The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Farshid Guilak - One of the best experts on this subject based on the ideXlab platform.
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osteoarthritis as a disease of the cartilage Pericellular Matrix
Matrix Biology, 2018Co-Authors: Farshid Guilak, Robert J Nims, Amanda Dicks, I MeulenbeltAbstract:Osteoarthritis is a painful joint disease characterized by progressive degeneration of the articular cartilage as well as associated changes to the subchondral bone, synovium, and surrounding joint tissues. While the effects of osteoarthritis on the cartilage extracellular Matrix (ECM) have been well recognized, it is now becoming apparent that in many cases, the onset of the disease may be initially reflected in the Matrix region immediately surrounding the chondrocytes, termed the Pericellular Matrix (PCM). Growing evidence suggests that the PCM - which along with the enclosed chondrocytes are termed the "chondron" - acts as a critical transducer or "filter" of biochemical and biomechanical signals for the chondrocyte, serving to help regulate the homeostatic balance of chondrocyte metabolic activity in response to environmental signals. Indeed, it appears that alterations in PCM properties and cell-Matrix interactions, secondary to genetic, epigenetic, metabolic, or biomechanical stimuli, could in fact serve as initiating or progressive factors for osteoarthritis. Here, we discuss recent advances in the understanding of the role of the PCM, with an emphasis on the reciprocity of changes that occur in this Matrix region with disease, as well as how alterations in PCM properties could serve as a driver of ECM-based diseases such as osteoarthritis. Further study of the structure, function, and composition of the PCM in normal and diseased conditions may provide new insights into the understanding of the pathogenesis of osteoarthritis, and presumably new therapeutic approaches for this disease.
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type vi collagen regulates Pericellular Matrix properties chondrocyte swelling and mechanotransduction in mouse articular cartilage
Arthritis & Rheumatism, 2015Co-Authors: Nicole A Zelenski, Holly A. Leddy, Johannah Sanchezadams, Jinzi Zhang, Paolo Bonaldo, Wolfgang Liedtke, Farshid GuilakAbstract:Objective Mechanical factors play a critical role in the physiology and pathology of articular cartilage, although the mechanisms of mechanical signal transduction are not fully understood. We undertook this study to test the hypothesis that type VI collagen is necessary for mechanotransduction in articular cartilage by determining the effects of type VI collagen knockout on the activation of the mechano-osmosensitive, calcium-permeable channel TRPV4 (transient receptor potential vanilloid channel 4) as well as on osmotically induced chondrocyte swelling and Pericellular Matrix (PCM) mechanical properties. Methods Confocal laser scanning microscopy was used to image TRPV4-mediated calcium signaling and osmotically induced cell swelling in intact femora from 2- and 9-month-old wild-type (WT) and type VI collagen–deficient (Col6a1−/−) mice. Immunofluorescence-guided atomic force microscopy was used to map PCM mechanical properties based on the presence of perlecan. Results Hypo-osmotic stress–induced TRPV4-mediated calcium signaling was increased in Col6a1−/− mice relative to WT controls at 2 months. Col6a1−/− mice exhibited significantly increased osmotically induced cell swelling and decreased PCM moduli relative to WT controls at both ages. Conclusion In contrast to our original hypothesis, type VI collagen was not required for TRPV4-mediated Ca2+ signaling; however, knockout of type VI collagen altered the mechanical properties of the PCM, which in turn increased the extent of cell swelling and osmotically induced TRPV4 signaling in an age-dependent manner. These findings emphasize the role of the PCM as a transducer of mechanical and physicochemical signals, and they suggest that alterations in PCM properties, as may occur with aging or osteoarthritis, can influence mechanotransduction via TRPV4 or other ion channels.
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high resistance of the mechanical properties of the chondrocyte Pericellular Matrix to proteoglycan digestion by chondroitinase aggrecanase or hyaluronidase
Journal of The Mechanical Behavior of Biomedical Materials, 2014Co-Authors: Rebecca E. Wilusz, Farshid GuilakAbstract:In articular cartilage, the extracellular Matrix (ECM) and chondrocyte-associated Pericellular Matrix (PCM) are characterized by a high concentration of proteoglycans (PGs) and their associated glycosaminoglycans (GAGs). These molecules serve important biochemical, structural, and biomechanical roles in the tissue and differences in their regional distributions suggest that different GAG/PG species contribute to the specific biomechanical properties of the ECM and PCM. The objective of this study was to investigate region-specific contributions of aggrecan, chondroitin and dermatan sulfate, and hyaluronan to the micromechanical properties of articular cartilage PCM and ECM in situ. Cryosections of porcine cartilage underwent digestion with ADAMTS-4, chondroitinase ABC, bacterial hyaluronidase or human leukocyte elastase. Guided by immunofluorescence for type VI collagen, AFM stiffness mapping was used to evaluate the elastic properties of matched PCM and ECM regions in paired control and digested cartilage sections. These methods were used to test the hypotheses that specific enzymatic digestion of GAGs or PGs would reduce both PCM and ECM elastic moduli. Elastase, which digests a number of PGs, some types of collagen, and non-collagenous proteins, was used as a positive control. ECM elastic moduli were significantly reduced by all enzyme treatments. However, PCM micromechanical properties were unaffected by enzymatic digestion of aggrecan, chondroitin/dermatan sulfate, and hyaluronan but were significantly reduced by 24% following elastase digestion. Our results provide new evidence for high resistance of PCM micromechanical properties to PG digestion and suggest a potential role for elastase in the degradation of the ECM and PCM.
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The structure and function of the Pericellular Matrix of articular cartilage
Matrix Biology, 2014Co-Authors: Rebecca E. Wilusz, Johannah Sanchez-adams, Farshid GuilakAbstract:Chondrocytes in articular cartilage are surrounded by a narrow Pericellular Matrix (PCM) that is both biochemically and biomechanically distinct from the extracellular Matrix (ECM) of the tissue. While the PCM was first observed nearly a century ago, its role is still under investigation. In support of early hypotheses regarding its function, increasing evidence indicates that the PCM serves as a transducer of biochemical and biomechanical signals to the chondrocyte. Work over the past two decades has established that the PCM in adult tissue is defined biochemically by several molecular components, including type VI collagen and perlecan. On the other hand, the biomechanical properties of this structure have only recently been measured. Techniques such as micropipette aspiration, in situ imaging, computational modeling, and atomic force microscopy have determined that the PCM exhibits distinct mechanical properties as compared to the ECM, and that these properties are influenced by specific PCM components as well as disease state. Importantly, the unique relationships among the mechanical properties of the chondrocyte, PCM, and ECM in different zones of cartilage suggest that this region significantly influences the stress-strain environment of the chondrocyte. In this review, we discuss recent advances in the measurement of PCM mechanical properties and structure that further increase our understanding of PCM function. Taken together, these studies suggest that the PCM plays a critical role in controlling the mechanical environment and mechanobiology of cells in cartilage and other cartilaginous tissues, such as the meniscus or intervertebral disc.
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micromechanical mapping of early osteoarthritic changes in the Pericellular Matrix of human articular cartilage
Osteoarthritis and Cartilage, 2013Co-Authors: Rebecca E. Wilusz, Stefan Zauscher, Farshid GuilakAbstract:Summary Objective Osteoarthritis (OA) is a degenerative joint disease characterized by the progressive loss of articular cartilage. While macroscale degradation of the cartilage extracellular Matrix (ECM) has been extensively studied, microscale changes in the chondrocyte Pericellular Matrix (PCM) and immediate microenvironment with OA are not fully understood. The objective of this study was to quantify osteoarthritic changes in the micromechanical properties of the ECM and PCM of human articular cartilage in situ using atomic force microscopy (AFM). Method AFM elastic mapping was performed on cryosections of human cartilage harvested from both condyles of macroscopically normal and osteoarthritic knee joints. This method was used to test the hypotheses that both ECM and PCM regions exhibit a loss of mechanical properties with OA and that the size of the PCM is enlarged in OA cartilage as compared to normal tissue. Results Significant decreases were observed in both ECM and PCM moduli of 45% and 30%, respectively, on the medial condyle of OA knee joints as compared to cartilage from macroscopically normal joints. Enlargement of the PCM, as measured biomechanically, was also observed in medial condyle OA cartilage, reflecting the underlying distribution of type VI collagen in the region. No significant differences were observed in elastic moduli or their spatial distribution on the lateral condyle between normal and OA joints. Conclusion Our findings provide new evidence of significant site-specific degenerative changes in the chondrocyte micromechanical environment with OA.
Rebecca E. Wilusz - One of the best experts on this subject based on the ideXlab platform.
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high resistance of the mechanical properties of the chondrocyte Pericellular Matrix to proteoglycan digestion by chondroitinase aggrecanase or hyaluronidase
Journal of The Mechanical Behavior of Biomedical Materials, 2014Co-Authors: Rebecca E. Wilusz, Farshid GuilakAbstract:In articular cartilage, the extracellular Matrix (ECM) and chondrocyte-associated Pericellular Matrix (PCM) are characterized by a high concentration of proteoglycans (PGs) and their associated glycosaminoglycans (GAGs). These molecules serve important biochemical, structural, and biomechanical roles in the tissue and differences in their regional distributions suggest that different GAG/PG species contribute to the specific biomechanical properties of the ECM and PCM. The objective of this study was to investigate region-specific contributions of aggrecan, chondroitin and dermatan sulfate, and hyaluronan to the micromechanical properties of articular cartilage PCM and ECM in situ. Cryosections of porcine cartilage underwent digestion with ADAMTS-4, chondroitinase ABC, bacterial hyaluronidase or human leukocyte elastase. Guided by immunofluorescence for type VI collagen, AFM stiffness mapping was used to evaluate the elastic properties of matched PCM and ECM regions in paired control and digested cartilage sections. These methods were used to test the hypotheses that specific enzymatic digestion of GAGs or PGs would reduce both PCM and ECM elastic moduli. Elastase, which digests a number of PGs, some types of collagen, and non-collagenous proteins, was used as a positive control. ECM elastic moduli were significantly reduced by all enzyme treatments. However, PCM micromechanical properties were unaffected by enzymatic digestion of aggrecan, chondroitin/dermatan sulfate, and hyaluronan but were significantly reduced by 24% following elastase digestion. Our results provide new evidence for high resistance of PCM micromechanical properties to PG digestion and suggest a potential role for elastase in the degradation of the ECM and PCM.
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The structure and function of the Pericellular Matrix of articular cartilage
Matrix Biology, 2014Co-Authors: Rebecca E. Wilusz, Johannah Sanchez-adams, Farshid GuilakAbstract:Chondrocytes in articular cartilage are surrounded by a narrow Pericellular Matrix (PCM) that is both biochemically and biomechanically distinct from the extracellular Matrix (ECM) of the tissue. While the PCM was first observed nearly a century ago, its role is still under investigation. In support of early hypotheses regarding its function, increasing evidence indicates that the PCM serves as a transducer of biochemical and biomechanical signals to the chondrocyte. Work over the past two decades has established that the PCM in adult tissue is defined biochemically by several molecular components, including type VI collagen and perlecan. On the other hand, the biomechanical properties of this structure have only recently been measured. Techniques such as micropipette aspiration, in situ imaging, computational modeling, and atomic force microscopy have determined that the PCM exhibits distinct mechanical properties as compared to the ECM, and that these properties are influenced by specific PCM components as well as disease state. Importantly, the unique relationships among the mechanical properties of the chondrocyte, PCM, and ECM in different zones of cartilage suggest that this region significantly influences the stress-strain environment of the chondrocyte. In this review, we discuss recent advances in the measurement of PCM mechanical properties and structure that further increase our understanding of PCM function. Taken together, these studies suggest that the PCM plays a critical role in controlling the mechanical environment and mechanobiology of cells in cartilage and other cartilaginous tissues, such as the meniscus or intervertebral disc.
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micromechanical mapping of early osteoarthritic changes in the Pericellular Matrix of human articular cartilage
Osteoarthritis and Cartilage, 2013Co-Authors: Rebecca E. Wilusz, Stefan Zauscher, Farshid GuilakAbstract:Summary Objective Osteoarthritis (OA) is a degenerative joint disease characterized by the progressive loss of articular cartilage. While macroscale degradation of the cartilage extracellular Matrix (ECM) has been extensively studied, microscale changes in the chondrocyte Pericellular Matrix (PCM) and immediate microenvironment with OA are not fully understood. The objective of this study was to quantify osteoarthritic changes in the micromechanical properties of the ECM and PCM of human articular cartilage in situ using atomic force microscopy (AFM). Method AFM elastic mapping was performed on cryosections of human cartilage harvested from both condyles of macroscopically normal and osteoarthritic knee joints. This method was used to test the hypotheses that both ECM and PCM regions exhibit a loss of mechanical properties with OA and that the size of the PCM is enlarged in OA cartilage as compared to normal tissue. Results Significant decreases were observed in both ECM and PCM moduli of 45% and 30%, respectively, on the medial condyle of OA knee joints as compared to cartilage from macroscopically normal joints. Enlargement of the PCM, as measured biomechanically, was also observed in medial condyle OA cartilage, reflecting the underlying distribution of type VI collagen in the region. No significant differences were observed in elastic moduli or their spatial distribution on the lateral condyle between normal and OA joints. Conclusion Our findings provide new evidence of significant site-specific degenerative changes in the chondrocyte micromechanical environment with OA.
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immunofluorescence guided atomic force microscopy to measure the micromechanical properties of the Pericellular Matrix of porcine articular cartilage
Journal of the Royal Society Interface, 2012Co-Authors: Rebecca E. Wilusz, Louis E. Defrate, Farshid GuilakAbstract:The Pericellular Matrix (PCM) is a narrow region that is rich in type VI collagen that surrounds each chondrocyte within the extracellular Matrix (ECM) of articular cartilage. Previous studies have demonstrated that the chondrocyte micromechanical environment depends on the relative properties of the chondrocyte, its PCM and the ECM. The objective of this study was to measure the influence of type VI collagen on site-specific micromechanical properties of cartilage in situ by combining atomic force microscopy stiffness mapping with immunofluorescence imaging of PCM and ECM regions in cryo-sectioned tissue samples. This method was used to test the hypotheses that PCM biomechanical properties correlate with the presence of type VI collagen and are uniform with depth from the articular surface. Control experiments verified that immunolabelling did not affect the properties of the ECM or PCM. PCM biomechanical properties correlated with the presence of type VI collagen, and Matrix regions lacking type VI collagen immediately adjacent to the PCM exhibited higher elastic moduli than regions positive for type VI collagen. PCM elastic moduli were similar in all three zones. Our findings provide further support for type VI collagen in defining the chondrocyte PCM and contributing to its biological and biomechanical properties.
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A biomechanical role for perlecan in the Pericellular Matrix of articular cartilage.
Matrix Biology, 2012Co-Authors: Rebecca E. Wilusz, Louis E. Defrate, Farshid GuilakAbstract:Chondrocytes are surrounded by a narrow Pericellular Matrix (PCM) that is biochemically, structurally, and biomechanically distinct from the bulk extracellular Matrix (ECM) of articular cartilage. While the PCM is often defined by the presence of type VI collagen, other macromolecules such as perlecan, a heparan sulfate (HS) proteoglycan, are also exclusively localized to the PCM in normal cartilage and likely contribute to PCM structural integrity and biomechanical properties. Though perlecan is essential for normal cartilage development, its exact role in the PCM is unknown. The objective of this study was to determine the biomechanical role of perlecan in the articular cartilage PCM in situ and its potential as a defining factor of the PCM. To this end, atomic force microscopy (AFM) stiffness mapping was combined with dual immunofluorescence labeling of cryosectioned porcine cartilage samples for type VI collagen and perlecan. While there was no difference in overall PCM mechanical properties between type VI collagen- and perlecan-based definitions of the PCM, within the PCM, interior regions containing both type VI collagen and perlecan exhibited lower elastic moduli than more peripheral regions rich in type VI collagen alone. Enzymatic removal of HS chains from perlecan with heparinase III increased PCM elastic moduli both overall and locally in interior regions rich in both perlecan and type VI collagen. Heparinase III digestion had no effect on ECM elastic moduli. Our findings provide new evidence for perlecan as a defining factor in both the biochemical and biomechanical properties of the PCM.
Thomas N Wight - One of the best experts on this subject based on the ideXlab platform.
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Hyaluronan-dependent Pericellular Matrix.
Advanced drug delivery reviews, 2007Co-Authors: Stephen P Evanko, Raija Tammi, Markku Tammi, Thomas N WightAbstract:Hyaluronan is a multifunctional glycosaminoglycan that forms the structural basis of the Pericellular Matrix. Hyaluronan is extruded directly through the plasma membrane by one of three hyaluronan synthases and anchored to the cell surface by the synthase or cell surface receptors such as CD44 or RHAMM. Aggregating proteoglycans and other hyaluronan-binding proteins, contribute to the material and biological properties of the Matrix and regulate cell and tissue function. The Pericellular Matrix plays multiple complex roles in cell adhesion/de-adhesion, and cell shape changes associated with proliferation and locomotion. Time-lapse studies show that Pericellular Matrix formation facilitates cell detachment and mitotic cell rounding. Hyaluronan crosslinking occurs through various proteins, such as tenascin, TSG-6, inter-alpha-trypsin inhibitor, pentraxin and TSP-1. This creates higher order levels of structured hyaluronan that may regulate inflammation and other biological processes. Microvillous or filopodial membrane protrusions are created by active hyaluronan synthesis, and form the scaffold of hyaluronan coats in certain cells. The importance of the Pericellular Matrix in cellular mechanotransduction and the response to mechanical strain are also discussed.
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platelet derived growth factor stimulates the formation of versican hyaluronan aggregates and Pericellular Matrix expansion in arterial smooth muscle cells
Archives of Biochemistry and Biophysics, 2001Co-Authors: Stephen P Evanko, Kathleen R Braun, Pamela Y Johnson, Charles B Underhill, Jayesh Dudhia, Thomas N WightAbstract:Hyaluronan and versican-rich Pericellular matrices form around arterial smooth muscle cells (ASMC) preferentially during the detachment phase of proliferation and migration. PDGF is a potent mitogen and chemotactic agent for ASMC and also stimulates the production of extracellular Matrix molecules which may regulate the proliferative and migratory capacity of the cells. We have examined the effect of PDGF on the formation of hyaluronan-dependent Pericellular matrices, and on the synthesis and interaction of several major Pericellular coat constituents. As demonstrated using a particle exclusion assay, PDGF stimulated the formation of Pericellular matrices and was seen both in an increased proportion of cells with a coat and a greater coat size. This increase was accompanied by a transient increase in hyaluronan synthase 2 (HAS2) expression and an increase in hyaluronan synthesis and polymer length. PDGF also increased the synthesis of versican and link protein as measured at the mRNA and protein levels. The amount of native versican-hyaluronan aggregates and link-stabilized aggregate was also increased following PDGF treatment. Time lapse imaging showed that Pericellular Matrix formation occurred around trailing cell processes prior to their detachment. These data suggest that PDGF modulates the synthesis and organization of ASMC Pericellular coat-forming molecules such as versican, hyaluronan, and link protein, which leads to extracellular Matrix expansion and alterations in ASMC phenotype.
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formation of hyaluronan and versican rich Pericellular Matrix is required for proliferation and migration of vascular smooth muscle cells
Arteriosclerosis Thrombosis and Vascular Biology, 1999Co-Authors: Stephen P Evanko, John C Angello, Thomas N WightAbstract:The accumulation of hyaluronan (HA) and the HA-binding proteoglycan versican around smooth muscle cells in lesions of atherosclerosis suggests that together these molecules play an important role in the events of atherogenesis. In this study we have examined the formation of HA- and versican-rich Pericellular matrices by human aortic smooth muscle cells in vitro, using a particle-exclusion assay, and the role of the Pericellular Matrix in cell proliferation and migration. The structural dependence of the Pericellular Matrix on HA can be demonstrated by the complete removal of the Matrix with Streptomyces hyaluronidase. The presence of versican in the Pericellular Matrix was confirmed immunocytochemically. By electron microscopy, the cell coat was seen as a tangled network of hyaluronidase-sensitive filaments decorated with ruthenium red-positive proteoglycan granules. Ninety percent of migrating cells in wounded cultures, and virtually all mitotic cells, displayed abundant HA- and versican-rich coats. Time-lapse video imaging revealed that HA- and versican-rich Pericellular Matrix formation is dynamic and rapid, and coordinated specifically with cell detachment and mitotic cell rounding. HA oligosaccharides, which inhibit the binding of HA to the cell surface and prevent Pericellular Matrix formation, significantly reduced proliferation and migration in response to platelet-derived growth factor, whereas larger HA fragments and high molecular weight HA had no effect. Treatment with HA oligosaccharides also led to changes in cell shape from a typical fusiform morphology to a more spread and flattened appearance. These data suggest that organization of HA- and versican-rich Pericellular matrices may facilitate migration and mitosis by diminishing cell surface adhesivity and affecting cell shape through steric exclusion and the viscous properties of HA proteoglycan gels.
A Grodzinsky - One of the best experts on this subject based on the ideXlab platform.
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the spatial organisation and the Pericellular Matrix of human foetal chondrocytes are not inborn but instead acquired characteristics
Orthopaedic Proceedings, 2018Co-Authors: Tino Felka, S Zouhair, S Bast, K Struck, L Handl, M Votteler, Ulrich Stoeckle, V Schmidt, K Schenkelayland, A GrodzinskyAbstract:SummaryIn adult articular cartilage, the Pericellular Matrix (PCM) mediates chondrocyte-Matrix-interactions and is associated with the spatial cellular organization. Immunofluorescence microscopy, ...
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the spatial organisation and the Pericellular Matrix of human foetal chondrocytes are not inborn but instead acquired characteristics
Journal of Bone and Joint Surgery-british Volume, 2014Co-Authors: Tino Felka, S Zouhair, S Bast, K Struck, L Handl, M Votteler, Ulrich Stoeckle, V Schmidt, K Schenkelayland, A GrodzinskyAbstract:Summary In adult articular cartilage, the Pericellular Matrix (PCM) mediates chondrocyte-Matrix-interactions and is associated with the spatial cellular organization. Immunofluorescence microscopy, multiphoton-induced autofluorescence and second harmonic generation (SHG) imaging, as well as point pattern analyses revealed that both PCM and spatial organization were absent in fetal chondrocytes. Introduction In adult articular cartilage, the Pericellular Matrix mediates the biomechanical, biophysical and biomechanical interactions between the chondrocyte and the extracellular Matrix. The PCM is also associated with the spatial organization of human superficial chondrocytes, which are situated in four distinct patterns of strings, clusters, pairs or single cells. However, little is known about the PCM and the spatial organization during fetal development. In this study, we asked the question whether fetal chondrocytes display a spatial organization comparable to that of adult chondrocytes, and whether a PCM is present or absent in the early stages of fetal cartilage development. Methods Articular cartilage sections (100µm thickness) were prepared from the condyles of human fetal knee joints (pregnancy weeks 7–10) and from macroscopically intact condylar areas (knee joint replacement procedures). The samples were characterised by immunofluorescence microscopy and multiphoton-induced autofluorescence imaging combined with quantitative SHG signal profiling, a technique that allows quantifying the fibrillar collagen content. The spatial organization was analyzed by point pattern analyses as we described previously. For these analyses the Cartesian coordinates of each nucleus were determined by converting the immunofluorescence images into gray-scale images and finding the local gray-scale maxima with ImageJ (NIH). Results In adult superficial cartilage, the PCM surrounded single or groups of chondrocytes and defined their spatial organization of groups of cellular strings. PCM was characterised by a strong collagen VI staining signal and high collagen intensity as measured by SHG (156.7±12.4). In fetal cartilage, the condyles were characterised by a high cellular density, no recognizable spatial organization, and little amounts of extracellular Matrix. No collagen VI was detected in the Matrix surrounding the fetal chondrocytes. Furthermore, SHG imaging revealed that the fibrillar collagen intensity was significantly weaker (4.7±0.8; p Discussion/Conclusion In human fetal articular cartilage the spatial organization typical for adult chondrocytes was not present. Instead, the chondrocytes were situated densely and in close proximity. This study determined for the first time that the collagenous components that are typical for the adult PCM were not present in fetal cartilage. In conclusion, the PCM and also the spatial organization of superficial human chondrocytes develop with cartilage maturation and thus are not inborn but instead acquired characteristics.
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structure of Pericellular Matrix around agarose embedded chondrocytes
Osteoarthritis and Cartilage, 2007Co-Authors: John D Kisiday, Michael A Dimicco, Haiyan Gong, A GrodzinskyAbstract:Summary Objective Determine whether the structure of the type VI collagen component of the chondrocyte Pericellular Matrix (PCM) generated by agarose-embedded chondrocytes in culture is similar to that found in native articular cartilage. Methods Confocal microscopy, quick-freeze deep-etch electron microscopy, and real-time polymerase chain reaction (PCR) were used to investigate temporal and spatial patterns of type VI collagen protein deposition and gene expression by bovine chondrocytes during 4 weeks of culture within a 2% agarose hydrogel. Similar analyses were performed on chondrocytes within samples of intact cartilage obtained from the same joint surfaces as those used for cell isolation for comparison. Results Type VI collagen accumulated uniformly around cells embedded in agarose, with the rate of deposition slowing after the second week. After 1 week, PCM fibrils were observed to be oriented perpendicular to the cell surface, in contrast with the primarily tangential fibrillar arrangement observed in native articular cartilage. Expression of col6 in agarose-embedded cells was initially much higher (∼400%) than that in chondrocytes within cartilage. Expression of col6 in the cultured chondrocytes declined by ∼60% after 1 week, and remained stable thereafter. Conclusions PCM structure and composition around cells in a hydrogel scaffold may be different than that in native cartilage, with potential implications for mass transport, mechanotransduction, and ultimately, the success of tissue engineering approaches.
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nanomechanical properties of individual chondrocytes and their developing growth factor stimulated Pericellular Matrix
Journal of Biomechanics, 2007Co-Authors: Hanhwa Hung, Alexander Sprunt, Susan Chubinskaya, Christine Ortiz, A GrodzinskyAbstract:The nanomechanical properties of individual cartilage cells (chondrocytes) and their aggrecan and collagen-rich Pericellular Matrix (PCM) were measured via atomic force microscope nanoindentation using probe tips of two length scales (nanosized and micron-sized). The properties of cells freshly isolated from cartilage tissue (devoid of PCM) were compared to cells that were cultured for selected times (up to 28 days) in 3-D alginate gels which enabled PCM assembly and accumulation. Cells were immobilized and kept viable in pyramidal wells microfabricated into an array on silicon chips. Hertzian contact mechanics and finite element analyses were employed to estimate apparent moduli from the force versus depth curves. The effects of culture conditions on the resulting PCM properties were studied by comparing 10% fetal bovine serum to medium containing a combination of insulin growth factor-1 (IGF-1)+osteogenic protein-1 (OP-1). While both systems showed increases in stiffness with time in culture between days 7 and 28, the IGF-1+OP-1 combination resulted in a higher stiffness for the cell-PCM composite by day 28 and a higher apparent modulus of the PCM which is compared to the FBS cultured cells. These studies give insight into the temporal evolution of the nanomechanical properties of the pericellar Matrix relevant to the biomechanics and mechanobiology of tissue-engineered constructs for cartilage repair.
David G. Fernig - One of the best experts on this subject based on the ideXlab platform.
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selectivity in glycosaminoglycan binding dictates the distribution and diffusion of fibroblast growth factors in the Pericellular Matrix
Open Biology, 2016Co-Authors: Changye Sun, Marco Marcello, David Mason, Raphael Levy, David G. FernigAbstract:The range of biological outcomes generated by many signalling proteins in development and homeostasis is increased by their interactions with glycosaminoglycans, particularly heparan sulfate (HS). This interaction controls the localization and movement of these signalling proteins, but whether such control depends on the specificity of the interactions is not known. We used five fibroblast growth factors with an N-terminal HaloTag (Halo-FGFs) for fluorescent labelling, with well-characterized and distinct HS-binding properties, and measured their binding and diffusion in Pericellular Matrix of fixed rat mammary 27 fibroblasts. Halo-FGF1, Halo-FGF2 and Halo-FGF6 bound to HS, whereas Halo-FGF10 also interacted with chondroitin sulfate/dermatan sulfate, and FGF20 did not bind detectably. The distribution of bound FGFs in the Pericellular Matrix was not homogeneous, and for FGF10 exhibited striking clusters. Fluorescence recovery after photobleaching showed that FGF2 and FGF6 diffused faster, whereas FGF1 diffused more slowly, and FGF10 was immobile. The results demonstrate that the specificity of the interactions of proteins with glycosaminoglycans controls their binding and diffusion. Moreover, cells regulate the spatial distribution of different protein-binding sites in glycosaminoglycans independently of each other, implying that the extracellular Matrix has long-range structure.
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Transport of fibroblast growth factor 2 in the Pericellular Matrix is controlled by the spatial distribution of its binding sites in heparan sulfate
PLoS Biology, 2012Co-Authors: Laurence Duchesne, Vivien Octeau, Alison Beckett, Brahim Lounis, Ian A. Prior, Rachel N Bearon, David G. FernigAbstract:The heparan sulfate (HS) chains of proteoglycans are a key regulatory component of the extracellular matrices of animal cells, including the Pericellular Matrix around the plasma membrane. In these matrices they regulate transport, gradient formation, and effector functions of over 400 proteins central to cell communication. HS from different matrices differs in its selectivity for its protein partners. However, there has been no direct test of how HS in the Matrix regulates the transport of its partner proteins. We address this issue by single molecule imaging and tracking in fibroblast Pericellular Matrix of fibroblast growth factor 2 (FGF2), stoichiometrically labelled with small gold nanoparticles. Transmission electron microscopy and photothermal heterodyne imaging (PHI) show that the spatial distribution of the HS-binding sites for FGF2 in the Pericellular Matrix is heterogeneous over length scales ranging from 22 nm to several µm. Tracking of individual FGF2 by PHI in the Pericellular Matrix of living cells demonstrates that they undergo five distinct types of motion. They spend much of their time in confined motion (∼110 nm diameter), but they are not trapped and can escape by simple diffusion, which may be slow, fast, or directed. These substantial translocations (µm) cover distances far greater than the length of a single HS chain. Similar molecular motion persists in fixed cells, where the movement of membrane PGs is impeded. We conclude that FGF2 moves within the Pericellular Matrix by translocating from one HS-binding site to another. The binding sites on HS chains form non-random, heterogeneous networks. These promote FGF2 confinement or substantial translocation depending on their spatial organisation. We propose that this spatial organisation, coupled to the relative selectivity and the availability of HS-binding sites, determines the transport of FGF2 in matrices. Similar mechanisms are likely to underpin the movement of many other HS-binding effectors.