John Sandy | Rush University Medical Center (original) (raw)

Papers by John Sandy

Arthritis & Rheumatism, 2002

To study the chondroprotective effect of constitutively expressed TSG-6 protein (tumor necrosis f... more To study the chondroprotective effect of constitutively expressed TSG-6 protein (tumor necrosis factor ␣-induced protein 6; Tnfip6) in cartilage, using antigen-induced arthritis (AIA) in mice.

Matrix Biology, May 31, 2007

Articular cartilage undergoes matrix degradation and loss of mechanical properties when stimulate... more Articular cartilage undergoes matrix degradation and loss of mechanical properties when stimulated with proinflammatory cytokines such as interleukin-1 (IL-1). Aggrecanases and matrix metalloproteinases (MMPs) are thought to be principal downstream effectors of cytokine-induced matrix catabolism, and aggrecanase- or MMP-selective inhibitors reduce or block matrix destruction in several model systems. The objective of this study was to use metalloproteinase inhibitors to perturb IL-1-induced matrix catabolism in bovine cartilage explants and examine their effects on changes in tissue compression and shear properties. Explanted tissue was stimulated with IL-1 for up to 24 days in the absence or presence of inhibitors that were aggrecanase-selective, MMP-selective, or non-selective. Analysis of conditioned media and explant digests revealed that aggrecanase-mediated aggrecanolysis was delayed to varying extents with all inhibitor treatments, but that aggrecan release persisted. Collagen degradation was abrogated by MMP- and non-selective inhibitors and reduced by the aggrecanase inhibitor. The inhibitors delayed but did not reduce loss of the equilibrium compression modulus, whereas the losses of dynamic compression and shear moduli were delayed and reduced. The data suggest that non-metalloproteinase mechanisms participate in IL-1-induced matrix degradation and loss of tissue material properties.

Matrix biology : journal of the International Society for Matrix Biology, Jan 31, 2015

ADAMTS5 (TS5), a member of the aggrecanase clade (TS1, 4, 5, 8, 9, 15) of ADAMTS-proteases, has b... more ADAMTS5 (TS5), a member of the aggrecanase clade (TS1, 4, 5, 8, 9, 15) of ADAMTS-proteases, has been considered largely responsible for the proteolysis of the hyalectans, aggrecan (Acan) and versican (Vcan), in vivo. However, we have reported that ts5-knockout (KO) mice show joint protection after injury due to inhibition of synovial scarring and enhanced Acan deposition. Also, KO mice have an impaired wound healing phenotype in skin and tendons which is associated with Acan/Vcan-rich deposits at the wound sites. Moreover, the Acan and Vcan deposited was aggrecanase-cleaved, even in the absence of TS5. In this study, we have used adipose-derived stromal cell (ADSC) and epiphyseal chondrocyte cultures from wild type and KO mice to further study the role of TS5 in Acan and Vcan turnover. We have confirmed with both cell types that the aggrecanase-mediated degradation of these hyalectans is not due to TS5, but an aggrecanase which primarily cleaves them before they are secreted. We als...

The Biochemical journal, 2004

Human brain tissue from cerebellum and hippocampus was obtained between 2 h and 24 h post mortem ... more Human brain tissue from cerebellum and hippocampus was obtained between 2 h and 24 h post mortem and, after extraction in the presence of proteinase inhibitors, proteoglycans were purified by anion-exchange chromatography. The versican component was characterized by Western analysis with antibodies to the N-terminal peptide (LF99), the N-terminal globular domain (12C5) and the two GAG (glycosaminoglycan) attachment regions (anti-GAG-alpha and anti-GAG-beta). The results indicated that versican V2 is the major variant in all brain samples, and that it exists as the full-length form and also as at least six C-terminally truncated forms. The major immunoreactive species present is a 64 kDa product, which we identified by biochemical and immunological analysis as the brain protein previously termed GHAP (glial hyaluronate binding protein) [Perides, Lane, Andrews, Dahl and Bignami (1989) J. Biol. Chem. 264, 5981-5987]. Immunological analysis of purified human GHAP using a new anti-neoepi...

Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society, 2010

To quantify the structural characteristics and nanomechanical properties of aggrecan produced by ... more 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. 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 microscopy-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). BMSC-aggrecan was primarily full-length while cartilage-aggrecan had many fragments. Single molecule measurem...

Tissue Engineering Part A, 2011

Self-assembling peptide hydrogels were modified to deliver transforming growth factor β1 (TGF-β1)... more Self-assembling peptide hydrogels were modified to deliver transforming growth factor β1 (TGF-β1) to encapsulated bone-marrow-derived stromal cells (BMSCs) for cartilage tissue engineering applications using two different approaches: (i) biotin-streptavidin tethering; (ii) adsorption to the peptide scaffold. Initial studies to determine the duration of TGF-β1 medium supplementation necessary to stimulate chondrogenesis showed that 4 days of transient soluble TGF-β1 to newborn bovine BMSCs resulted in 10-fold higher proteoglycan accumulation than TGF-β1-free culture after 3 weeks. Subsequently, BMSC-seeded peptide hydrogels with either tethered TGF-β1 (Teth-TGF) or adsorbed TGF-β1 (Ads-TGF) were cultured in the TGF-β1-free medium, and chondrogenesis was compared to that for BMSCs encapsulated in unmodified peptide hydrogels, both with and without soluble TGF-β1 medium supplementation. Ads-TGF peptide hydrogels stimulated chondrogenesis of BMSCs as demonstrated by cell proliferation and cartilage-like extracellular matrix accumulation, whereas Teth-TGF did not stimulate chondrogenesis. In parallel experiments, TGF-β1 adsorbed to agarose hydrogels stimulated comparable chondrogenesis. Full-length aggrecan was produced by BMSCs in response to Ads-TGF in both peptide and agarose hydrogels, whereas medium-delivered TGF-β1 stimulated catabolic aggrecan cleavage product formation in agarose but not peptide scaffolds. Smad2/3 was transiently phosphorylated in response to Ads-TGF but not Teth-TGF, whereas medium-delivered TGF-β1 produced sustained signaling, suggesting that dose and signal duration are potentially important for minimizing aggrecan cleavage product formation. Robustness of this technology for use in multiple species and ages was demonstrated by effective chondrogenic stimulation of adult equine BMSCs, an important translational model used before the initiation of human clinical studies.

Osteoarthritis and Cartilage, 2010

Objective: As part of the National Institutes of Health (NIH)-sponsored Glucosamine/Chondroitin s... more Objective: As part of the National Institutes of Health (NIH)-sponsored Glucosamine/Chondroitin sulfate Arthritis Intervention Trial (GAIT) our objective here was to examine (1) the pharmacokinetics (PK) of glucosamine (GlcN) and chondroitin sulfate (CS) when taken separately or in combination as a single dose in normal individuals (n ¼ 29) and (2) the PK of GlcN and CS when taken as a single dose after 3 months daily dosing with GlcN, CS or GlcN þ CS, in patients with symptomatic knee pain (n ¼ 28).

Osteoarthritis and Cartilage, 2007

Osteoarthritis and Cartilage, 2010

Osteoarthritis and Cartilage, 2010

Objective: Oral glucosamine (GlcN) has been widely studied for its potential therapeutic benefits... more Objective: Oral glucosamine (GlcN) has been widely studied for its potential therapeutic benefits in alleviating the pain and disability of osteoarthritis (OA). Its popularity has grown despite ongoing controversy regarding its effectiveness vs placebo in clinical trials, and lack of information regarding possible mechanisms of action. Here, we review the state of knowledge concerning the biology of GlcN as it relates to OA, and discuss a framework for future research directions.

Journal of Structural Biology, 2003

Atomic force microscopy was used in ambient conditions to directly image dense and sparse monolay... more Atomic force microscopy was used in ambient conditions to directly image dense and sparse monolayers of bovine fetal epiphyseal and mature nasal cartilage aggrecan macromolecules adsorbed on mica substrates. Distinct resolution of the non-glycosylated N-terminal region from the glycosaminoglycan (GAG) brush of individual aggrecan monomers was achieved, as well as nanometer-scale resolution of individual GAG chain conformation and spacing. Fetal aggrecan core protein trace length (398+/-57 nm) and end-to-end length (257+/-87 nm) were both larger than that of mature aggrecan (352+/-88 and 226+/-81 nm, respectively). Similarly, fetal aggrecan GAG chain trace length (41+/-7 nm) and end-to-end (32+/-8 nm) length were both larger than that of mature aggrecan GAG (32+/-5 and 26+/-7 nm, respectively). GAG-GAG spacing along the core protein was significantly smaller in fetal compared to mature aggrecan (3.2+/-0.8 and 4.4+/-1.2nm, respectively). Together, these differences between the two aggrecan types were likely responsible for the greater persistence length of the fetal aggrecan (110 nm) compared to mature aggrecan (82 nm) calculated using the worm-like chain model. Measured dimensions and polymer statistical analyses were used in conjunction with the results of Western analyses, chromatographic, and carbohydrate electrophoresis measurements to better understand the dependence of aggrecan structure and properties on its constituent GAG chains.

Journal of Orthopaedic Research, 2011

To investigate the role of ADAMTS5 in murine osteoarthritis (OA), resulting from destabilization ... more To investigate the role of ADAMTS5 in murine osteoarthritis (OA), resulting from destabilization of the medial meniscus (DMM model) or from TGFb1 injection and enforced uphill treadmill running (TTR model). Wild-type (WT) and ADAMTS5-/- mice were subjected to either DMM or TTR and joints were evaluated for meniscal damage, cartilage changes, and fibrotic ingrowths from the joint margins. Cartilage lesions were quantified on an 8-point scoring system. Cartilage chondroitin sulfate (CS) content was evaluated by SafraninO staining and by quantitative electrophoresis (FACE). The abundance of aggrecan, versican, and specific aggrecanase-generated products was determined by Western analysis. Joint changes were similar for WT mice taken through either the DMM or the TTR model. ADAMTS5 ablation essentially eliminated cartilage erosion and fibrous overgrowth in both models. In the TTR model, ADAMTS5 ablation did not eliminate aggrecanase activity from the articular cartilage but blocked fibrosis and resulted in the accumulation of aggrecan in the articular cartilage. The cartilage protection provided by ADAMTS5 ablation in the mouse does not result from prevention of aggrecanase activity per se, but it appears to be due to a blockade of joint tissue fibrosis and a concomitant increase in cartilage aggrecan content.

Journal of Orthopaedic Research, 2013

A recently developed murine model of tendinopathy, induced by TGF-b1 injection, has been used to ... more A recently developed murine model of tendinopathy, induced by TGF-b1 injection, has been used to examine the reparative capacity of tendinopathic Achilles in Adamts5 À/À mice. After TGF-b1 injection and 2 weeks of treadmill exercise, the Achilles from Adamts5 À/À mice exhibited a reduction in maximum tensile stress of approximately 60%. However, in contrast to wild type mice previously characterized by this model, Adamts5 À/À mice subjected to further treadmill exercise were unable to reverse this biomechanical deficit. This nonreparative phenotype was accompanied by a major deficiency, relative to wild-type, in expression of Col1a1 and Col3a1 and an abnormally elevated expression of a wide range of integrins. In addition, the tendinopathic Adamts5 À/À mice showed a persistent accumulation of chondrogenic cells in the tendon body and an aggrecan-rich fibrocartilaginous matrix within disorganized collagen fiber bundles. Moreover, consistent with the compromised biomechanical properties of the Achilles in the Adamts5 À/À mice, in vivo gait analysis revealed a strong trend (p ¼ 0.07) towards increased swing time of the injected limb in Adamts5 À/À relative to wild-type mice. These findings demonstrate that a deficiency in ADAMTS5 promotes a chondrogenic response to TGF-b1 injection that is not reversed by treadmill exercise. Hence, repair of biomechanically compromised tendons exhibiting midsubstance chondroid accumulation requires ADAMTS5. ß

Journal of Histochemistry & Cytochemistry, 2005

The composition of extracellular matrix during growth and regression of the neointima was analyze... more The composition of extracellular matrix during growth and regression of the neointima was analyzed during healing in a baboon aorto-iliac polytetrafluoroethylene graft. Graft neointimal thickening can be modulated by altering blood flow by construction of downstream arteriovenous fistulas. Normal flow with normal shear stress induces neointimal thickening, whereas high flow with high shear stress upstream of a fistula induces regression of established neointima. The neointima formed under normal shear stress is enriched in hyaluronan and proteoglycans, particularly versican. On the other hand, the neointima near the graft material is enriched in collagen and biglycan. Neointimal regression in response to high shear stress is associated with a loss of proteoglycans as detected by histochemical staining. Immunostaining with an antibody against an ADAMTS cleavage neoepitope of versican increases after switching to high flow, although immunostaining for versican core protein is not appreciably changed by high flow. The present data demonstrate that the graft neointima is enriched with proteoglycans, particularly versican and hyaluronan, as well as collagen, and there is a differential distribution of each. Neointimal atrophy occurs with an apparent loss of proteoglycans and evidence of versican degradation. (J Histochem Cytochem 53:131-140, 2005) K E Y W O R D S neointima proteoglycans shear stress smooth muscle cells versican

Journal of Biomechanics, 2013

Tendinopathy is a widespread and disabling condition characterized by collagen fiber disruption a... more Tendinopathy is a widespread and disabling condition characterized by collagen fiber disruption and accumulation of a glycosaminoglycan-rich chondroid matrix. Recent clinical reports have illustrated the potential of mechanical loading (exercise) therapies to successfully treat chronic tendinopathies. We have developed a new murine tendinopathy model which requires a single injection of TGF-β1 into the Achilles tendon midsubstance followed by normal cage activity for 2 weeks. At this time, tendon maximum stress showed a dramatic (66%) reduction relative to that of normal controls and this persisted at four weeks. Loss of material properties was accompanied by abundant chondroid cells within the tendon (closely resembling the changes observed in human samples obtained intra-operatively) and increased expression of Acan, Col1a1, Col2a1, Col3a1, Fn1 and Mmp3. Mice subjected to two weeks of daily treadmill exercise following TGF-β1 injection showed a similar reduction in tendon material properties as the caged group. However, in mice subjected to 4 weeks of treadmill exercise, tendon maximum stress values were similar to those of naive controls. Tendons from the mice exercised for 4 weeks showed essentially no chondroid cells and the expression of Acan, Col1a1, Col2a1, Col3a1, and Mmp3 was significantly reduced relative to the 4-week cage group. This technically simple murine tendinopathy model is highly amenable to detailed mechanistic and translational studies of the biomechanical and cell biological pathways, that could be targeted to enhance healing of tendinopathy.

Journal of Biological Chemistry, 2004

1 The abbreviations used are: ECM, extracellular matrix; MMP, matrix metalloproteinase; MT, membr... more 1 The abbreviations used are: ECM, extracellular matrix; MMP, matrix metalloproteinase; MT, membrane type; ADAMTS, a disintegrin and metalloproteinase with thrombospondin-like motif; IL-1, interleukin 1; TIMP, tissue inhibitor of metalloproteinase; PIPLC, phosphatidylinositol-specific phospholipase C; GPI, glycosylphosphatidyl inositol; HS, heparan sulfate; 2-DFG, 2-deoxyfluoroglucose; GAG, glycosaminoglycan; CS, chondroitin sulfate.

IEEE Journal of Quantum Electronics, 2000

The ultrafast limit of the response of arrayed waveguide gratings is studied both theoretically a... more The ultrafast limit of the response of arrayed waveguide gratings is studied both theoretically and experimentally. We present new experimental results that show that interference occurs inside of an arrayed waveguide pulse shaper even when pulses that travel through different paths do not overlap. A comprehensive discussion of this extreme case is presented.

Brain Research, 2005

Introduction: To test the hypothesis that matrix metalloprotease-13 (MMP-13) and aggrecan may pla... more Introduction: To test the hypothesis that matrix metalloprotease-13 (MMP-13) and aggrecan may play roles in post-ischemic neuronal pathophysiology, we examined the impact of middle cerebral artery occlusion/reperfusion (MCAO/R) on the abundance of these proteins in different regions of the infarct by immunohistochemistry (IHC) and Western blotting (WB). Methods: The effect of MCAO/R on the abundance of MMP-13 and aggrecan was examined in 23 Wistar rats using antibodies against MMP-13 and aggrecan. BrdU was administered the last 2 days of the experiment. The cellular source of the respective antigens was examined with fluorescent double labeling using the neuronal marker NeuN. Sections were also stained for BrdU. The ischemic zone was defined by MRI on T2-weighted images and also on the tissue sections with the help of H & E counterstain. WB was performed for MMP-13. Results: MMP-13 protein is highly induced in ischemic brain and is associated with neurons, whereas aggrecan is associated with the perineuronal matrix in non-ischemic brain. After 3 days of cerebral ischemia, the number of MMP-13 positive neurons in the periphery of the ischemic lesion increased compared to the respective area in the non-ischemic brain with a peak on day 7. A stronger staining for aggrecan was observed around MMP-13 positive neurons compared with other neurons. The majority of the MMP-13 positive neurons in normal non-ischemic brain were also NeuN positive. BrdU was incorporated into MMP-13 positive neurons in the periphery of the infarct. WB confirmed this results by detecting MMP-13 bands in ischemic brains and activated MMP-13 up to 14 days after ischemia. Conclusions: There is a close spatial association of MMP-13 and aggrecan around individual neurons. Both MMP-13 and aggrecan appear to be involved in perineuronal matrix remodeling suggesting a role in neuronal reorganization after cerebral ischemia. D

Biology of Reproduction, 2005

Protease cascades are essential for many biological events, including the LH-induced process of o... more Protease cascades are essential for many biological events, including the LH-induced process of ovulation. ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin-like repeats-1) is expressed and hormonally regulated in the ovary by LH and the progesterone receptor. To determine whether other family members might be expressed and regulated in the rodent ovary, those closely related to ADAMTS1 (ADAMTS4 and ADAMTS5) were analyzed in the mouse ovary by reverse transcription-polymerase chain reaction as well as by Western blot, immunohistochemical, and immunocytochemical analyses using highly specific antibodies. Prior to ovulation, ADAMTS4 and ADAMTS5 were coexpressed in granulosa cells of most follicles, whereas ADAMTS5 was also present in granulosa cells of atretic follicles. Following ovulation, ADAMTS1 and ADAMTS4 (but not ADAMTS5) were expressed in multiple cell types, including those within the highly vascular ovulation cone that marks the site of follicle rupture, endothelial cells of newly forming corpora lutea, and cumulus cells within the ovulated cumulus cell-oocyte complex (COC). Versican, a substrate for ADAMTS1 and ADAMTS4, colocalized with these proteases and hylauronan on the cumulus cell surface. To further characterize induction of these proteases and associated molecules, COCs and granulosa cells were isolated from preovulatory follicles and treated with FSH. In expanded COCs and differentiated granulosa cells, FSH induced expression of ADAMTS4 and versican message and protein, whereas increased levels of ADAMTS1 protein was observed in the media of granulosa cells where it was stabilized by heparin in this in vitro system. These studies provide the first evidence that ADAMTS1, ADAMTS4, and ADAMTS5 are expressed in spatiotemporal patterns that suggest distinct as well as some overlapping functions that relate to the broad expression pattern of versican in granulosa cells of small follicles, expanded COCs, and endothelial cells of the mouse ovary.

Arthritis Research & Therapy, 2009

Little is known about endogenous or cytokinestimulated aggrecan catabolism in the meniscal fibroc... more Little is known about endogenous or cytokinestimulated aggrecan catabolism in the meniscal fibrocartilage of the knee. The objectives of this study were to characterize the structure, distribution, and processing of aggrecan in menisci from immature bovines, and to identify mechanisms of extracellular matrix degradation that lead to changes in the mechanical properties of meniscal fibrocartilage.

Arthritis & Rheumatism, 2002

To study the chondroprotective effect of constitutively expressed TSG-6 protein (tumor necrosis f... more To study the chondroprotective effect of constitutively expressed TSG-6 protein (tumor necrosis factor ␣-induced protein 6; Tnfip6) in cartilage, using antigen-induced arthritis (AIA) in mice.

Matrix Biology, May 31, 2007

Articular cartilage undergoes matrix degradation and loss of mechanical properties when stimulate... more Articular cartilage undergoes matrix degradation and loss of mechanical properties when stimulated with proinflammatory cytokines such as interleukin-1 (IL-1). Aggrecanases and matrix metalloproteinases (MMPs) are thought to be principal downstream effectors of cytokine-induced matrix catabolism, and aggrecanase- or MMP-selective inhibitors reduce or block matrix destruction in several model systems. The objective of this study was to use metalloproteinase inhibitors to perturb IL-1-induced matrix catabolism in bovine cartilage explants and examine their effects on changes in tissue compression and shear properties. Explanted tissue was stimulated with IL-1 for up to 24 days in the absence or presence of inhibitors that were aggrecanase-selective, MMP-selective, or non-selective. Analysis of conditioned media and explant digests revealed that aggrecanase-mediated aggrecanolysis was delayed to varying extents with all inhibitor treatments, but that aggrecan release persisted. Collagen degradation was abrogated by MMP- and non-selective inhibitors and reduced by the aggrecanase inhibitor. The inhibitors delayed but did not reduce loss of the equilibrium compression modulus, whereas the losses of dynamic compression and shear moduli were delayed and reduced. The data suggest that non-metalloproteinase mechanisms participate in IL-1-induced matrix degradation and loss of tissue material properties.

Matrix biology : journal of the International Society for Matrix Biology, Jan 31, 2015

ADAMTS5 (TS5), a member of the aggrecanase clade (TS1, 4, 5, 8, 9, 15) of ADAMTS-proteases, has b... more ADAMTS5 (TS5), a member of the aggrecanase clade (TS1, 4, 5, 8, 9, 15) of ADAMTS-proteases, has been considered largely responsible for the proteolysis of the hyalectans, aggrecan (Acan) and versican (Vcan), in vivo. However, we have reported that ts5-knockout (KO) mice show joint protection after injury due to inhibition of synovial scarring and enhanced Acan deposition. Also, KO mice have an impaired wound healing phenotype in skin and tendons which is associated with Acan/Vcan-rich deposits at the wound sites. Moreover, the Acan and Vcan deposited was aggrecanase-cleaved, even in the absence of TS5. In this study, we have used adipose-derived stromal cell (ADSC) and epiphyseal chondrocyte cultures from wild type and KO mice to further study the role of TS5 in Acan and Vcan turnover. We have confirmed with both cell types that the aggrecanase-mediated degradation of these hyalectans is not due to TS5, but an aggrecanase which primarily cleaves them before they are secreted. We als...

The Biochemical journal, 2004

Human brain tissue from cerebellum and hippocampus was obtained between 2 h and 24 h post mortem ... more Human brain tissue from cerebellum and hippocampus was obtained between 2 h and 24 h post mortem and, after extraction in the presence of proteinase inhibitors, proteoglycans were purified by anion-exchange chromatography. The versican component was characterized by Western analysis with antibodies to the N-terminal peptide (LF99), the N-terminal globular domain (12C5) and the two GAG (glycosaminoglycan) attachment regions (anti-GAG-alpha and anti-GAG-beta). The results indicated that versican V2 is the major variant in all brain samples, and that it exists as the full-length form and also as at least six C-terminally truncated forms. The major immunoreactive species present is a 64 kDa product, which we identified by biochemical and immunological analysis as the brain protein previously termed GHAP (glial hyaluronate binding protein) [Perides, Lane, Andrews, Dahl and Bignami (1989) J. Biol. Chem. 264, 5981-5987]. Immunological analysis of purified human GHAP using a new anti-neoepi...

Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society, 2010

To quantify the structural characteristics and nanomechanical properties of aggrecan produced by ... more 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. 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 microscopy-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). BMSC-aggrecan was primarily full-length while cartilage-aggrecan had many fragments. Single molecule measurem...

Tissue Engineering Part A, 2011

Self-assembling peptide hydrogels were modified to deliver transforming growth factor β1 (TGF-β1)... more Self-assembling peptide hydrogels were modified to deliver transforming growth factor β1 (TGF-β1) to encapsulated bone-marrow-derived stromal cells (BMSCs) for cartilage tissue engineering applications using two different approaches: (i) biotin-streptavidin tethering; (ii) adsorption to the peptide scaffold. Initial studies to determine the duration of TGF-β1 medium supplementation necessary to stimulate chondrogenesis showed that 4 days of transient soluble TGF-β1 to newborn bovine BMSCs resulted in 10-fold higher proteoglycan accumulation than TGF-β1-free culture after 3 weeks. Subsequently, BMSC-seeded peptide hydrogels with either tethered TGF-β1 (Teth-TGF) or adsorbed TGF-β1 (Ads-TGF) were cultured in the TGF-β1-free medium, and chondrogenesis was compared to that for BMSCs encapsulated in unmodified peptide hydrogels, both with and without soluble TGF-β1 medium supplementation. Ads-TGF peptide hydrogels stimulated chondrogenesis of BMSCs as demonstrated by cell proliferation and cartilage-like extracellular matrix accumulation, whereas Teth-TGF did not stimulate chondrogenesis. In parallel experiments, TGF-β1 adsorbed to agarose hydrogels stimulated comparable chondrogenesis. Full-length aggrecan was produced by BMSCs in response to Ads-TGF in both peptide and agarose hydrogels, whereas medium-delivered TGF-β1 stimulated catabolic aggrecan cleavage product formation in agarose but not peptide scaffolds. Smad2/3 was transiently phosphorylated in response to Ads-TGF but not Teth-TGF, whereas medium-delivered TGF-β1 produced sustained signaling, suggesting that dose and signal duration are potentially important for minimizing aggrecan cleavage product formation. Robustness of this technology for use in multiple species and ages was demonstrated by effective chondrogenic stimulation of adult equine BMSCs, an important translational model used before the initiation of human clinical studies.

Osteoarthritis and Cartilage, 2010

Objective: As part of the National Institutes of Health (NIH)-sponsored Glucosamine/Chondroitin s... more Objective: As part of the National Institutes of Health (NIH)-sponsored Glucosamine/Chondroitin sulfate Arthritis Intervention Trial (GAIT) our objective here was to examine (1) the pharmacokinetics (PK) of glucosamine (GlcN) and chondroitin sulfate (CS) when taken separately or in combination as a single dose in normal individuals (n ¼ 29) and (2) the PK of GlcN and CS when taken as a single dose after 3 months daily dosing with GlcN, CS or GlcN þ CS, in patients with symptomatic knee pain (n ¼ 28).

Osteoarthritis and Cartilage, 2007

Osteoarthritis and Cartilage, 2010

Osteoarthritis and Cartilage, 2010

Objective: Oral glucosamine (GlcN) has been widely studied for its potential therapeutic benefits... more Objective: Oral glucosamine (GlcN) has been widely studied for its potential therapeutic benefits in alleviating the pain and disability of osteoarthritis (OA). Its popularity has grown despite ongoing controversy regarding its effectiveness vs placebo in clinical trials, and lack of information regarding possible mechanisms of action. Here, we review the state of knowledge concerning the biology of GlcN as it relates to OA, and discuss a framework for future research directions.

Journal of Structural Biology, 2003

Atomic force microscopy was used in ambient conditions to directly image dense and sparse monolay... more Atomic force microscopy was used in ambient conditions to directly image dense and sparse monolayers of bovine fetal epiphyseal and mature nasal cartilage aggrecan macromolecules adsorbed on mica substrates. Distinct resolution of the non-glycosylated N-terminal region from the glycosaminoglycan (GAG) brush of individual aggrecan monomers was achieved, as well as nanometer-scale resolution of individual GAG chain conformation and spacing. Fetal aggrecan core protein trace length (398+/-57 nm) and end-to-end length (257+/-87 nm) were both larger than that of mature aggrecan (352+/-88 and 226+/-81 nm, respectively). Similarly, fetal aggrecan GAG chain trace length (41+/-7 nm) and end-to-end (32+/-8 nm) length were both larger than that of mature aggrecan GAG (32+/-5 and 26+/-7 nm, respectively). GAG-GAG spacing along the core protein was significantly smaller in fetal compared to mature aggrecan (3.2+/-0.8 and 4.4+/-1.2nm, respectively). Together, these differences between the two aggrecan types were likely responsible for the greater persistence length of the fetal aggrecan (110 nm) compared to mature aggrecan (82 nm) calculated using the worm-like chain model. Measured dimensions and polymer statistical analyses were used in conjunction with the results of Western analyses, chromatographic, and carbohydrate electrophoresis measurements to better understand the dependence of aggrecan structure and properties on its constituent GAG chains.

Journal of Orthopaedic Research, 2011

To investigate the role of ADAMTS5 in murine osteoarthritis (OA), resulting from destabilization ... more To investigate the role of ADAMTS5 in murine osteoarthritis (OA), resulting from destabilization of the medial meniscus (DMM model) or from TGFb1 injection and enforced uphill treadmill running (TTR model). Wild-type (WT) and ADAMTS5-/- mice were subjected to either DMM or TTR and joints were evaluated for meniscal damage, cartilage changes, and fibrotic ingrowths from the joint margins. Cartilage lesions were quantified on an 8-point scoring system. Cartilage chondroitin sulfate (CS) content was evaluated by SafraninO staining and by quantitative electrophoresis (FACE). The abundance of aggrecan, versican, and specific aggrecanase-generated products was determined by Western analysis. Joint changes were similar for WT mice taken through either the DMM or the TTR model. ADAMTS5 ablation essentially eliminated cartilage erosion and fibrous overgrowth in both models. In the TTR model, ADAMTS5 ablation did not eliminate aggrecanase activity from the articular cartilage but blocked fibrosis and resulted in the accumulation of aggrecan in the articular cartilage. The cartilage protection provided by ADAMTS5 ablation in the mouse does not result from prevention of aggrecanase activity per se, but it appears to be due to a blockade of joint tissue fibrosis and a concomitant increase in cartilage aggrecan content.

Journal of Orthopaedic Research, 2013

A recently developed murine model of tendinopathy, induced by TGF-b1 injection, has been used to ... more A recently developed murine model of tendinopathy, induced by TGF-b1 injection, has been used to examine the reparative capacity of tendinopathic Achilles in Adamts5 À/À mice. After TGF-b1 injection and 2 weeks of treadmill exercise, the Achilles from Adamts5 À/À mice exhibited a reduction in maximum tensile stress of approximately 60%. However, in contrast to wild type mice previously characterized by this model, Adamts5 À/À mice subjected to further treadmill exercise were unable to reverse this biomechanical deficit. This nonreparative phenotype was accompanied by a major deficiency, relative to wild-type, in expression of Col1a1 and Col3a1 and an abnormally elevated expression of a wide range of integrins. In addition, the tendinopathic Adamts5 À/À mice showed a persistent accumulation of chondrogenic cells in the tendon body and an aggrecan-rich fibrocartilaginous matrix within disorganized collagen fiber bundles. Moreover, consistent with the compromised biomechanical properties of the Achilles in the Adamts5 À/À mice, in vivo gait analysis revealed a strong trend (p ¼ 0.07) towards increased swing time of the injected limb in Adamts5 À/À relative to wild-type mice. These findings demonstrate that a deficiency in ADAMTS5 promotes a chondrogenic response to TGF-b1 injection that is not reversed by treadmill exercise. Hence, repair of biomechanically compromised tendons exhibiting midsubstance chondroid accumulation requires ADAMTS5. ß

Journal of Histochemistry & Cytochemistry, 2005

The composition of extracellular matrix during growth and regression of the neointima was analyze... more The composition of extracellular matrix during growth and regression of the neointima was analyzed during healing in a baboon aorto-iliac polytetrafluoroethylene graft. Graft neointimal thickening can be modulated by altering blood flow by construction of downstream arteriovenous fistulas. Normal flow with normal shear stress induces neointimal thickening, whereas high flow with high shear stress upstream of a fistula induces regression of established neointima. The neointima formed under normal shear stress is enriched in hyaluronan and proteoglycans, particularly versican. On the other hand, the neointima near the graft material is enriched in collagen and biglycan. Neointimal regression in response to high shear stress is associated with a loss of proteoglycans as detected by histochemical staining. Immunostaining with an antibody against an ADAMTS cleavage neoepitope of versican increases after switching to high flow, although immunostaining for versican core protein is not appreciably changed by high flow. The present data demonstrate that the graft neointima is enriched with proteoglycans, particularly versican and hyaluronan, as well as collagen, and there is a differential distribution of each. Neointimal atrophy occurs with an apparent loss of proteoglycans and evidence of versican degradation. (J Histochem Cytochem 53:131-140, 2005) K E Y W O R D S neointima proteoglycans shear stress smooth muscle cells versican

Journal of Biomechanics, 2013

Tendinopathy is a widespread and disabling condition characterized by collagen fiber disruption a... more Tendinopathy is a widespread and disabling condition characterized by collagen fiber disruption and accumulation of a glycosaminoglycan-rich chondroid matrix. Recent clinical reports have illustrated the potential of mechanical loading (exercise) therapies to successfully treat chronic tendinopathies. We have developed a new murine tendinopathy model which requires a single injection of TGF-β1 into the Achilles tendon midsubstance followed by normal cage activity for 2 weeks. At this time, tendon maximum stress showed a dramatic (66%) reduction relative to that of normal controls and this persisted at four weeks. Loss of material properties was accompanied by abundant chondroid cells within the tendon (closely resembling the changes observed in human samples obtained intra-operatively) and increased expression of Acan, Col1a1, Col2a1, Col3a1, Fn1 and Mmp3. Mice subjected to two weeks of daily treadmill exercise following TGF-β1 injection showed a similar reduction in tendon material properties as the caged group. However, in mice subjected to 4 weeks of treadmill exercise, tendon maximum stress values were similar to those of naive controls. Tendons from the mice exercised for 4 weeks showed essentially no chondroid cells and the expression of Acan, Col1a1, Col2a1, Col3a1, and Mmp3 was significantly reduced relative to the 4-week cage group. This technically simple murine tendinopathy model is highly amenable to detailed mechanistic and translational studies of the biomechanical and cell biological pathways, that could be targeted to enhance healing of tendinopathy.

Journal of Biological Chemistry, 2004

1 The abbreviations used are: ECM, extracellular matrix; MMP, matrix metalloproteinase; MT, membr... more 1 The abbreviations used are: ECM, extracellular matrix; MMP, matrix metalloproteinase; MT, membrane type; ADAMTS, a disintegrin and metalloproteinase with thrombospondin-like motif; IL-1, interleukin 1; TIMP, tissue inhibitor of metalloproteinase; PIPLC, phosphatidylinositol-specific phospholipase C; GPI, glycosylphosphatidyl inositol; HS, heparan sulfate; 2-DFG, 2-deoxyfluoroglucose; GAG, glycosaminoglycan; CS, chondroitin sulfate.

IEEE Journal of Quantum Electronics, 2000

The ultrafast limit of the response of arrayed waveguide gratings is studied both theoretically a... more The ultrafast limit of the response of arrayed waveguide gratings is studied both theoretically and experimentally. We present new experimental results that show that interference occurs inside of an arrayed waveguide pulse shaper even when pulses that travel through different paths do not overlap. A comprehensive discussion of this extreme case is presented.

Brain Research, 2005

Introduction: To test the hypothesis that matrix metalloprotease-13 (MMP-13) and aggrecan may pla... more Introduction: To test the hypothesis that matrix metalloprotease-13 (MMP-13) and aggrecan may play roles in post-ischemic neuronal pathophysiology, we examined the impact of middle cerebral artery occlusion/reperfusion (MCAO/R) on the abundance of these proteins in different regions of the infarct by immunohistochemistry (IHC) and Western blotting (WB). Methods: The effect of MCAO/R on the abundance of MMP-13 and aggrecan was examined in 23 Wistar rats using antibodies against MMP-13 and aggrecan. BrdU was administered the last 2 days of the experiment. The cellular source of the respective antigens was examined with fluorescent double labeling using the neuronal marker NeuN. Sections were also stained for BrdU. The ischemic zone was defined by MRI on T2-weighted images and also on the tissue sections with the help of H & E counterstain. WB was performed for MMP-13. Results: MMP-13 protein is highly induced in ischemic brain and is associated with neurons, whereas aggrecan is associated with the perineuronal matrix in non-ischemic brain. After 3 days of cerebral ischemia, the number of MMP-13 positive neurons in the periphery of the ischemic lesion increased compared to the respective area in the non-ischemic brain with a peak on day 7. A stronger staining for aggrecan was observed around MMP-13 positive neurons compared with other neurons. The majority of the MMP-13 positive neurons in normal non-ischemic brain were also NeuN positive. BrdU was incorporated into MMP-13 positive neurons in the periphery of the infarct. WB confirmed this results by detecting MMP-13 bands in ischemic brains and activated MMP-13 up to 14 days after ischemia. Conclusions: There is a close spatial association of MMP-13 and aggrecan around individual neurons. Both MMP-13 and aggrecan appear to be involved in perineuronal matrix remodeling suggesting a role in neuronal reorganization after cerebral ischemia. D

Biology of Reproduction, 2005

Protease cascades are essential for many biological events, including the LH-induced process of o... more Protease cascades are essential for many biological events, including the LH-induced process of ovulation. ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin-like repeats-1) is expressed and hormonally regulated in the ovary by LH and the progesterone receptor. To determine whether other family members might be expressed and regulated in the rodent ovary, those closely related to ADAMTS1 (ADAMTS4 and ADAMTS5) were analyzed in the mouse ovary by reverse transcription-polymerase chain reaction as well as by Western blot, immunohistochemical, and immunocytochemical analyses using highly specific antibodies. Prior to ovulation, ADAMTS4 and ADAMTS5 were coexpressed in granulosa cells of most follicles, whereas ADAMTS5 was also present in granulosa cells of atretic follicles. Following ovulation, ADAMTS1 and ADAMTS4 (but not ADAMTS5) were expressed in multiple cell types, including those within the highly vascular ovulation cone that marks the site of follicle rupture, endothelial cells of newly forming corpora lutea, and cumulus cells within the ovulated cumulus cell-oocyte complex (COC). Versican, a substrate for ADAMTS1 and ADAMTS4, colocalized with these proteases and hylauronan on the cumulus cell surface. To further characterize induction of these proteases and associated molecules, COCs and granulosa cells were isolated from preovulatory follicles and treated with FSH. In expanded COCs and differentiated granulosa cells, FSH induced expression of ADAMTS4 and versican message and protein, whereas increased levels of ADAMTS1 protein was observed in the media of granulosa cells where it was stabilized by heparin in this in vitro system. These studies provide the first evidence that ADAMTS1, ADAMTS4, and ADAMTS5 are expressed in spatiotemporal patterns that suggest distinct as well as some overlapping functions that relate to the broad expression pattern of versican in granulosa cells of small follicles, expanded COCs, and endothelial cells of the mouse ovary.

Arthritis Research & Therapy, 2009

Little is known about endogenous or cytokinestimulated aggrecan catabolism in the meniscal fibroc... more Little is known about endogenous or cytokinestimulated aggrecan catabolism in the meniscal fibrocartilage of the knee. The objectives of this study were to characterize the structure, distribution, and processing of aggrecan in menisci from immature bovines, and to identify mechanisms of extracellular matrix degradation that lead to changes in the mechanical properties of meniscal fibrocartilage.