mechanical properties. Finally, we combined the top-down and bottom-up approaches in a unique way to introduce native cartilage signals to a stem cell population. Microparticulated and decellularized cartilage was introduced to collagen-suspended human mesenchymal stem cells in a 3D composite matrix. Additional composites formed from Gu•HCl reductions gave further insight into the contribution of the various ECM components of articular cartilage on the differentiation potential of human MSCs for cartilage regeneration. The culmination of these studies gives unique information on the separation and combination of top-down and bottom-up approaches to tissue engineering in the field of cartilage and osteoarthritis
- …
