Holly A. Leddy

Surface protein characterization of human adipose tissue-derived stromal cells.

Human bone marrow stromal cells are a multipotent population of cells capable of differentiating into a number of mesodermal lineages as well as supporting hematopoeisis. Their distinct protein and gene expression phenotype is well characterized in the literature. Human adipose tissue presents an alternative source of multipotent stromal cells. In this study, we have defined the phenotype of the human adipose tissue‐derived stromal cells in both the differentiated and undifferentiated states. Flow cytometry and immunohistochemistry show that human adipose tissue‐derived stromal cells have a protein expression phenotype that is similar to that of human bone marrow stromal cells. Expressed proteins include CD9, CD10, CD13, CD29, CD34, CD44, CD 49d, CD 49e, CD54, CD55, CD59, CD105, CD106, CD146, and CD166. Expression of some of these proteins was further confirmed by PCR and immunoblot detection. Unlike human bone marrow‐derived stromal cells, we did not detect the STRO‐1 antigen on human adipose tissue‐derived stromal cells. Cells cultured under adipogenic conditions uniquely expressed C/EBPα and PPARδ, two transcriptional regulators of adipogenesis. Cells cultured under osteogenic conditions were more likely to be in the proliferative phases of the cell cycle based on flow cytometric analysis of PCNA and Ki67. The similarities between the phenotypes of human adipose tissue‐derived and human bone marrow‐derived stromal cells could have broad implications for human tissue engineering.

TRPV4-mediated mechanotransduction regulates the metabolic response of chondrocytes to dynamic loading.

Significance Physiologic joint loading plays a critical role in the maintenance of articular cartilage structure and function, whereas abnormal loading can lead to pathologic changes in joint tissues. However, the mechanisms by which mechanical loading is transduced into intracellular signals that regulate chondrocyte homeostasis are not fully understood. In this study, we show that the mechanosensitive cation channel transient receptor potential vanilloid 4 (TRPV4) plays a critical role in the physiological link between mechanical loading and chondrocyte function. Specifically, TRPV4 acts a transducer of mechanical loading to regulate cartilage extracellular matrix biosynthesis. A better understanding of the mechanisms involved in chondrocyte mechanotransduction could enable the development of novel therapies for joint diseases such as osteoarthritis. Mechanical loading of joints plays a critical role in maintaining the health and function of articular cartilage. The mechanism(s) of chondrocyte mechanotransduction are not fully understood, but could provide important insights into new physical or pharmacologic therapies for joint diseases. Transient receptor potential vanilloid 4 (TRPV4), a Ca2+-permeable osmomechano-TRP channel, is highly expressed in articular chondrocytes, and loss of TRPV4 function is associated with joint arthropathy and osteoarthritis. The goal of this study was to examine the hypothesis that TRPV4 transduces dynamic compressive loading in articular chondrocytes. We first confirmed the presence of physically induced, TRPV4-dependent intracellular Ca2+ signaling in agarose-embedded chondrocytes, and then used this model system to study the role of TRPV4 in regulating the response of chondrocytes to dynamic compression. Inhibition of TRPV4 during dynamic loading prevented acute, mechanically mediated regulation of proanabolic and anticatabolic genes, and furthermore, blocked the loading-induced enhancement of matrix accumulation and mechanical properties. Furthermore, chemical activation of TRPV4 by the agonist GSK1016790A in the absence of mechanical loading similarly enhanced anabolic and suppressed catabolic gene expression, and potently increased matrix biosynthesis and construct mechanical properties. These findings support the hypothesis that TRPV4-mediated Ca2+ signaling plays a central role in the transduction of mechanical signals to support cartilage extracellular matrix maintenance and joint health. Moreover, these insights raise the possibility of therapeutically targeting TRPV4-mediated mechanotransduction for the treatment of diseases such as osteoarthritis, as well as to enhance matrix formation and functional properties of tissue-engineered cartilage as an alternative to bioreactor-based mechanical stimulation.

Site-specific molecular diffusion in articular cartilage measured using fluorescence recovery after photobleaching.

AbstractDiffusive transport of solutes is critical to the normal function of articular cartilage. The diffusion of macromolecules through cartilage may be affected by the local composition and structure, which vary with depth from the tissue surface. We hypothesized that the diffusion coefficient of uncharged molecules also varies with depth and molecular size. We used fluorescence recovery after photobleaching (FRAP) to measure site-specific diffusion coefficients of fluorescent dextran molecules (3, 40, 70, and 500 kDa) in porcine articular cartilage. The diffusion coefficients measured using FRAP exhibited an inverse size dependence and were in general agreement with those measured using other techniques. The diffusion coefficients for all molecules varied significantly with depth in a manner that depended upon the size of the diffusing molecule. The diffusion coefficients for the 3 and 500 kDa dextrans were 1.6 and 2.4 times greater, respectively, in the surface zone as compared to the middle and deep zones, whereas the diffusion coefficients of the 40 and 70 kDa dextrans were 0.3 and 0.2 times lower in the surface zone as compared to the middle and deep zones. These differences may reflect variations in the structure and composition of collagen, proteoglycans, and other macromolecules among the zones.© 2003 Biomedical Engineering Society. PAC2003: 8715Vv, 8719Rr, 8764Tt

Transient receptor potential vanilloid 4: The sixth sense of the musculoskeletal system?

The critical discovery in the past two decades of the transient receptor potential (TRP) superfamily of ion channels has revealed the potential mechanisms by which cells sense diverse stimuli beyond the prototypical “five senses,” identifying ion channels that are gated by heat, cold, mechanical loading, osmolarity, and other physical and chemical stimuli. TRP vanilloid 4 (TRPV4) is a Ca2+‐permeable nonselective cation channel that appears to play a mechanosensory or osmosensory role in several musculoskeletal tissues. In articular cartilage, TRPV4 exhibits osmotic sensitivity, controlling cellular volume recovery, and other physiologic responses to osmotic stress. TRPV4 is expressed in both osteoblasts and osteoclasts, and the absence of TRPV4 prevents disuse‐induced bone loss. TRPV4 activation promotes chondrogenesis by inducing SOX9 transcription, whereas a TRPV4 gain‐of‐function mutation leads to a developmental skeletal dysplasia, suggesting a critical role for TRPV4 in skeletal development. These studies provide mounting evidence for a regulatory role for the sensory channel TRPV4 in control of musculoskeletal tissues.

Synergy between Piezo1 and Piezo2 channels confers high-strain mechanosensitivity to articular cartilage.

Significance Cartilage, a mechanically sensitive tissue that covers joints, is essential for vertebrate locomotion by sustaining skeletal mobility. Transduction of mechanical stimuli by cartilage cells, chondrocytes, leads to biochemical–metabolic responses. Such mechanotransduction can be beneficial for tissue maintenance when evoked by low-level mechanical stimuli, or can have health-adverse effects via cartilage-damaging high-strain mechanical stress. Thus, high-strain mechanotransduction by cartilage mechanotrauma is relevant for the pathogenesis of osteoarthritis. Molecular mechanisms of high-strain mechanotransduction of chondrocytes have been elusive. Here we identify Piezo1 and Piezo2 mechanosensitive ion channels in chondrocytes as transduction channels for high-strain mechanical stress. We verify their functional link to the cytoskeleton as important for their concerted function and offer a remedial strategy by application of a Piezo1/2 blocking peptide, GsMTx4, from tarantula venom. Diarthrodial joints are essential for load bearing and locomotion. Physiologically, articular cartilage sustains millions of cycles of mechanical loading. Chondrocytes, the cells in cartilage, regulate their metabolic activities in response to mechanical loading. Pathological mechanical stress can lead to maladaptive cellular responses and subsequent cartilage degeneration. We sought to deconstruct chondrocyte mechanotransduction by identifying mechanosensitive ion channels functioning at injurious levels of strain. We detected robust expression of the recently identified mechanosensitive channels, PIEZO1 and PIEZO2. Combined directed expression of Piezo1 and -2 sustained potentiated mechanically induced Ca2+ signals and electrical currents compared with single-Piezo expression. In primary articular chondrocytes, mechanically evoked Ca2+ transients produced by atomic force microscopy were inhibited by GsMTx4, a PIEZO-blocking peptide, and by Piezo1- or Piezo2-specific siRNA. We complemented the cellular approach with an explant-cartilage injury model. GsMTx4 reduced chondrocyte death after mechanical injury, suggesting a possible therapy for reducing cartilage injury and posttraumatic osteoarthritis by attenuating Piezo-mediated cartilage mechanotransduction of injurious strains.

Synovial Fluid Concentrations and Relative Potency of Interleukin-1 alpha and beta in Cartilage and Meniscus Degradation

Cartilage degeneration with osteoarthritis (OA) is believed to involve the activities of interleukin‐1 (IL‐1), which exists as alpha and beta isoforms. The goal of this study was to measure the concentrations of both isoforms of IL‐1 in the synovial fluid of normal and spontaneously osteoarthritic porcine knees, and to test the hypothesis that physiologic concentrations of IL‐1α and IL‐1β exhibit different potencies in activating calcium signaling, the production of matrix metalloproteinases and nitric oxide, and the loss of proteoglycans and tissue mechanical properties in cartilage and meniscus. Median concentrations of IL‐1α were 0.043 ng/ml with mild OA and 0.288 ng/ml with moderate OA, whereas IL‐1β concentrations were 0.109 ng/ml with mild OA and 0.122 ng/ml with moderate OA. Both isoforms induced calcium signaling in chondrocytes and meniscal cells at all concentrations. Overall, cartilage and meniscus catabolism was significantly more sensitive to IL‐1α than IL‐1β at concentrations of 1 ng/ml or less, while few differences were observed between the two forms at 10 ng/ml. These data provide a range of physiologic IL‐1 concentrations that can serve as a framework for the comparison of various in vitro studies, as well as providing further insight for the development of anti‐cytokine therapies for OA.

Diurnal Variations in Articular Cartilage Thickness and Strain in the Human Knee

Due to the biphasic viscoelastic nature of cartilage, joint loading may result in deformations that require times on the order of hours to fully recover. Thus, cartilaginous tissues may exhibit cumulative strain over the course of each day. The goal of this study was to assess the magnitude and spatial distribution of strain in the articular cartilage of the knee with daily activity. Magnetic resonance (MR) images of 10 asymptomatic subjects (six males and four females) with mean age of 29 years were obtained at 8:00 AM and 4:00 PM on the same day using a 3T magnet. These images were used to create 3D models of the femur, tibia, and patella from which cartilage thickness distributions were quantified. Cartilage thickness generally decreased from AM to PM in all areas except the patellofemoral groove and was associated with significant compressive strains in the medial condyle and tibial plateau. From AM to PM, cartilage of the medial tibial plateau exhibited a compressive strain of -5.1±1.0% (mean±SEM) averaged over all locations, while strains in the lateral plateau were slightly lower (-3.1±0.6%). Femoral cartilage showed an average strain of -1.9±0.6%. The findings of this study show that human knee cartilage undergoes diurnal changes in strain that vary with site in the joint. Since abnormal joint loading can be detrimental to cartilage homeostasis, these data provide a baseline for future studies investigating the effects of altered biomechanics on diurnal cartilage strains and cartilage physiology.

The Mechanobiology of Articular Cartilage: Bearing the Burden of Osteoarthritis

Articular cartilage injuries and degenerative joint diseases are responsible for progressive pain and disability in millions of people worldwide, yet there is currently no treatment available to restore full joint functionality. As the tissue functions under mechanical load, an understanding of the physiologic or pathologic effects of biomechanical factors on cartilage physiology is of particular interest. Here, we highlight studies that have measured cartilage deformation at scales ranging from the macroscale to the microscale, as well as the responses of the resident cartilage cells, chondrocytes, to mechanical loading using in vitro and in vivo approaches. From these studies, it is clear that there exists a complex interplay among mechanical, inflammatory, and biochemical factors that can either support or inhibit cartilage matrix homeostasis under normal or pathologic conditions. Understanding these interactions is an important step toward developing tissue engineering approaches and therapeutic interventions for cartilage pathologies, such as osteoarthritis.

High Body Mass Index is Associated with Increased Diurnal Strains in the Articular Cartilage of the Knee

OBJECTIVE Obesity is an important risk factor for osteoarthritis (OA) and is associated with changes in both the biomechanical and inflammatory environments within the joint. However, the relationship between obesity and cartilage deformation is not fully understood. The goal of this study was to determine the effects of body mass index (BMI) on the magnitude of diurnal cartilage strain in the knee. METHODS Three-dimensional maps of knee cartilage thickness were developed from 3T magnetic resonance images of the knees of asymptomatic age- and sex-matched subjects with normal BMI (18.5-24.9 kg/m2) or high BMI (25-31 kg/m2). Site-specific magnitudes of diurnal cartilage strain were determined using aligned images recorded at 8:00 AM and 4:00 PM on the same day. RESULTS Subjects with high BMI had significantly thicker cartilage on both the patella and femoral groove, as compared to subjects with normal BMI. Diurnal cartilage strains were dependent on location in the knee joint, as well as BMI. Subjects with high BMI, compared to those with normal BMI, exhibited significantly higher compressive strains in the tibial cartilage. Cartilage thickness on both femoral condyles decreased significantly from the AM to the PM time point; however, there was no significant effect of BMI on diurnal cartilage strain in the femur. CONCLUSION Increased BMI is associated with increased diurnal strains in articular cartilage of both the medial and lateral compartments of the knee. The increased cartilage strains observed in individuals with high BMI may, in part, explain the elevated risk of OA associated with obesity or may reflect alterations in the cartilage mechanical properties in subjects with high BMI.

Site-Specific Effects of Compression on Macromolecular Diffusion in Articular Cartilage

Articular cartilage is the connective tissue that lines joints and provides a smooth surface for joint motion. Because cartilage is avascular, molecular transport occurs primarily via diffusion or convection, and cartilage matrix structure and composition may affect diffusive transport. Because of the inhomogeneous compressive properties of articular cartilage, we hypothesized that compression would decrease macromolecular diffusivity and increase diffusional anisotropy in a site-specific manner that depends on local tissue strain. We used two fluorescence photobleaching methods, scanning microphotolysis and fluorescence imaging of continuous point photobleaching, to measure diffusion coefficients and diffusional anisotropy of 70 kDa dextran in cartilage during compression, and measured local tissue strain using texture correlation. For every 10% increase in normal strain, the fractional change in diffusivity decreased by 0.16 in all zones, and diffusional anisotropy increased 1.1-fold in the surface zone and 1.04-fold in the middle zone, and did not change in the deep zone. These results indicate that inhomogeneity in matrix structure and composition may significantly affect local diffusive transport in cartilage, particularly in response to mechanical loading. Our findings suggest that high strains in the surface zone significantly decrease diffusivity and increase anisotropy, which may decrease transport between cartilage and synovial fluid during compression.