The Experts below are selected from a list of 20496 Experts worldwide ranked by ideXlab platform
Peter J. Roughley - One of the best experts on this subject based on the ideXlab platform.
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Aggrecan nanoscale solid-fluid interactions are a primary determinant of cartilage dynamic mechanical properties.
ACS nano, 2015Co-Authors: Hadi Tavakoli Nia, Peter J. Roughley, Alan J Grodzinsky, Lin Han, Iman Soltani Bozchalooi, Kamal Youcef-toumi, Christine OrtizAbstract:Poroelastic interactions between interstitial fluid and the extracellular matrix of connective tissues are critical to biological and pathophysiological functions involving solute transport, energy dissipation, self-stiffening and lubrication. However, the molecular origins of poroelasticity at the nanoscale are largely unknown. Here, the broad-spectrum dynamic nanomechanical behavior of cartilage Aggrecan monolayer is revealed for the first time, including the equilibrium and instantaneous moduli and the peak in the phase angle of the complex modulus. By performing a length scale study and comparing the experimental results to theoretical predictions, we confirm that the mechanism underlying the observed dynamic nanomechanics is due to solid–fluid interactions (poroelasticity) at the molecular scale. Utilizing finite element modeling, the molecular-scale hydraulic permeability of the Aggrecan assembly was quantified (kAggrecan = (4.8 ± 2.8) × 10–15 m4/N·s) and found to be similar to the nanoscale hydraul...
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Structure, function, aging and turnover of Aggrecan in the intervertebral disc.
Biochimica et biophysica acta, 2014Co-Authors: Sarit Sivan, Ellen Wachtel, Peter J. RoughleyAbstract:Abstract Background Aggrecan is the major non-collagenous component of the intervertebral disc. It is a large proteoglycan possessing numerous glycosaminoglycan chains and the ability to form aggregates in association with hyaluronan. Its abundance and unique molecular features provide the disc with its osmotic properties and ability to withstand compressive loads. Degradation and loss of Aggrecan result in impairment of disc function and the onset of degeneration. Scope of review This review summarizes current knowledge concerning the structure and function of Aggrecan in the normal intervertebral disc and how and why these change in aging and degenerative disc disease. It also outlines how supplementation with Aggrecan or a biomimetic may be of therapeutic value in treating the degenerate disc. Major conclusions Aggrecan abundance reaches a plateau in the early twenties, declining thereafter due to proteolysis, mainly by matrix metalloproteinases and Aggrecanases, though degradation of hyaluronan and non-enzymic glycation may also participate. Aggrecan loss is an early event in disc degeneration, although it is a lengthy process as degradation products may accumulate in the disc for decades. The low turnover rate of the remaining Aggrecan is an additional contributing factor, preventing protein renewal. It may be possible to retard the degenerative process by restoring the Aggrecan content of the disc, or by supplementing with a bioimimetic possessing similar osmotic properties. General significance This review provides a basis for scientists and clinicians to understand and appreciate the central role of Aggrecan in the function, degeneration and repair of the intervertebral disc.
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The role of Aggrecan in normal and osteoarthritic cartilage
Journal of Experimental Orthopaedics, 2014Co-Authors: Peter J. Roughley, John S. MortAbstract:Aggrecan is a large proteoglycan bearing numerous chondroitin sulfate and keratan sulfate chains that endow articular cartilage with its ability to withstand compressive loads. It is present in the extracellular matrix in the form of proteoglycan aggregates, in which many Aggrecan molecules interact with hyaluronan and a link protein stabilizes each interaction. Aggrecan structure is not constant throughout life, but changes due to both synthetic and degradative events. Changes due to synthesis alter the structure of the chondroitin sulfate and keratan sulfate chains, whereas those due to degradation cause cleavage of all components of the aggregate. These latter changes can be viewed as being detrimental to cartilage function and are enhanced in osteoarthritic cartilage, resulting in Aggrecan depletion and predisposing to cartilage erosion. Matrix metalloproteinases and Aggrecanases play a major role in Aggrecan degradation and their production is upregulated by mediators associated with joint inflammation and overloading. The presence of increased levels of Aggrecan fragments in synovial fluid has been used as a marker of ongoing cartilage destruction in osteoarthritis. During the early stages of osteoarthritis it may be possible to retard the destructive process by enhancing the production of Aggrecan and inhibiting its degradation. Aggrecan production also plays a central role in cartilage repair techniques involving stem cell or chondrocyte implantation into lesions. Thus Aggrecan participates in both the demise and survival of articular cartilage.
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age related nanostructural and nanomechanical changes of individual human cartilage Aggrecan monomers and their glycosaminoglycan side chains
PMC, 2012Co-Authors: Peter J. Roughley, Alan J Grodzinsky, Christine OrtizAbstract:The nanostructure and nanomechanical properties of Aggrecan monomers extracted and purified from human articular cartilage from donors of different ages (newborn, 29 and 38 year old) were directly visualized and quantified via atomic force microscopy (AFM)-based imaging and force spectroscopy. AFM imaging enabled direct comparison of full length monomers at different ages. The higher proportion of Aggrecan fragments observed in adult versus newborn populations is consistent with the cumulative proteolysis of Aggrecan known to occur in vivo. The decreased dimensions of adult full length Aggrecan (including core protein and glycosaminoglycan (GAG) chain trace length, end-to-end distance and extension ratio) reflect altered Aggrecan biosynthesis. The demonstrably shorter GAG chains observed in adult full length Aggrecan monomers, compared to newborn monomers, also reflects markedly altered biosynthesis with age. Direct visualization of Aggrecan subjected to chondroitinase and/or keratanase treatment revealed conformational properties of Aggrecan monomers associated with chondroitin sulfate (CS) and keratan sulfate (KS) GAG chains. Furthermore, compressive stiffness of chemically end-attached layers of adult and newborn Aggrecan was measured in various ionic strength aqueous solutions. Adult Aggrecan was significantly weaker in compression than newborn Aggrecan even at the same total GAG density and bath ionic strength, suggesting the importance of both electrostatic and non-electrostatic interactions in nanomechanical stiffness. These results provide molecular-level evidence of the effects of age on the conformational and nanomechanical properties of Aggrecan, with direct implications for the effects of Aggrecan nanostructure on the age-dependence of cartilage tissue biomechanical and osmotic properties.
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MMPs are less efficient than ADAMTS5 in cleaving Aggrecan core protein
Matrix biology : journal of the International Society for Matrix Biology, 2010Co-Authors: Michaela Durigova, Hideaki Nagase, John S. Mort, Peter J. RoughleyAbstract:Aggrecan degradation in articular cartilage occurs predominantly through proteolysis and has been attributed to the action of members of the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) families. Both families of enzymes cleave Aggrecan at specific sites within the Aggrecan core protein. One cleavage site within the interglobular domain (IGD), between Glu373–374Ala and five additional sites in the chondroitin sulfate-2 (CS-2) region of Aggrecan were characterized as “Aggrecanase” (ADAMTS) cleavage sites, while cleavage between Ser341–342Phe within the IGD of bovine Aggrecan is attributed to MMP action. The objective of this study was to assess the cleavage efficiency of MMPs relative to ADAMTS and their contribution to Aggrecan proteolysis in vitro. The analysis of Aggrecan IGD degradation in bovine articular cartilage explants treated with catabolic cytokines over a 19-day period showed that MMP-mediated degradation of Aggrecan within the IGD can only be observed following day 12 of culture. This delay is associated with the lack of activation of proMMPs during the first 12 days of culture. Analysis of MMP1, 2, 3, 7, 8, 9, 12, 13 and ADAMTS5 efficiencies at cleaving within the Aggrecan IGD and CS-2 region in vitro was carried out by the digestion of bovine Aggrecan with the various enzymes and Western blot analysis using Aggrecan anti-G1 and anti-G3 antibodies. Of these MMPs, MMP12 was the most efficient at cleaving within the Aggrecan IGD. In addition to cleavage in the IGD, MMP, 3, 7, 8 and 12 were also able to degrade the Aggrecan CS-2 region. MMP3 and MMP12 were able to degrade Aggrecan at the very C-terminus of the CS-2 region, cleaving the Glu2047–2048Ala bond which was previously shown to be cleaved by ADAMTS5. However, in comparison to ADAMTS5, MMP3 was about 100 times and 10 times less efficient at cleaving within the Aggrecan IGD and CS-2 regions, respectively. Collectively, our results showed that the delayed activation of proMMPs and the relatively low cleavage efficiency of MMPs can explain the minor contribution of these enzymes to Aggrecan catabolism in vivo. This study also uncovered a potential role for MMPs in the C-terminal truncation of Aggrecan.
Christine Ortiz - One of the best experts on this subject based on the ideXlab platform.
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Aggrecan nanoscale solid-fluid interactions are a primary determinant of cartilage dynamic mechanical properties.
ACS nano, 2015Co-Authors: Hadi Tavakoli Nia, Peter J. Roughley, Alan J Grodzinsky, Lin Han, Iman Soltani Bozchalooi, Kamal Youcef-toumi, Christine OrtizAbstract:Poroelastic interactions between interstitial fluid and the extracellular matrix of connective tissues are critical to biological and pathophysiological functions involving solute transport, energy dissipation, self-stiffening and lubrication. However, the molecular origins of poroelasticity at the nanoscale are largely unknown. Here, the broad-spectrum dynamic nanomechanical behavior of cartilage Aggrecan monolayer is revealed for the first time, including the equilibrium and instantaneous moduli and the peak in the phase angle of the complex modulus. By performing a length scale study and comparing the experimental results to theoretical predictions, we confirm that the mechanism underlying the observed dynamic nanomechanics is due to solid–fluid interactions (poroelasticity) at the molecular scale. Utilizing finite element modeling, the molecular-scale hydraulic permeability of the Aggrecan assembly was quantified (kAggrecan = (4.8 ± 2.8) × 10–15 m4/N·s) and found to be similar to the nanoscale hydraul...
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Aggrecan: Approaches to Study Biophysical and Biomechanical Properties
Methods in molecular biology (Clifton N.J.), 2014Co-Authors: Hadi Tavakoli Nia, Christine Ortiz, Alan J GrodzinskyAbstract:Aggrecan, the most abundant extracellular proteoglycan in cartilage (~35 % by dry weight), plays a key role in the biophysical and biomechanical properties of cartilage. Here, we review several approaches based on atomic force microscopy (AFM) to probe the physical, mechanical, and structural properties of Aggrecan at the molecular level. These approaches probe the response of Aggrecan over a wide time (frequency) scale, ranging from equilibrium to impact dynamic loading. Experimental and theoretical methods are described for the investigation of electrostatic and fluid-solid interactions that are key mechanisms underlying the biomechanical and physicochemical functions of Aggrecan. Using AFM-based imaging and nanoindentation, ultrastructural features of Aggrecan are related to its mechanical properties, based on Aggrecans harvested from human vs. bovine, immature vs. mature, and healthy vs. osteoarthritic cartilage.
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age related nanostructural and nanomechanical changes of individual human cartilage Aggrecan monomers and their glycosaminoglycan side chains
PMC, 2012Co-Authors: Peter J. Roughley, Alan J Grodzinsky, Christine OrtizAbstract:The nanostructure and nanomechanical properties of Aggrecan monomers extracted and purified from human articular cartilage from donors of different ages (newborn, 29 and 38 year old) were directly visualized and quantified via atomic force microscopy (AFM)-based imaging and force spectroscopy. AFM imaging enabled direct comparison of full length monomers at different ages. The higher proportion of Aggrecan fragments observed in adult versus newborn populations is consistent with the cumulative proteolysis of Aggrecan known to occur in vivo. The decreased dimensions of adult full length Aggrecan (including core protein and glycosaminoglycan (GAG) chain trace length, end-to-end distance and extension ratio) reflect altered Aggrecan biosynthesis. The demonstrably shorter GAG chains observed in adult full length Aggrecan monomers, compared to newborn monomers, also reflects markedly altered biosynthesis with age. Direct visualization of Aggrecan subjected to chondroitinase and/or keratanase treatment revealed conformational properties of Aggrecan monomers associated with chondroitin sulfate (CS) and keratan sulfate (KS) GAG chains. Furthermore, compressive stiffness of chemically end-attached layers of adult and newborn Aggrecan was measured in various ionic strength aqueous solutions. Adult Aggrecan was significantly weaker in compression than newborn Aggrecan even at the same total GAG density and bath ionic strength, suggesting the importance of both electrostatic and non-electrostatic interactions in nanomechanical stiffness. These results provide molecular-level evidence of the effects of age on the conformational and nanomechanical properties of Aggrecan, with direct implications for the effects of Aggrecan nanostructure on the age-dependence of cartilage tissue biomechanical and osmotic properties.
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adult bone marrow stromal cell based tissue engineered Aggrecan exhibits ultrastructure and nanomechanical properties superior to native cartilage
Osteoarthritis and Cartilage, 2010Co-Authors: Hsuyi Lee, Alan J Grodzinsky, Anna Plaas, Paul W Kopesky, John D Sandy, John D Kisiday, David D Frisbie, Christine OrtizAbstract:Summary Objective To quantify the structural characteristics and nanomechanical properties of Aggrecan produced by adult bone marrow stromal cells (BMSCs) in peptide hydrogel scaffolds and compare to Aggrecan from adult articular cartilage. Design Adult equine BMSCs were encapsulated in 3D-peptide hydrogels and cultured for 21 days with TGF-β1 to induce chondrogenic differentiation. BMSC-Aggrecan was extracted and compared with Aggrecan from age-matched adult equine articular cartilage. Single molecules of Aggrecan were visualized by atomic force microcopy-based imaging and Aggrecan nanomechanical stiffness was quantified by high resolution force microscopy. Population-averaged measures of Aggrecan hydrodynamic size, core protein structures and CS sulfation compositions were determined by size-exclusion chromatography, Western analysis, and fluorescence-assisted carbohydrate electrophoresis (FACE). Results BMSC-Aggrecan was primarily full-length while cartilage-Aggrecan had many fragments. Single molecule measurements showed that core protein and GAG chains of BMSC-Aggrecan were markedly longer than those of cartilage-Aggrecan. Comparing full-length Aggrecan of both species, BMSC-Aggrecan had longer GAG chains, while the core protein trace lengths were similar. FACE analysis detected a ∼1:1 ratio of chondroitin-4-sulfate to chondroitin-6-sulfate in BMSC-GAG, a phenotype consistent with Aggrecan from skeletally-immature cartilage. The nanomechanical stiffness of BMSC-Aggrecan was demonstrably greater than that of cartilage-Aggrecan at the same total sGAG (fixed charge) density. Conclusions The higher proportion of full-length monomers, longer GAG chains and greater stiffness of the BMSC-Aggrecan makes it biomechanically superior to adult cartilage-Aggrecan. Aggrecan stiffness was not solely dependent on fixed charge density, but also on GAG molecular ultrastructure. These results support the use of adult BMSCs for cell-based cartilage repair.
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compressive nanomechanics of opposing Aggrecan macromolecules
Journal of Biomechanics, 2006Co-Authors: Delphine Dean, Lin Han, Alan J Grodzinsky, Christine OrtizAbstract:In this study, we have measured the nanoscale compressive interactions between opposing Aggrecan macromolecules in near-physiological conditions, in order to elucidate the molecular origins of tissue-level cartilage biomechanical behavior. Aggrecan molecules from fetal bovine epiphyseal cartilage were chemically end-grafted to planar substrates, standard nanosized atomic force microscopy (AFM) probe tips (R(tip) approximately 50 nm), and larger colloidal probe tips (R(tip) approximately 2.5 microm). To assess normal nanomechanical interaction forces between opposing Aggrecan layers, substrates with microcontact printed Aggrecan were imaged using contact mode AFM, and Aggrecan layer height (and hence deformation) was measured as a function of solution ionic strength (IS) and applied normal load. Then, using high-resolution force spectroscopy, nanoscale compressive forces between opposing Aggrecan on the tip and substrate were measured versus tip-substrate separation distance in 0.001-1M NaCl. Nanosized tips enabled measurement of the molecular stiffness of 2-4 Aggrecan while colloidal tips probed the nanomechanical properties of larger assemblies (approximately 10(4) molecules). The compressive stiffness of Aggrecan was much higher when using a densely packed colloidal tip than the stiffness measured for using the nanosized tip with a few Aggrecan, demonstrating the importance of lateral interactions to the normal nanomechanical properties. The measured stress at 0.1M NaCl (near-physiological ionic strength) increased sharply at Aggrecan densities under the tip of approximately 40 mg/ml (physiological densities are approximately 20-80 mg/ml), corresponding to an average inter-GAG spacing of 4-5 Debye lengths (4-5 nm); this characteristic spacing is consistent with the onset of significant electrostatic interactions between GAG chains of opposing Aggrecan molecules. Comparison of nanomechanical data to the predictions of Poisson-Boltzmann-based models further elucidated the regimes over which electrostatic and nonelectrostatic interactions affect Aggrecan stiffness in compression. The most important aspects of this study include: the incorporation of experiments at two different length scales, the use of microcontact printing to enable quantification of Aggrecan deformation and the corresponding nanoscale compressive stress vs. strain curve, the use of tips of differing functionality to provide insights into the molecular mechanisms of deformation, and the comparison of experimental data to the predictions of three increasingly refined Poisson-Boltzmann (P-B)-based theoretical models for the electrostatic double layer component of the interaction.
Dwight Xuan - One of the best experts on this subject based on the ideXlab platform.
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Association between the expression of Aggrecan and the distribution of Aggrecan gene variable number of tandem repeats with symptomatic lumbar disc herniation in Chinese Han of Northern China.
Spine, 2010Co-Authors: Lin Cong, Hao Pang, Dwight XuanAbstract:STUDY DESIGN Case-control study. OBJECTIVE To examine the association between the expression of Aggrecan and the Aggrecan gene variable number of tandem repeats (VNTR) polymorphism with symptomatic lumbar disc herniation (LDH) in Chinese Han of Northern China. SUMMARY OF BACKGROUND DATA Aggrecan fragments have been found in human degenerated discs, and an association between the Aggrecan VNTR polymorphism and intervertebral disc degeneration has been previously reported in middle-aged Finnish men. However, the relationship between the munity of symptomatic LDH with the expression of Aggrecan and Aggrecan gene VNTR has not been well studied. METHODS The disease group consisted of 70 patients already diagnosed with symptomatic LDH. The control group consisted of 14 patients restricted to spinal trauma and 113 healthy blood donors without symptoms of LDH who were not diagnosed with LDH. Disc tissue samples were obtained from surgical operations, and blood samples were donated from all participants. The Aggrecan expression in isolated tissues was assessed by Western blot using specific antibodies. The Aggrecan gene VNTR region was analyzed by polymerase chain reaction. RESULTS The Aggrecan expression positive rate of control group was statistically and significantly higher (P 25 repeats, subjects with 1 or 2 alleles < or = 25 repeats statistically and significantly overrepresented the disease group without the expression of Aggrecan (P < 0.001). CONCLUSION The findings suggest a relation between Aggrecan and symptomatic LDH, where symptomatic LDH has a lower tendency of allele repeats. In addition, this study observed an association between the distribution of Aggrecan gene VNTR polymorphism and the expression of Aggrecan in symptomatic LDH.
Alan J Grodzinsky - One of the best experts on this subject based on the ideXlab platform.
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Coculture of bovine cartilage with synovium and fibrous joint capsule increases Aggrecanase and matrix metalloproteinase activity
BMC, 2017Co-Authors: Per Swärd, Alan J Grodzinsky, Yang Wang, Maria Hansson, Stefan L. Lohmander, André StruglicsAbstract:Abstract Background A hallmark of osteoarthritis is increased proteolytic cleavage of Aggrecan. Cross talk between cartilage and the synovium + joint capsule (SJC) can drive cartilage degradation by activating proteases in both tissues. We investigated Aggrecan proteolysis patterns in cartilage explants using a physiologically relevant explant model of joint injury combining cartilage mechanical compression and coincubation with SJC. Methods Bovine cartilage explants were untreated; coincubated with SJC; or subjected to mechanical injury and coincubated with SJC, mechanical injury alone, or mechanical injury and incubated with tumor necrosis factor-α (TNF-α). To compare the patterns of Aggrecan proteolysis between 6 h and 16 days, release of sulfated glycosaminoglycans and specific proteolytic Aggrecan fragments into medium or remaining in cartilage explants was measured by dimethylmethylene blue and Western blot analysis. Results Aggrecanase activity toward Aggrecan was observed in all conditions, but it was directed toward the TEGE↓ARGS interglobular domain (IGD) site only when cartilage was coincubated with SJC or TNF-α. Matrix metalloproteinase (MMP) activity at the Aggrecan IGD site (IPES↓FFGV) was not detected when cartilage was exposed to TNF-α (up to 6 days), but it was in all other conditions. Compared with when bovine cartilage was left untreated or subjected to mechanical injury alone, additional Aggrecan fragment types were released into medium and proteolysis of Aggrecan started at an earlier time when SJC was present. Conclusions Indicative of different proteolytic pathways for Aggrecan degradation, the SJC increases both Aggrecanase and MMP activity toward Aggrecan, whereas TNF-α inhibits MMP activity against the IGD of Aggrecan
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Aggrecan nanoscale solid-fluid interactions are a primary determinant of cartilage dynamic mechanical properties.
ACS nano, 2015Co-Authors: Hadi Tavakoli Nia, Peter J. Roughley, Alan J Grodzinsky, Lin Han, Iman Soltani Bozchalooi, Kamal Youcef-toumi, Christine OrtizAbstract:Poroelastic interactions between interstitial fluid and the extracellular matrix of connective tissues are critical to biological and pathophysiological functions involving solute transport, energy dissipation, self-stiffening and lubrication. However, the molecular origins of poroelasticity at the nanoscale are largely unknown. Here, the broad-spectrum dynamic nanomechanical behavior of cartilage Aggrecan monolayer is revealed for the first time, including the equilibrium and instantaneous moduli and the peak in the phase angle of the complex modulus. By performing a length scale study and comparing the experimental results to theoretical predictions, we confirm that the mechanism underlying the observed dynamic nanomechanics is due to solid–fluid interactions (poroelasticity) at the molecular scale. Utilizing finite element modeling, the molecular-scale hydraulic permeability of the Aggrecan assembly was quantified (kAggrecan = (4.8 ± 2.8) × 10–15 m4/N·s) and found to be similar to the nanoscale hydraul...
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Aggrecan: Approaches to Study Biophysical and Biomechanical Properties
Methods in molecular biology (Clifton N.J.), 2014Co-Authors: Hadi Tavakoli Nia, Christine Ortiz, Alan J GrodzinskyAbstract:Aggrecan, the most abundant extracellular proteoglycan in cartilage (~35 % by dry weight), plays a key role in the biophysical and biomechanical properties of cartilage. Here, we review several approaches based on atomic force microscopy (AFM) to probe the physical, mechanical, and structural properties of Aggrecan at the molecular level. These approaches probe the response of Aggrecan over a wide time (frequency) scale, ranging from equilibrium to impact dynamic loading. Experimental and theoretical methods are described for the investigation of electrostatic and fluid-solid interactions that are key mechanisms underlying the biomechanical and physicochemical functions of Aggrecan. Using AFM-based imaging and nanoindentation, ultrastructural features of Aggrecan are related to its mechanical properties, based on Aggrecans harvested from human vs. bovine, immature vs. mature, and healthy vs. osteoarthritic cartilage.
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age related nanostructural and nanomechanical changes of individual human cartilage Aggrecan monomers and their glycosaminoglycan side chains
PMC, 2012Co-Authors: Peter J. Roughley, Alan J Grodzinsky, Christine OrtizAbstract:The nanostructure and nanomechanical properties of Aggrecan monomers extracted and purified from human articular cartilage from donors of different ages (newborn, 29 and 38 year old) were directly visualized and quantified via atomic force microscopy (AFM)-based imaging and force spectroscopy. AFM imaging enabled direct comparison of full length monomers at different ages. The higher proportion of Aggrecan fragments observed in adult versus newborn populations is consistent with the cumulative proteolysis of Aggrecan known to occur in vivo. The decreased dimensions of adult full length Aggrecan (including core protein and glycosaminoglycan (GAG) chain trace length, end-to-end distance and extension ratio) reflect altered Aggrecan biosynthesis. The demonstrably shorter GAG chains observed in adult full length Aggrecan monomers, compared to newborn monomers, also reflects markedly altered biosynthesis with age. Direct visualization of Aggrecan subjected to chondroitinase and/or keratanase treatment revealed conformational properties of Aggrecan monomers associated with chondroitin sulfate (CS) and keratan sulfate (KS) GAG chains. Furthermore, compressive stiffness of chemically end-attached layers of adult and newborn Aggrecan was measured in various ionic strength aqueous solutions. Adult Aggrecan was significantly weaker in compression than newborn Aggrecan even at the same total GAG density and bath ionic strength, suggesting the importance of both electrostatic and non-electrostatic interactions in nanomechanical stiffness. These results provide molecular-level evidence of the effects of age on the conformational and nanomechanical properties of Aggrecan, with direct implications for the effects of Aggrecan nanostructure on the age-dependence of cartilage tissue biomechanical and osmotic properties.
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adult bone marrow stromal cell based tissue engineered Aggrecan exhibits ultrastructure and nanomechanical properties superior to native cartilage
Osteoarthritis and Cartilage, 2010Co-Authors: Hsuyi Lee, Alan J Grodzinsky, Anna Plaas, Paul W Kopesky, John D Sandy, John D Kisiday, David D Frisbie, Christine OrtizAbstract:Summary Objective To quantify the structural characteristics and nanomechanical properties of Aggrecan produced by adult bone marrow stromal cells (BMSCs) in peptide hydrogel scaffolds and compare to Aggrecan from adult articular cartilage. Design Adult equine BMSCs were encapsulated in 3D-peptide hydrogels and cultured for 21 days with TGF-β1 to induce chondrogenic differentiation. BMSC-Aggrecan was extracted and compared with Aggrecan from age-matched adult equine articular cartilage. Single molecules of Aggrecan were visualized by atomic force microcopy-based imaging and Aggrecan nanomechanical stiffness was quantified by high resolution force microscopy. Population-averaged measures of Aggrecan hydrodynamic size, core protein structures and CS sulfation compositions were determined by size-exclusion chromatography, Western analysis, and fluorescence-assisted carbohydrate electrophoresis (FACE). Results BMSC-Aggrecan was primarily full-length while cartilage-Aggrecan had many fragments. Single molecule measurements showed that core protein and GAG chains of BMSC-Aggrecan were markedly longer than those of cartilage-Aggrecan. Comparing full-length Aggrecan of both species, BMSC-Aggrecan had longer GAG chains, while the core protein trace lengths were similar. FACE analysis detected a ∼1:1 ratio of chondroitin-4-sulfate to chondroitin-6-sulfate in BMSC-GAG, a phenotype consistent with Aggrecan from skeletally-immature cartilage. The nanomechanical stiffness of BMSC-Aggrecan was demonstrably greater than that of cartilage-Aggrecan at the same total sGAG (fixed charge) density. Conclusions The higher proportion of full-length monomers, longer GAG chains and greater stiffness of the BMSC-Aggrecan makes it biomechanically superior to adult cartilage-Aggrecan. Aggrecan stiffness was not solely dependent on fixed charge density, but also on GAG molecular ultrastructure. These results support the use of adult BMSCs for cell-based cartilage repair.
L. Stefan Lohmander - One of the best experts on this subject based on the ideXlab platform.
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An ARGS-Aggrecan assay for analysis in blood and synovial fluid
Osteoarthritis and cartilage, 2013Co-Authors: Staffan Larsson, L. Stefan Lohmander, André StruglicsAbstract:Summary Objective To validate a modified ligand-binding assay for the detection of Aggrecanase generated Aggrecan fragments with the ARGS neoepitope in synovial fluid (SF) and blood, and to verify the identity of Aggrecan fragments found in blood. Design An enzyme-linked immunosorbent assay (ELISA) on the Meso Scale Discovery (MSD) platform for detection of ARGS-Aggrecan was validated, using a standard made from recombinant human Aggrecan. Matched samples of SF, serum, plasma, and urine were obtained from 36 subjects at different time points after knee injury, and analysed for ARGS-Aggrecan content. Aggrecan was purified from serum and plasma pools and analysed by Western blot. Results The limits of quantification for the ARGS-Aggrecan assay was between 0.2 and 0.025 pmol ARGS/ml, and the sensitivity of the assay was improved two-fold compared to when using a standard purified from human donors. The ARGS concentrations were highest in SF (mean, range; 3.02, 0.36–30.22 pmol/ml), 20 times lower in the blood samples (0.14, 0.055–0.28 pmol/ml serum and 0.13, 0.053–0.28 pmol/ml plasma), and 80 times lower in urine (0.036, below detection – 0.087 pmol/ml). Serum-ARGS and plasma-ARGS concentrations were similar, and correlated ( r S = 0.773, P r S = 0.420, P = 0.011). In blood, we identified 129–138 kDa Aggrecan fragments containing the ARGS neoepitope. Conclusions This novel ARGS-Aggrecan assay is highly sensitive and suited for analysis of SF and blood samples. Both SF and blood contains ARGS-Aggrecan, and ARGS concentrations in SF and serum are correlated.
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Aggrecanase cleavage in juvenile idiopathic arthritis patients is minimally detected in the Aggrecan interglobular domain but robust at the Aggrecan C-terminus
Arthritis and rheumatism, 2012Co-Authors: André Struglics, L. Stefan Lohmander, Jonathan D Akikusa, Roger C. Allen, Amanda J FosangAbstract:OBJECTIVE: To understand Aggrecan degradation in juvenile idiopathic arthritis (JIA), the pattern and abundance of Aggrecan fragments in synovial fluid aspirates from JIA patients were analysed and compared with Aggrecan fragments in synovial fluids from patients with other arthritides, juvenile knee injury and a knee-healthy reference group. METHODS: The concentration of sulphated glycosaminoglycans in synovial fluid was measured by the Alcian blue precipitation assay. Aggrecan fragments were purified by dissociative CsCl density gradient centrifugation, deglycosylated and analysed by Western blot using antibodies specific for either Aggrecanase-derived ARGS, SELE and KEEE neoepitopes, or the Aggrecan G3-domain. RESULTS: The concentration of sulphated glycosaminoglycans in JIA synovial fluids was significantly lower compared with the levels in fluids from OA (P
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elevated Aggrecanase activity in a rat model of joint injury is attenuated by an Aggrecanase specific inhibitor
Osteoarthritis and Cartilage, 2011Co-Authors: Priya S Chockalingam, D. R. Dufield, L. Stefan Lohmander, Katy E Georgiadis, Weilan Zeng, Carl R Flannery, M A Riverabermudez, S S Glasson, S Larsson, Elisabeth A MorrisAbstract:Summary Objective To evaluate Aggrecanase activity after traumatic knee injury in a rat model by measuring the level of Aggrecanase-generated Ala-Arg-Gly-Aggrecan (ARG-Aggrecan) fragments in synovial fluid, and compare with ARG-Aggrecan release into joint fluid following human knee injury. To evaluate the effect of small molecule inhibitors on induced Aggrecanase activity in the rat model. Method An enzyme-linked immunosorbent assay (ELISA) was developed to measure ARG-Aggrecan levels in animal and human joint fluids. A rat model of meniscal tear (MT)-induced joint instability was used to assess ARG-Aggrecan release into joint fluid and the effects of Aggrecanase inhibition. Synovial fluids were also obtained from patients with acute joint injury or osteoarthritis and assayed for ARG-Aggrecan. Results Joint fluids from human patients after knee injury showed significantly enhanced levels of ARG-Aggrecan compared to uninjured reference subjects. Similarly, synovial fluid ARG-Aggrecan levels increased following surgically-induced joint instability in the rat MT model, which was significantly attenuated by orally dosing the animals with AGG-523, an Aggrecanase specific inhibitor. Conclusions Aggrecanase-generated Aggrecan fragments were rapidly released into human and rat joint fluids after injury to the knee and remained elevated over a prolonged period. Our findings in human and preclinical models strengthen the connection between Aggrecanase activity in joints and knee injury and disease. The ability of a small molecule Aggrecanase inhibitor to reduce the release of Aggrecanase-generated Aggrecan fragments into rat joints suggests that pharmacologic inhibition of Aggrecanase activity in humans may be an effective treatment for slowing cartilage degradation following joint injury.
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development and characterization of a highly specific and sensitive sandwich elisa for detection of Aggrecanase generated Aggrecan fragments
Osteoarthritis and Cartilage, 2006Co-Authors: M A Pratta, L. Stefan Lohmander, S Larsson, A Struglics, J L Su, M A Leesnitzer, Sanjay KumarAbstract:Objective: To develop an enzyme linked immunosorbent assay (ELISA) to quantify the levels of specific Aggrecan fragments generated by Aggrecanase-mediated cleavage at the (373)Glu-(374)Ala bond within the Aggrecan interglobular domain. Methods: The ELISA employs a commercially available monoclonal antibody to capture Aggrecan fragments containing keratan sulfate (KS). Aggrecan fragments generated by cleavage at the Glu-Ala bond were then detected using a monoclonal neoepitope antibody (mAb OA-1) that specifically recognizes the N-terminal sequence 'ARGSVIL'. Results: The mAb OA-1 antibody was highly specific for the immunizing neoepitope peptide since neither peptides spanning the cleavage site nor mutated peptides were detected. Aggrecan fragments generated by ADAMTS-4 digested human Aggrecan monomers and from IL-1-stimulated human cartilage explants were quantified by the ELISA, and we observed increased sensitivity of the ELISA compared to mAb OA-1 Western analysis. We also observed that the basal, as well as IL-1-stimulated production of ARGS Aggrecan fragments from human articular cartilage explants was blocked by a selective Aggrecanase inhibitor, consistent with generation of the ARGS neoepitope in human articular cartilage being mediated by Aggrecanase. Using purified human Aggrecan digested by ADAMTS-4 as standard to quantify ARGS Aggrecan fragments in human synovial fluids, we determined that the calculated amount of ARGSVIL-Aggrecan fragments by ELISA measurement is in agreement with the published levels of these fragments, supporting its potential utility as a biomarker assay for osteoarthritis. Conclusion: We have developed an assay that detects and quantifies specific Aggrecan fragments generated by Aggrecanase-mediated cleavage. Because Aggrecanase mediates degradation of human articular Aggrecan in joint disease, the KS/mAb OA-1 ELISA may serve as a biomarker assay for evaluation of preclinical and clinical samples. (C) 2006 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
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human osteoarthritis synovial fluid and joint cartilage contain both Aggrecanase and matrix metalloproteinase generated Aggrecan fragments
Osteoarthritis and Cartilage, 2006Co-Authors: A Struglics, Michael A Pratta, S Larsson, Sanjay Kumar, M W Lark, L. Stefan LohmanderAbstract:Summary Objective To identify the major Aggrecanase- and matrix metalloproteinase (MMP)-generated Aggrecan fragments in human osteoarthritis (OA) synovial fluid and in human OA joint cartilage. Method Aggrecan fragments were prepared by CsCl gradient centrifugation. Fragment distributions were compared with Aggrecanase-1 (ADAMTS-4) and MMP-3 digested human Aggrecan by analysis with neoepitope antibodies and an anti-G1 domain antibody, using Western immuno-blots. Results The overall fragment pattern of OA synovial fluid Aggrecan was similar to the fragment pattern of cartilage Aggrecan cleaved in vitro by ADAMTS-4. However, multiple glycosaminoglycan (GAG) containing Aggrecanase and MMP-generated Aggrecan fragments were identified in OA synovial fluid and some of these fragments were produced by the action of both types of proteinases. The synovial fluid content of large size Aggrecan fragments with 374 ARGS- and 342 FFGV- N-terminals was about 107 and 40pmoles per ml, respectively, out of a total concentration of Aggrecan fragments of about 185pmoles per ml. OA synovial fluid contained insignificant amounts of the G1-IPEN 341 fragment as compared to the G1-TEGE 373 fragment, while OA cartilage contained significant amounts of both fragments. OA cartilage contained several GAG-containing Aggrecan fragments with N-terminals of G1- or 342 FFGV- but no fragments with an N-terminal of 374 ARGS-. Conclusions The overall pattern of Aggrecan fragments in human OA synovial fluid and cartilage supports an important role for Aggrecanase in Aggrecan degradation. However, the fragment patterns and their differential distribution between cartilage and synovial fluid are consistent with the existence of at least two proteolytic pathways for Aggrecan degradation in human OA, generating both 342 FFGV- and 374 ARGS-fragments.