3D distribution of perlecan within intervertebral disc chondrons suggests novel regulatory roles for this multifunctional modular heparan sulphate proteoglycan.

Perlecan is a modular, multifunctional heparan sulphate-proteoglycan (HS-PG) that is present in the pericellular and wider extracellular matrix of connective tissues. In the present study, confocal microscopy was used to study perlecan distribution within intervertebral disc chondrons. Perlecan immunolabel was demonstrated intracellularly and in close association with the cell nucleus within chondrons of both the annulus fibrosus (AF) and nucleus pulposus (NP). This observation is consistent with earlier studies that have localised HS-PGs with nuclear cytoskeletal components. Nuclear HS-PGs have been proposed to transport fibroblast growth factor (FGF)-1, FGF-2 and FGFR-1 into the cell nucleus, influencing cell proliferation and the cell-cycle. Perlecan has well-known interactive properties with FGF family members in the pericellular and extracellular matrix. Perinuclear perlecan may also participate in translocation events with FGFs. The glycosaminoglycan side chains of HS-PGs can modulate chromatin structure by regulating the access of transcription factors to DNA. These mechanisms are consistent with the distribution patterns identified here and previously reported for other HS-PGs, introducing a potentially-novel arena for perlecan in gene regulation. Whilst much is known of the structure and function of perlecan in the pericellular and extracellular matrix, very little is known of any intracellular forms of perlecan. The perlecan labelling patterns described here suggest the possibility of involvement of this HS-PG in an intracrine regulatory system. Future studies should further explore this possibility and the potential for this HS-PG as a novel therapeutic target.

Genetic murine models of spinal development and degeneration provide valuable insights into intervertebral disc pathobiology.

Disc degeneration and associated back and neck pain elicits a substantial burden on healthcare systems and the individuals affected, necessitating the development of novel therapeutic strategies. This goal can only be achieved by a better understanding of intervertebral disc development, homeostasis and pathogenesis. A number of genetic and in-bred murine models are reviewed to underscore the importance of the mouse as an animal model of choice for the assessment of intervertebral disc pathobiology. Appraisals of the differences between mouse and human musculoskeletal systems and proteoglycan structures are also included. A number of important target pathways and molecules have been identified, many of which are worthy of further examination, requiring that the activity of these be confirmed in large animal models and assessed in the context of therapeutic intervention.

Harnessing chondroitin sulphate in composite scaffolds to direct progenitor and stem cell function for tissue repair.

The development of bioscaffolds that incorporate chondroitin sulphate (CS) and their applications with progenitor and stem cells in cartilage, bone, cornea, skin, and neural repair are reviewed. CS is a heterogeneous structure due to the organisation of multiple CS disaccharide sulphation motifs, giving rise to a vast range of CS chain structures, and hence the wide range of biological activity. The incorporation of this biological molecule represents a significant advance in bioscaffold design and performance in tissue repair strategies. The intrinsic stem-cell directive properties of CS are covered in the context of tissue development, and the differing CS disaccharide motifs, referred to as the 'glyco-code'. These structural motifs contribute to stem cell proliferation and differentiation in the scaffold environment and improve outcomes in terms of tissue repair or regeneration worthy of future research.

Pericellular colocalisation and interactive properties of type VI collagen and perlecan in the intervertebral disc.

The aim of this study was to immunolocalise type VI collagen and perlecan and determine their interactive properties in the intervertebral disc (IVD). Confocal laser scanning microscopy co-localised perlecan with type VI collagen as pericellular components of IVD cells and translamellar cross-bridges in ovine and murine IVDs. These cross-bridges were significantly less abundant in the heparin sulphate deficient Hspg2 exon 3 null mouse IVD than in wild type. This association of type VI collagen with elastic components provides clues as to its roles in conveying elastic recoil properties to annular tissues. Perlecan and type VI collagen were highly interactive in plasmon resonance studies. Pericellular colocalisation of perlecan and type VI collagen provides matrix stabilisation and cell-matrix communication which allows IVD cells to perceive and respond to perturbations in their biomechanical microenvironment. Perlecan, at the cell surface, provides an adhesive interface between the cell and its surrounding extracellular matrix. Elastic microfibrillar structures regulate tensional connective tissue development and function. The 2010 Global Burden of Disease study examined 291 disorders and identified disc degeneration and associated low back pain as the leading global musculoskeletal disorder emphasising its massive socioeconomic impact and the need for more effective treatment strategies. A greater understanding of how the IVD achieves its unique biomechanical functional properties is of great importance in the development of such therapeutic measures.

The knee joint loose body as a source of viable autologous human chondrocytes.

Loose bodies are fragments of cartilage or bone present in the synovial fluid. In the present study we assessed if loose bodies could be used as a source of autologous human chondrocytes for experimental purposes. Histochemical examination of loose bodies and differential enzymatic digestions were undertaken, the isolated cells were cultured in alginate bead microspheres and immunolocalisations were undertaken for chondrogenic markers such as aggrecan, and type II collagen. Isolated loose body cells had high viability (≥90% viable), expressed chondrogenic markers (aggrecan, type II collagen) but no type I collagen. Loose bodies may be a useful source of autologous chondrocytes of high viability.

Zonal differences in meniscus matrix turnover and cytokine response.

OBJECTIVE: To determine the mechanisms of meniscal degeneration and whether this varied zonally and from articular cartilage. DESIGN: Normal ovine menisci were dissected into inner and outer zones and along with cartilage cultured ±IL-1α and TNFα. Glycosaminoglycan (GAG) and collagen release, and gene expression were quantified. Aggrecan proteolysis was analysed by Western blotting with neoepitope-specific antibodies. Matrix metalloproteinase (MMP)2, MMP9 and MMP13 activity was evaluated by gelatin zymography or fluorogenic assay. RESULTS: Inner meniscus was more cartilaginous containing more GAG and expressing more ACAN and COL2A1 than outer zones. Higher expression of VCAN and ADAMTS4 in medial outer and both zones of the lateral meniscus reflected their embryologic origin from cells outside the cartilage anlagen. All meniscal regions released a greater % GAG in response to cytokines; only outer zones had cytokine-stimulated collagenolysis. Cytokine-induced aggrecanolysis was primarily due to increased ADAMTS cleavage in cartilage and inner menisci but MMPs in the outer menisci. Outer menisci always released more active MMP2 than other tissues and more active MMP13 in basal and TNF-stimulated cultures. Expression of ACAN, COL1A1 and COL2A1 was decreased by both cytokines in all tissues, while VCAN was increased by IL-1α in cartilage and inner menisci. Metalloproteinase expression was differentially regulated by IL-1α and TNFα: ADAMTS4, MMP1, MMP3 were upregulated more by IL-1α in inner zones whereas ADAMTS5, MMP13 and MMP9 were more upregulated by TNFα in outer zones. CONCLUSIONS: Meniscal degeneration mechanisms are zonally-dependent, and may contribute to the enzymatic burden in the joint.

The use of Histochoice for histological examination of articular and growth plate cartilages, intervertebral disc and meniscus.

Histochoice is a proprietary nontoxic, non-cross-linking fixative designed by the manufacturer to replace formaldehyde based fixation protocols. We compared Histochoice and formalin fixation for several cartilaginous tissues including, articular and growth plate cartilage, meniscus and intervertebral disc. The tissues were stained with general histology stains including toluidine blue for tissue proteoglycans, picrosirius red to evaluate collagenous organization, and hematoxylin and eosin to assess cell morphology. The chondroitin sulfate and heparin sulfate substituted proteoglycans aggrecan and perlecan were also immunolocalized in some of the tissues to provide a comparison. Histochoice did not fix deep into the tissue blocks resulting in focal loss of aggrecan and other matrix components from the more central regions of the blocks. This was evident in toluidine blue stained sections of immature tibial articular cartilage where loss of glycosaminoglycan was significant in Histochoice fixed tissues. Histochoice fixation worked well, however, in the aggrecan and perlecan immunohistology applications where its non-cross-linking traits were conducive to epitope retrieval and identification by primary antibodies to extracellular matrix components.

Modulation of aggrecan and ADAMTS expression in ovine tendinopathy induced by altered strain.

OBJECTIVE: To evaluate histologic, immunohistochemical, and molecular changes in tendon induced by altered strain in a large-animal model. METHODS: A full-thickness partial-width laceration of the infraspinatus tendon was created in 5 sheep, while 5 sham-operated sheep were used as controls. Sheep were killed after 4 weeks, and 4 differentially stressed tendon regions (tensile or near bone attachment from overstressed or stress-deprived halves) were evaluated for histopathology, proteoglycan (PG) accumulation, and characterization of glycosaminoglycans and aggrecan catabolites. Gene expression of matrix components, enzymes, and inhibitors was analyzed by reverse transcriptase-polymerase chain reaction. RESULTS: Histopathologic changes were detected in both overstressed and stress-deprived tensile tendon, but only in stress-deprived tendon near bone. In overstressed and stress-deprived tensile tendon, levels of keratan sulfate, chondroitin 4-sulfate, and chondroitin 6-sulfate were increased. In overstressed tensile tendon, levels of ADAMTS-generated aggrecan catabolites were increased. There was increased matrix metalloproteinase 13 (MMP-13) and decreased fibromodulin and decorin expression in all regions. Increased MMP-1, MMP-9, MMP-14, and ADAMTS-1 expression, and decreased type II collagen expression were restricted to stress-deprived tendon. In stress-deprived bone-attachment regions, messenger RNA (mRNA) for aggrecan was decreased, and ADAMTS was increased. In overstressed tensile tendon, aggrecan mRNA was increased, and ADAMTS was decreased. CONCLUSION: The distinct molecular changes in adjacent tissue implicate altered strain rather than humoral factors in controlling abnormal tenocyte metabolism, and highlight the importance of regional sampling. Tendon abnormalities induced by increased strain are accompanied by increased aggrecan, decreased ADAMTS, and low PG expression, which may negatively impact the structural integrity of the tissue and predispose to rupture.

Perlecan from human epithelial cells is a hybrid heparan/chondroitin/keratan sulfate proteoglycan.

Perlecan is a multidomain proteoglycan, usually substituted with heparan sulphate (HS), and sometimes substituted with both HS and chondroitin sulphate (CS). In this paper, we describe perlecan purified from HEK-293 cells substituted with HS, CS and keratan sulphate (KS). KS substitution was confirmed by immunoreactivity with antibody 5D4, sensitivity to keratanase treatment, and fluorophore-assisted carbohydrate electrophoresis. HEK-293 perlecan failed to promote FGF-dependent cell growth in an in vitro assay. This study is the first to report perlecan containing KS, and makes perlecan one of only a very few proteoglycans substituted with three distinct types of glycosaminoglycan chains.

A mechanics model for the compression of plant and vegetative tissues.

The mechanics analysis of plant or vegetable tissue under a compressive stress has been developed based on large deformation elasticity theory. The tissue was treated as a lattice of regular perfect three-dimensional hexagonal cells. The cell walls were assumed to be impermeable under the time-scale of the loading. The cell walls of plants and vegetables are polymeric composite materials, consisting of a relatively amorphous matrix and a highly structured network of microfibrils embedded in the cell wall matrix. The micromechanical features of the individual cells have been related to the macroscopic properties of the whole tissue. The effects of microfibrillar stiffening factors k(1) and k(2), the cell wall matrix property alpha and the initial cell expansion ratio nu(i) on the compressive behaviour of a plant or vegetable tissue have been investigated. The predicted results have also been related to some experimental evidence.

Induction of matrix metalloproteinase-2 and -3 activity in ovine nucleus pulposus cells grown in three-dimensional agarose gel culture by interleukin-1beta: a potential pathway of disc degeneration.

Degeneration of the intervertebral disc is an important clinical problem, which often contributes to low back pain. Since approximately 80-90% of the general population will be subject to back pain at some stage during their lifetime, this has major socioeconomic consequences. Matrix metalloproteinases (MMPs) have been implicated in the excessive breakdown of extracellular matrix components during disc degeneration. The aim of the present study was to evaluate the regulation of MMP-2 (gelatinase-A) and MMP-3 (stromelysin) produced by cultured ovine nucleus pulposus (NP) cells stimulated with interleukin-1beta (IL-1beta). NP cells were established in three-dimensional agarose culture and stimulated with IL-1beta under serum-free conditions. Conditioned media samples were evaluated by gelatin and casein zymography and by fluorimetry using an MMP-specific substrate. Time-course and dose dependencies were established for MMP-2, -3 production by the NP cells in response to the IL-1beta. Gelatin and casein zymography indicated that elevated levels of proMMP-2 and proMMP-3 were present in media samples in response to the IL-1beta treatment. After 24-96 h culture, levels of the active 43 and 45 kDa active MMP-3 were significantly elevated, whereas MMP-2 was present mainly as its 72 kDa pro-form. Additional 36, 28 and 21 kDa MMP species were also present after prolonged incubation with IL-1beta, probably representing MMP breakdown species. IL-1beta was a potent catabolic mediator for the NP cells, resulting in the production of elevated levels of MMP-2 and -3 in culture. However, approximately 70% of the MMP-2 was present as the 72 kDa pro-form, which suggests that some additional steps are involved in its activation in vivo.

Visualisation of hyaluronan and hyaluronan-binding proteins within ovine vertebral cartilages using biotinylated aggrecan G1-link complex and biotinylated hyaluronan oligosaccharides.

The aim of this study was to localise hyaluronan (HA)-binding proteins (HABPs) in ovine vertebral tissues using biotinylated HA oligosaccharides (bHA oligos) as novel affinity probes and to compare this with the distribution of tissue HA visualised using biotinylated aggrecan G1 domain-link protein complex. The bHA oligos, with a size of 6-18 disaccharides were prepared by partial digestion of HA with ovine testicular hyaluronidase, labelled with biotin hydrazide and purified by a combination of aggrecan G1 domain and avidin affinity chromatography. Hyaluronan and HABPs were both prominent pericellular components of hypertrophic cells of the vertebral epiphyseal growth plate and enlarged cells in the cartilaginous end plate of the disc. The bHA oligo probe also visualised HABPs intracellularly in hypertrophic cells, which also contained intracellular HA. Monolayer cultures of ovine annulus fibrosus and nucleus pulposus cells rapidly internalised the bHA oligo affinity probe which was subsequently visualised by indirect fluorescence using avidin-FITC, to cytoplasm and discrete nuclear regions. The results indicate that the abundant pericellular and intracellular HA associated with cartilaginous cells in the vertebral tissues is colocalised with HABPs. The bHA oligo affinity probe may have further applications in investigations of intracellular HABPs, HA endocytosis and the roles they play in cellular regulatory processes.

Affinity and Western blotting reveal homologies between ovine intervertebral disc serine proteinase inhibitory proteins and bovine pancreatic trypsin inhibitor.

The objective of this study was to assess any similarities between ovine intervertebral disc (IVD) serine proteinase inhibitory proteins (SPIs) and known mammalian IVD SPIs. Ovine IVDs were dissected into the annulus fibrosus and nucleus pulposus and the tissue finely diced then extracted with 4 M guanidine hydrochloride. The tissue extracts were subjected to caesium chloride density gradient ultracentrifugation to separate the large high buoyant density (rho > 1.5 g/mL) proteoglycans from the SPI proteins of low buoyant density (rho < 1.33 g/mL). The top two ultracentrifuge fractions containing the SPIs of interest were subjected to enzyme linked immunosorbent analysis (ELISA) and also examined by Western and Affinity blotting using an antibody to bovine pancreatic trypsin inhibitor and biotinylated trypsin respectively for detection and an alkaline phosphatase 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium system for visualisation. The major SPI proteins present in the Western and Affinity blots were 34-36 kDa species, minor 12 and 16, and 85 and 120 kDa species were also present. Qualitatively similar results were obtained for each respective tissue zone of the lumbar and lumbosacral disc specimens examined. Densitometric analysis of the major 34-36 kDa SPI bands visualised on Western and Affinity blots using NIH 1.61.1 image analysis software indicated that lumbar IVD samples contained higher levels of this SPI species than lumbosacral IVD samples. ELISA confirmed that lumbar IVD extracts contained quantitatively higher levels of BPTI equivalents per g of tissue extracted than lumbosacral IVDs. This study therefore has demonstrated that the ovine disc contains a range of SPI species which share some homology with bovine pancreatic trypsin inhibitor and in this respect are similar to SPIs previously demonstrated in canine IVDs.

Electrophoretic, biosensor, and bioactivity analyses of perlecans of different cellular origins.

Three cellular sources of perlecan were examined in this study, namely human umbilical arterial endothelial cells (HUAEC), a transformed human umbilical venous endothelial cell line (C 1 1 STH) and a human colon carcinoma cell line (WiDr). Perlecans were immunopurified from conditioned media of the above cells and the purity of the perlecan preparations was examined by composite agarose polyacrylamide gel electrophoresis (CAPAGE) and semi-dry immunoblotting with monoclonal antibodies directed to either the perlecan core protein (mAb A76) or heparan sulphate (HS) side-chain (mAb10E4). The ability of each perlecan species to bind fibroblast growth factor-l (FGF-1) was examined using a biosensor (BIAcore). The bioactivity of perlecan FGF-1 interactions was also analysed using BaF3 cells transfected with fibroblast growth factor receptors FGFR1b and 1c. CAPAGE demonstrated subtle differences between the perlecans, indicating they had differing charge to mass ratios with C 11 STH perlecan being slightly more mobile in CAPAGE than the HUAEC and WiDr sample. BIAcore biosensor analysis demonstrated distinct differences in the ability of perlecan preparations to bind FGF-1; HUAEC and C 11 STH perlecan showed similar high binding responses as compared to WiDr perlecan, which bound FGF-1 very poorly. Binding of FGF-1 to endothelial perlecans was shown to be HS-dependent. Interestingly, HUAEC perlecan stimulated the growth of FGFR1b and FGFR1c expressing cells in the presence of FGF-1 comparable to heparin, whereas C 11 STH perlecan showed only very limited stimulation of FGFR 1b cells and was incapable of stimulating FGFR1c cells. WiDr perlecan exhibited no stimulation of growth in either cell line. Collectively the data presented herein indicate that. different cell types express perlecans which vary in the growth factor binding capabilities, which may suggest differences in their HS chain substructure. This may represent a subtle mechanism whereby cells can modulate the responsiveness of perlecan to a range of biologically important ligands and thus in a broader context may have important implications for cell signalling.

Regulation of gelatinase-A (MMP-2) production by ovine intervertebral disc nucleus pulposus cells grown in alginate bead culture by Transforming Growth Factor-beta(1)and insulin like growth factor-I.

The aim of this study was to gain information relevant to disc repair processes. Limited degradation of the collagen matrix by matrix metalloproteases (MMPs) may facilitate the loosening of cell-cell and cell-matrix interactions within the injured intervertebral disc (IVD) to favour the penetration of blood vessels and migration of fibroblasts into the defect to promote repair processes. Gelatinase A (MMP-2) has a particularly important role to play in angiogenesis, in the present study we investigated the in vitro regulation of MMP-2 by Transforming Growth Factor-beta 1 (TGF-beta 1) and Insulin-like Growth Factor-1 (beta IGF-I) in cells from the nucleus pulposus (NP) of the ovine IVD. Ovine NP cells were grown in alginate bead cultures in complete medium (10% foetal calf serum) for 7 days, established in serum-free conditions for 24 h, then stimulated with TGF-beta 1 (0.1 or 10 ng/ml) or IGF-I (2 or 50 ng/ml) +/-Concanavalin A (20 microg/ml) for an additional 48 h. Conditioned medium was examined for matrix metalloproteases using gelatin zymography, Tissue Inhibitor of Metalloproteinase 2 (TIMP-2) and Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) were immunolocalised in beads. Pro (72 kDa) and active (59 kDa) MMP-2 were the major gelatinolytic MMPs detected in control cultures, the TGF-beta 1 and IGF-I treatments significantly decreased levels of the active MMP-2, inclusion of Concanavalin A resulted in a complete reversal of this trend with IGF-I, and to a lesser extent with TGF-beta 1. Cell surface levels of TIMP-2 and MT1-MMP were decreased by the TGF-beta 1 treatment while IGF-I only appeared to decrease TIMP-2 expression. The findings of this study provide some insight as to why dense avascular connective tissues such as the intervertebral disc have such a poor healing potential.

E-selectin, but not P-selectin, is required for development of adjuvant-induced arthritis in the rat.

OBJECTIVE: To determine the role of the endothelial cell adhesion molecules E- and P-selectin in the development and severity of adjuvant-induced arthritis (AIA) in the rat. METHODS: Lewis rats were immunized subcutaneously with Mycobacterium butyricum (Mb), and blocking monoclonal antibodies (mAb) to rat E- and P-selectin were administered. Clinical score, radiolabeled (51Cr and 111In) blood polymorphonuclear leukocyte (PMN) and monocyte migration to joints, and histologic features were monitored. RESULTS: When mAb treatment was started on day 5 postimmunization with Mb (preclinical stage), development of AIA was significantly (P < 0.01) inhibited by mAb to E- but not to P-selectin (mean score on day 14 control 10.2, anti-E 2.8, anti-P 9.1). This was associated with markedly decreased migration (by 66-94%) of PMN and monocytes to arthritic joints and diminished cartilage degradation. When treatment was delayed until animals showed signs of arthritis (day 10 postimmunization), only a marginal and variable effect was observed as compared with blockade during the preclinical (day 5) stage. E-selectin blockade on day 5 and day 7 postimmunization resulted in inhibition of antigen-dependent T cell-mediated inflammation, since it decreased T cell migration to sites of dermal-delayed hypersensitivity induced by Mb without affecting migration to concanavalin A or cytokines. The proliferative response of T cells to Mb in vitro was not altered. CONCLUSION: E-selectin plays an important role early in the development of AIA. This adhesion molecule may contribute to the migration of antigen-reactive T cells to peripheral tissues, including the joints where T cells initiate the arthritis.

HuM291(Nuvion), a humanized Fc receptor-nonbinding antibody against CD3, anergizes peripheral blood T cells as partial agonist of the T cell receptor.

BACKGROUND: Humanized Fc receptor (FcR)-nonbinding antibodies against CD3 are promising immunosuppressive agents that may overcome both the neutralizing response to and the cytokine release syndrome seen with conventional monoclonal antibodies against CD3. In addition, evidence from several murine models suggests that these recombinant antibodies may actively induce T cell unresponsiveness by a mechanism other than modulation of the T cell receptor (TCR) or T cell depletion. We hypothesized that FcR-nonbinding antibodies against CD3 could induce T cell unresponsiveness by acting as partial agonist ligands of the TCR and thus, inducing T cell anergy. METHODS: To test this hypothesis, we examined the signaling and functional effects of HuM291 (Nuvion), a FcR-nonbinding humanized antibody against CD3, on primary human T cells. RESULTS: Short exposure of human peripheral blood T lymphocytes to HuM291 caused a partial agonist type of signaling through the TCR characterized by incomplete phosphorylation of TCR zeta, failure to activate ZAP-70 and to phosphorylate LAT but activation of ERK-1/-2 and subsequent up-regulation of CD69 expression. These changes correlated with a dose-dependent induction of anergy in human, primary resting T cells, which was reversed by exogenous interleukin-2. CONCLUSIONS: The tolerogenic properties of FcR-nonbinding monoclonal antibodies against CD3 correlate with its ability to reproduce the biochemical and functional effects of TCR partial agonist ligands. Thus, generation of engineered antibodies against CD3 with low TCR oligomerization potential may provide a clinically applicable partial agonist-based strategy for the prevention of polyclonal T cell responses.