Load-induced proteoglycan orientation in bone tissuein vivo andin vitro

Summary Previous studies of Alcian blue-induced birefringence in adult avian cortical bone showed that a short period of intermittent loading rapidly produces an increased level of orientation of proteoglycans within the bone tissue. In the absence of further loading, this persists for over 24 hours. We have proposed that this phenomenon could provide a means for “capturing” the effects of transient strains, and so provide a persistent, constantly updated strain-related influence on osteocyte populations related to the bones' averaged recent strain history, in effect, a “strain memory” in bone tissue. In our present study, we use the Alcian blue-induced birefringence technique to demonstrate that proteoglycan orientation also occurs after intermittent loading of both cortical and cancellous mammalian bonein vivo andin vitro. We also show that the change in birefringence is proportional to the magnitude of the applied strain, and that the reorientation occurs rapidly, reaching a maximal value after only 50 loading cycles. Examination of electron micrographs of bone tissue after staining with cupromeronic blue allows direct visualization and quantification of the change in proteoglycan orientation produced by loading. This shows that intermittent loading is associated with a realignment of the proteoglycan protein cores, bringing them some 5 degrees closer to the direction of collagen fibrils in the bone matrix.     

Site-related variations in glycosaminoglycan content and swelling properties of bovine flexor tendon

The presence of morphologically distinct tissues within the adult bovine deep flexor tendon presented a model which we examined for correlations between proteoglycan content and tissue swelling properties. The proximal portion which experiences only tensile forces contained low levels of glycosaminoglycan (0.2% glycosaminoglycan hexosamine as percentage of dry weight) that were evenly distributed throughout its length and thickness. Collagen accounted for 80% of tissue mass, and collagen fibers formed parallel arrays running longitudinal to the direction of tensile force. In equilibrium bulk swelling tests, the properties of proximal tissue were uniform throughout the tissue and typical of collagenous tissues in which the response to equilibration in low ionic strength buffers or acid pH is dominated by the collagen network. The anterior aspect of the distal deep flexor tendon articulates with the stiff paratendinous sheath and sesamoid bones and is subjected to compressive and frictional forces in addition to longitudinal tensile forces. Along this anterior surface and extending partially into the tendon is tissue that resembles fibrocartilage with collagen fibers organized as a random network. Greatest glycosaminoglycan hexosamine contents (2-3% of dry weight) were found in the distal surface layer, and large proteoglycans were concentrated at the site receiving direct compressive and frictional loads. Equilibrium bulk swelling tests on distal tissue showed that the articulating surface layer possessed unique material properties. Distal surface layers swelled when counter ions were washed from the tissue, whereas lowered pH had little effect on distal tissue volume. Swelling properties of distal tissue correlated directly with proteoglycan content and were similar to swelling properties of articular cartilage. These results suggest that the articulating layer of distal flexor tendon is adapted for a unique set of mechanical requirements and that an elevated proteoglycan content at the site of compressive and frictional forces contributes to meeting these functional needs.     

Matrix Vesicles Promote Mineralization in a Gelatin Gel

Extracellular matrix vesicles (MVs) are associated with initial calcification in a variety of tissues, but the mechanisms by which they promote mineralization are not certain. In this study, MVs isolated from fourth passage rat growth plate chondrocyte cultures were included within a gelatin gel into which calcium and phosphate ions diffused from opposite ends. In this gel, apatite formation occurs by 3.5 days in the absence of mineralization promoters, allowing measurement of the ability of different factors to ``nucleate' apatite before this time or to assess the effects of molecules which modulate the rate and extent of mineral deposition. Mineral ion accumulation and crystal type are assayed at 5 days. In this study, MV protein content in the central band of a 10% gelatin gel was varied by including 100 μl of a Tris-buffered solution containing 0–300 μg/ml MV protein. There was a concentration-dependent increase in mineral accretion. Whereas 10 μg MV protein in the gel did not significantly promote apatite formation as compared with vesicle-free gels, 20 and 30 μg MV protein in the gel did promote apatite deposition. Inclusion of 10 mM β-glycerophosphate in the gels, along with MVs, did not significantly increase apatite formation despite the demonstrable alkaline phosphatase activity of the MVs. In contrast, MVs at all concentrations significantly increased apatite accumulation when proteoglycan aggregates or ATP, inhibitors of apatite formation and proliferation, were included in the gel. Slight increases in calcium, but not phosphate accumulation, were also noted when an ionophore was included with the MVs to facilitate Ca ion transport into the vesicles. FT-IR analysis of the mineral formed in the vesicle-containing gels revealed the presence of a bone-like apatite. These data suggest that MVs facilitate mineralization by providing enzymes that modify inhibitory factors in the extracellular matrix, as well as by providing a protected environment in which mineral ions can accumulate. Received: 28 January 1996 / Accepted: 9 August 1996     

Effect of ultrafilterable fragments from chondroitinase and protease-treated aggrecan on calcium phosphate precipitation in liposomal suspensions

A liposome-centered endogenous precipitation method was used to investigate the effect of ultrafilterable fragments from the enzymatic digestion of rat chondrosarcoma aggrecan on the formation of insoluble calcium phosphate salts in buffered solutions at pH 7.4 and 22°C. Unlike the intact aggrecan and its major chondroitin sulfate and core protein components, disaccharide units from chondroitinase degradation of the aggrecan and small (<3kg/mol molecular weight) fragments from protease digestion of the core structure were found to be only weakly inhibitory toward mineral formation. Corresponding reductions in Ca²⁺-binding indicate that these fragments were unable to adsorb to active sites on the apatite surface for long enough periods to significantly hinder crystal growth. The data suggest that controlled enzymatic breakdown of aggrecan may be one possible mechanism by which the calcification of growth plate cartilage is allowed to advance in vivo.The commercial materials and equipment identified in this paper do not imply recommendation or endorsement by the National Institute of Standards and Technology nor is the material and equipment necessarily the best available for the purpose.     

Proteoglycans in the Control of Tumor Growth and Metastasis Formation

Proteoglycans (PGs) as a whole, or when considering their GAG chains as single entities, are emerging as key regulators of tumor progression. Expectations on using them as putative prognostic markers and potential therapeutic targets are increasing coincidentally. Due to the multitude of biological roles that they may invest and the ample spectrum of cellular processes that they may control, we still need to learn better how they regulate phenomena such as intracellular signaling, proliferation, apoptosis, motility, and drug resistance. Depending on the type, their expression pattern, and the accessibility of their molecular ligands, PGs can either promote or inhibit tumorigenesis. The structural and functional diversity of PGs coupled with their ubiquitous abundance place them at the crossroads of many critical steps within the metastatic cascade. As this phenomenon is the pivotal factor for patient survivals, particular attention should be given to the understanding of how PGs govern metastasis formation.     

硫酸软骨素蛋白多糖与中枢神经系统可塑性

硫酸软骨素蛋白多糖(chondroitin sulfate proteoglycan,CSPG)是在发育和成熟的中枢神经系统(central nervous syste,CNS)中广泛表达的一组细胞外基质分子,在胚胎CNS发育和成年期CNS可塑性中发挥着重要作用.CSPG作为一种主要的细胞外抑制性成分,可影响CNS损伤后轴突再生和神经功能恢复.     

The localisation of inflammatory cells and expression of associated proteoglycans in response to implanted chitosan

Implantation of a foreign material almost certainly results in the formation of a fibrous capsule around the implant however, mechanistic events leading to its formation are largely unexplored. Mast cells are an inflammatory cell type known to play a role in the response to material implants, through the release of pro-inflammatory proteases and cytokines from their α-granules following activation. This study examined the in vivo and in vitro response of mast cells to chitosan, through detection of markers known to be produced by mast cells or involved with the inflammatory response. Mast cells, identified as Leder stained positive cells, were shown to be present in response to material implants. Additionally, the mast cell receptor, c-kit, along with collagen, serglycin, perlecan and chondroitin sulphate were detected within the fibrous capsules, where distribution varied between material implants. In conjunction, rat mast cells (RBL-2H3) were shown to be activated following exposure to chitosan as indicated by the release of β-hexosaminidase. Proteoglycan and glycosaminoglycans produced by the cells showed similar expression and localisation when in contact with chitosan to when chemically activated. These data support the role that mast cells play in the inflammatory host response to chitosan implants, where mediators released from their α-granules impact on the formation of a fibrous capsule by supporting the production and organisation of collagen fibres.     

Biglycan protects cardiomyocytes against hypoxia/reoxygenation injury: Role of nitric oxide

Biglycan, a proteoglycan component of extracellular matrix, has been suspected to contribute to the development of atherosclerosis, but overexpression of biglycan in transgenic mice has been shown to induce cardioprotective genes including nitric oxide (NO) synthases in the heart. Therefore, here we hypothesized if exogenous administration of biglycan exerts cytoprotection. Primary cardiomyocytes from neonatal rats were subjected to 150 min hypoxia and 2 h reoxygenation. Mortality of cardiomyocytes was dose-dependently attenuated by pretreatment with 1-100 nM biglycan. Biglycan enhanced eNOS mRNA and protein, and significantly increased NO content of cardiomyocytes. The NO synthase inhibitor l-nitro-arginine-methyl-ester significantly attenuated the cytoprotective effect of biglycan. This is the first demonstration that biglycan leads to cytoprotection against hypoxia/reoxygenation injury, and that this phenomenon is partially mediated by an NO-dependent mechanism.     

Acute transplantation of glial-restricted precursor cells into spinal cord contusion injuries: survival, differentiation, and effects on lesion environment and axonal regeneration

Transplantation of stem cells and immature cells has been reported to ameliorate tissue damage, induce axonal regeneration, and improve locomotion following spinal cord injury. However, unless these cells are pushed down a neuronal lineage, the majority of cells become glia, suggesting that the alterations observed may be potentially glially mediated. Transplantation of glial-restricted precursor (GRP) cells--a precursor cell population restricted to oligodendrocyte and astrocyte lineages--offers a novel way to examine the effects of glial cells on injury processes and repair. This study examines the survival and differentiation of GRP cells, and their ability to modulate the development of the lesion when transplanted immediately after a moderate contusion injury of the rat spinal cord. GRP cells isolated from a transgenic rat that ubiquitously expresses heat-stable human placental alkaline phosphatase (PLAP) were used to unambiguously detect transplanted GRP cells. Following transplantation, some GRP cells differentiated into oligodendrocytes and astrocytes, retaining their differentiation potential after injury. Transplanted GRP cells altered the lesion environment, reducing astrocytic scarring and the expression of inhibitory proteoglycans. Transplanted GRP cells did not induce long-distance regeneration from corticospinal tract (CST) and raphe-spinal axons when compared to control animals. However, GRP cell transplants did alter the morphology of CST axons toward that of growth cones, and CST fibers were found within GRP cell transplants, suggesting that GRP cells may be able to support axonal growth in vivo after injury.     

The extracellular matrix of cartilage in the growth plate before and during calcification: Changes in composition and degradation of type II collagen

Summary Calcification occurs in the extracellular matrix of the hypertrophic zone of the growth plate when the extra-cellular matrix volume is reduced to a minimum and alkaline phosphatase content is maximal. The present study shows that significant quantitative and qualitative changes occur in the composition and structure of macromolecules in the extracellular matrix before and during calcification in the proximal tibial growth plate of the bovine fetus. These were detected in part by using microchemical and microimmuno-chemical analyses of sequential transverse frozen sections at chemical analyses of sequential transverse frozen sections at defined sites throughout the growth plate. Concentrations of matrix molecules in the extracellular matrix have not previously been determined biochemically. They were measured per unit matrix volume by using combined immunochemical/chemical-histomorphometric analyses. The concentrations within the extracellular matrix of the C-propeptide of type II collagen, aggregating proteoglycan (aggrecan), and hyaluronic acid all progressively increased in the maturing and hypertrophic zones, being maximal (or near maximal) at the time of initiation of mineralization. These results for proteoglycan are contrary to some earlier reports of a loss of proteoglycan prior to mineralization which measured the tissue content of proteoglycan rather than that present in the extracellular matrix, the volume of which is progressively reduced as the growth plate matures. The C-propeptide data provides a quantitative confirmation of previous immunohistochemical studies. Total collagen concentration (measured as hydroxyproline) in the extracellular matrix initially increased through the proliferating and maturing zones but then rapidly decreased in the hypertrophic zone. Immunohistochemical studies revealed that this is associated with the unwinding of the triple helix of type II collagen (previously shown to result from cleavage) which starts in pericellular sites in the zone of maturation (when type X collagen is first synthesized) and then extends throughout the hypertrophic zone. The significance of these matrix changes in the development and mineralization of the growth plate is discussed.