Does the thickness of the vertebral subchondral bone reflect the composition of the intervertebral disc?

Degeneration of the intervertebral disc, seen radiologically as loss of disc height, is often associated with apparent remodelling in the adjacent vertebral body. In contrast, maintenance or apparent increase in disc height is a common finding in osteoporosis, suggesting the properties of the intervertebral disc may be dependent on those of the vertebral body or vice versa. We have investigated this relationship by measuring the radiological thickness of the subchondral bone and comparing it to the chemical composition of the adjacent disc. Sagittal slabs were sampled from lumbar spines obtained at autopsy and X-rayed microfocally. The thickness of the subchondral bone was measured and correlated with the composition of the adjacent intervertebral disc. Eighty-three cadaveric endplates were studied from individuals aged 17–85 years. There was regional variation in thickness of the subchondral bone, being greater adjacent to the annulus than the nucleus, and the endplates cranial to the disc were thicker than those caudal. There was a positive correlation between the thickness of the subchondral bone and the proteoglycan content of the adjacent disc, particularly in the region of the nucleus. A weaker correlation was seen here between water content and thickness, whilst there was no significant correlation at the annulus or between the bone thickness and collagen content. The positive relationship between the radiographic thickness of vertebral subchondral bone and the proteoglycan content of the adjacent disc seen in human cadaveric material could be due to the bone responding to a greater hydrostatic pressure being exerted by discs with higher proteoglycan content than by those with less proteoglycan present. It is suggested that while this is true in normal specimens, the relationship becomes altered in disease states, possibly because of changes to the nutritional pathway of the disc, with resultant endplate-bone remodelling affecting the flow of solutes to and from the intervertebral disc.     

Proteoglycans are potent modulators of the biological responses of eosinophils to chemokines

Chemokines play critical roles in governing the recruitment and activation of eosinophils at sites of allergic inflammation, particularly the asthmatic lung. However, we know little of how chemokine function is regulated post-translationally. Proteoglycans, consisting of a core protein and glycosaminoglycan (GAG) side chains, are cell surface molecules and components of the extracellular matrix that are able to bind chemokines, whilst heparin is a GAG with therapeutic value in asthma. We examined whether soluble GAG could alter the actions of chemokines in assays of eosinophil activation. Heparin inhibited intracellular calcium flux, respiratory burst and chemotactic responses of eosinophils to CCL11, but not to the chemoattractant C5a, and inhibited binding of CCL11 to CCR3. Heparin also inhibited eosinophil stimulation by CCL11, CCL24, CCL7, CCL13 and CCL5 to differing degrees, which broadly correlated with their relative affinities for heparin. Heparan sulfate and dermatan sulfate, but not chondroitin sulfate, also inhibited the actions of CCL11 and CCL13 in assays of cellular shape change and chemotaxis. Following treatment with the sulfation inhibitor chlorate or proteoglycanases, no inhibition of CCL11-induced activity was observed using either eosinophils or a CCR3-expressing transfectant cell line. This suggests that cell surface proteoglycans are not necessary for signaling via CCR3. However, the GAG context in which chemokines are expressed is likely to represent an important level of regulation of allergic inflammation.     

Site-Specific Molecular Diffusion in Articular Cartilage Measured using Fluorescence Recovery after Photobleaching

Diffusive transport of solutes is critical to the normal function of articular cartilage. The diffusion of macromolecules through cartilage may be affected by the local composition and structure, which vary with depth from the tissue surface. We hypothesized that the diffusion coefficient of uncharged molecules also varies with depth and molecular size. We used fluorescence recovery after photobleaching (FRAP) to measure site-specific diffusion coefficients of fluorescent dextran molecules (3, 40, 70, and 500 kDa) in porcine articular cartilage. The diffusion coefficients measured using FRAP exhibited an inverse size dependence and were in general agreement with those measured using other techniques. The diffusion coefficients for all molecules varied significantly with depth in a manner that depended upon the size of the diffusing molecule. The diffusion coefficients for the 3 and 500 kDa dextrans were 1.6 and 2.4 times greater, respectively, in the surface zone as compared to the middle and deep zones, whereas the diffusion coefficients of the 40 and 70 kDa dextrans were 0.3 and 0.2 times lower in the surface zone as compared to the middle and deep zones. These differences may reflect variations in the structure and composition of collagen, proteoglycans, and other macromolecules among the zones.© 2003 Biomedical Engineering Society. PAC2003: 8715Vv, 8719Rr, 8764Tt     

Relationship Between Collagen Fibrils, Glycosaminoglycans, and Stress Relaxation in Mitral Valve Chordae Tendineae

The tensile properties of mitral valve chordae tendineae derive from their structural make-up. The objectives of this study were to compare the stress relaxation properties of different types of chordae and relate their variation to structural features. Fifty chordae from eight hearts were subjected to stress relaxation tests. The percent stress relaxation and the relaxation rates were found to increase in the order of marginal. basal, and strut chordae. The water content of the three types of chordae was the same (marginal 77.1+/-5.9%, basal 77.0+/-3.4%, strut 78.0+/-2.3% wet weight). The collagen, elastin, and glycosaminoglycan (GAG) content in chordae were quantified using hydroxyproline assay, fastin elastin assay, and fluorophore-assisted carbohydrate electrophoresis, respectively. Collagen content of marginal chordae was only slightly less than that of basal and strut chordae (marginal 56.6+/-8.2%, basal 61.4+/-5.6%, strut 63.8+/-3.9% dry weight). There was also no significant difference in elastin content between the chordae (marginal 5.3+/-3.2%, basal 5.4+/-2.7%, strut 4.6+/-1.7% dry weight). However, the concentrations of unsulfated chondroitin/dermatan sulfate, 6-sulfated chondroitin sulfate, and 4-sulfate chondroitin sulfate significantly decreased in the order of marginal, basal, and strut. The total GAG-content also decreased in the order of marginal, basal, and strut (p = 0.06). The greater amount of GAGs in marginal versus strut chordae is consistent with our previous observations that marginal chordae have a greater collagen fibril density and thus more GAG-mediated, fibril-to-fibril linkages. The greater number of proteoglycan linkages may prevent the slippage of fibrils with respect to each other, and thus reduce stress relaxation. The different viscoelastic properties of mitral valve chordae can thus be explained morphologically.     

Comparison of biochemical characteristics of cultured fibrochondrocytes isolated from the inner and outer regions of human meniscus

In order to evaluate the ability of fibrochondrocytes to synthesize collagen and proteoglycan, human medial meniscal cells were cultured in a monolayer. Meniscal cells were prepared from the two regions (outer 1/3 and inner 2/3) in consideration of the difference in vascular supply, and articular chondrocytes were also obtained from the same knee joint. Regarding total collagen synthesis, regional differences were not found, but age-related differences were found in human medial meniscus. In contrast, proteoglycan synthesis revealed significant regional differences; meniscal cells from the inner 2/3 synthesized a greater amount of proteoglycan. After long-term monolayer culturing, proteoglycan synthesis by meniscal cells decreased in a time-dependent manner, and morphological changes to fibroblast-like cells were found. In the presence of transforming growth factor (TGF)-β, proteoglycan synthesis increased in a dose-dependent manner. These findings suggest that the inner regions of the human meniscus contain cells with a chondrocytic phenotype. Received: 11 February 1998 Accepted: 14 September 1998     

Scanning Force Microscopy of the Fine Structure of Cartilage Irradiated with a CO2 Laser

. Alterations of the cartilage matrix structure under non-destructive laser irradiation have been investigated by scanning force microscopy. Porcine nasal septum cartilage was irradiated with a CO₂ laser with a power density of 50 W/cm² under two different time regimes: for 3 s and for 30 s. Short-time irradiation had little effect on the structure of the cartilage matrix. In comparison with non-irradiated cartilage, small channels of 100–400 nm in cross-section appeared. This observation gives evidence that the underlying mechanism of laser-induced stress relaxation of cartilage is based on short-time depolymerisation and subsequent re-formation of proteoglycan units. The 30 s laser treatment results in melting and denaturation of the matrix. For the first time, small crystals, 100–800 nm, were found on cut sections of the laser treated cartilage. The crystals mainly consist of resolvable sodium carbonate. Thus, they cannot be responsible for the formation of a stable cartilage configuration after laser treatment. Paper received 24 February 1998; accepted after revision 22 June 1999.     

Serum decorin measurement in prediction of the risk for preterm birth

Objective

To define serum decorin (sDEC) levels in healthy pregnants and in patients with preterm labor (PTL), and to introduce possible role of sDEC in predicting the risk for preterm birth (PTB).

Assessment of the stromal contribution to Sonic Hedgehog-dependent pancreatic adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. It is typically detected at an advanced stage, at which the therapeutic options are very limited. One remarkable feature of PDAC that contributes to its resilience to treatment is the extreme stromal activation seen in these tumors. Often, the vast majority of tumor bulk consists of non-tumor cells that together provide a tumor-promoting environment. One of the signals that maintains and activates the stroma is the developmental protein Sonic Hedgehog (SHH). As the disease progresses, tumor cells produce increasing amounts of SHH, which activates the surrounding stroma to aid in tumor progression. To better understand this response and identify targets for inhibition, we aimed to elucidate the proteins that mediate the SHH-driven stromal response in PDAC. For this a novel mixed-species coculture model was set up in which the cancer cells are human, and the stroma is modeled by mouse fibroblasts. In conjunction with next-generation sequencing we were able to use the sequence difference between these species to genetically distinguish between the epithelial and stromal responses to SHH. The stromal SHH-dependent genes from this analysis were validated and their relevance for human disease was subsequently determined in two independent patient cohorts. In non-microdissected tissue from PDAC patients, in which a large amount of stroma is present, the targets were confirmed to associate with tumor stroma versus normal pancreatic tissue. Patient survival analysis and immunohistochemistry identified CDA, EDIL3, ITGB4, PLAUR and SPOCK1 as SHH-dependent stromal factors that are associated with poor prognosis in PDAC patients. Summarizing, the presented data provide insight into the role of the activated stroma in PDAC, and how SHH acts to mediate this response. In addition, the study has yielded several candidates that are interesting therapeutic targets for a disease for which treatment options are still inadequate.     

Determination of β4-galactosyltransferase-7 activity using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry

Objectives

Patients with Ehlers-Danlos syndrome were described to contain reduced activities of β4-galactosyltransferase-7 (β4Gal-T7). Therefore, measurement of β4Gal-T7 activity can help to characterize defects in proteoglycan biosynthesis in patients with connective tissue diseases.

Design and methods

We developed a sensitive and specific method to assay β4Gal-T7 which is based on the transfer of galactose from UDP-galactose to the synthetic peptide Bio-BIK-F-Xyl.

Results

Calibration curves exhibited consistent linearity in the range of 10-2000 μg/L Bio-BIK-F-Xyl-Gal, corresponding to a β4Gal-T7 activity of 3.5-659 μU/L. The limit of detection and the lower limit of quantification were 3.70 μg/L (1.22 μU/L) and 4.50 μg/L Bio-BIK-F-Xyl-Gal (1.48 μU/L β4Gal-T7 activity), respectively. Interassay imprecision (CV) was 8.1-13.1% in the range from 15.9 to 659 μU/L, and mean recovery was 85.3% (range 61.7-106.3%).

Conclusions

This sensitive, robust and interference-free LC-MS/MS assay allows an accurate determination of β4Gal-T7 activity in human body fluids.     

大鼠肠系膜淋巴结高内皮微静脉内蛋白多糖的分布

目的　研究蛋白多糖在大鼠肠系膜淋巴结高内皮微静脉(HEVs)中的分布,探讨蛋白多糖在淋 巴细胞归巢过程中的调节作用。方法　阳性胶体铁染色—酶连续阻断法,光镜和电镜观察蛋白多糖于HEVs 内的淋巴细胞、内皮细胞、基膜上的分布。结果　HEVs的基膜和邻接基膜的淋巴细胞胞膜呈强阳性染色,能 被透明质酸酶、肝素酶、软骨素酶ABC阻断;电镜显示,胶体颗粒主要排列于基膜的内、外侧及穿越基膜的淋 巴细胞胞膜上,内皮细胞、穿内皮细胞的淋巴细胞和腔内的淋巴细胞不着色。结论　蛋白多糖于大鼠肠系膜 淋巴结HEVs内主要分布于基膜和穿基膜的淋巴细胞胞膜上,可能对归巢淋巴细胞穿越HEVs管壁有调节作 用。