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1.
A biotinylated complex of aggrecan G1-domain and link protein was used to characterize the distribution of hyaluronan in paraffin-embedded sections of adult human and canine intervertebral disc and cartilage endplate. Limited chondroitinase ABC and trypsin digestions of the sections before staining was utilized to expose hyaluronan potentially masked by aggrecan. Hyaluronan concentration and hyaluronan to uronic acid ratio in different parts of the discs were measured as a background for the histological analysis.Hyaluronan staining was strong in the nucleus pulposus and inner parts of annulus fibrosus of both species, corroborated by biochemical assays of the same compartments. Particularly in human samples, hyaluronan in the interterritorial matrix of nucleus pulposus and annulus fibrosus was readily accessible to the probe without enzyme treatments. In contrast, the cell-associated hyaluronan signal was enhanced after trypsin or limited chondroitinase ABC-treatment of the sections, suggesting that pericellular hyaluronan was more masked by aggrecan than in the distant matrix. A puzzling feature of canine cartilage endplate cells was their intensive cell-associated hyaluronan signal, part of which appeared intracellular. Hyaluronan was abundant between the collagenous lamellae in annulus fibrosus, perhaps important in the plasticity of this tissue. 相似文献
2.
The high-buoyant-density proteoglycans of the nucleus pulposus and annulus fibrosus of the beagle intervertebral disc have been isolated by CsCl density gradient ultracentrifugation. The sulphated proteoglycans were labelled in vivo with 35SO4, 24 h and 60 days prior to killing. The hydrodynamic size and aggregation of the 24 h, 60 day and resident (from hexuronic acid and hexosamine analysis) proteoglycan subunit populations were determined by Sepharose CL-2B chromatography in the presence or absence of excess hyaluronic acid. The hydrodynamic size of the keratan sulphate-proteoglycan core protein complexes were also determined by Sepharose CL-2B chromatography after chondroitinase ABC digestion of proteoglycans. When initially synthesised (24 h) or after 60 days, the percentage aggregation and hydrodynamic size of the proteoglycans derived from the annulus fibrosus were larger than those present in the nucleus pulposus. Hexosamine, hexuronic and protein determination of the high-buoyant-density fractions showed that the proteoglycans of the nucleus pulposus were richer in chondroitin sulphate than those in the annulus. However there was no difference in Mr of the chondroitin sulphate and keratan sulphate attached to the proteoglycans of the two disc regions, nor were differences detected by HPLC between the proportions of chondroitin 4-sulphate and chondroitin 6-sulphate present in these high-density fractions. In contrast, the low-buoyant-density (1.54 greater than p greater than 1.45) proteoglycan fractions and tissue residues remaining after 4 M GuHCl extraction were found to contain dermatan sulphate, suggesting the presence of a third proteoglycan species possibly associated with the collagen of the fibrocartilagenous matrix. 相似文献
3.
Intervertebral disc degeneration is the main cause of low back pain. In the past 20 years, the injection of mesenchymal stromal cells (MSCs) into the nucleus pulposus of the degenerative disc has become the main approach for the treatment of low back pain. Despite the progress made in this field, there are still many barriers to overcome. First, intervertebral disc is a highly complex load-bearing composite tissue composed of annulus fibrosus, nucleus pulposus and cartilaginous endplates. Any structural damage will change its overall biomechanical function, thereby causing progressive degeneration of the entire intervertebral disc. Therefore, MSC-based treatment strategies should not only target the degenerated nucleus pulposus but also include degenerated annulus fibrosus or cartilaginous endplates. Second, to date, there has been relatively little research on the basic biology of annulus fibrosus and cartilaginous endplates, although their pathological changes such as annular tears or fissures, Modic changes, or Schmorl's nodes are more commonly associated with low back pain. Given the high complexity of the structure and composition of the annulus fibrosus and cartilaginous endplates, it remains an open question whether any regeneration techniques are available to achieve their restorative regeneration. Finally, due to the harsh microenvironment of the degenerated intervertebral disc, the delivered MSCs die quickly. Taken together, current MSC-based regenerative medicine therapies to regenerate the entire disc complex by targeting the degenerated nucleus pulposus alone are unlikely to be successful. 相似文献
4.
The angled, lamellar structure of the annulus fibrosus is integral to its load-bearing function. Reorientation of this fiber structure with applied load may contribute to nonlinear mechanical behavior and to large increases in tensile modulus. Fiber reorientation has not yet been quantified for loaded non-degenerated and degenerated annulus fibrosus tissue. The objective of this study was to measure fiber reorientation and mechanical properties (toe- and linear-region modulus, transition strain, and Poisson's ratio) of loaded outer annulus fibrosus tissue using a new application of FFT image processing techniques. This method was validated for quantification of annulus fiber reorientation during loading in this study. We hypothesized that annulus fibrosus fibers would reorient under circumferential tensile load, and that fiber reorientation would be affine. Additionally, we hypothesized that degeneration would affect fiber reorientation, toe-region modulus and Poisson's ratio. Annulus fibrosus fibers were found to reorient toward the loading direction, and degeneration significantly decreased fiber reorientation (the fiber reorientation parameter, m(FFT)=-1.70 degrees /% strain for non-degenerated and -0.95 degrees /% strain for degenerated tissue). Toe-region modulus was significantly correlated with age (r=0.6). Paired t-tests showed no significant difference in the fiber reorientation parameter calculated experimentally with that calculated using an affine prediction. Thus, an affine prediction is a good approximation of fiber reorientation. The findings of this study add to the understanding of overall disc mechanical behavior and degeneration. 相似文献
5.
The main function of the intervertebral disc is biomechanical function, since it must resist repetitive high loadings, while giving the spine its flexibility and protecting the spinal cord from over-straining. It partially owes its mechanical characteristics to the lamellar architecture of its outer layer, the annulus fibrosus. Today, no non-invasive means exist to characterize annulus lamellar structure in vivo. The aim of this work was to test the feasibility of imaging annulus fibrosus microstructure in vivo with ultrasonography. Twenty-nine healthy adolescents were included. Ultrasonographies of L3–L4 disc were acquired with a frontal approach. Annulus fibrosus was segmented in the images to measure the thickness of the lamellae. To validate lamellar appearance in ultrasonographies, multimodality images of two cow tail discs were compared: ultrasonography, magnetic resonance and optical microscopy. In vivo average lamellar thickness was 229.7 ± 91.5 μm, and it correlated with patient body mass index and age. Lamellar appearance in the three imaging modalities in vitro was consistent. Lamellar measurement uncertainty was 7%, with good agreement between two operators. Feasibility of ultrasonography for the analysis of lumbar annulus fibrosus structure was confirmed. Further work should aim at validating measurement reliability, and to assess the relevance of the method to characterize annulus alterations, for instance in disc degeneration or scoliosis. 相似文献
6.
A group of young (2.0 +/- 0.6 years) (group 1) and old (9.7 +/- 1.5 years) (group 2) beagle dogs were given Na2 35SO4 (1.0 mCi/kg) intravenously 60 days prior to being killed to radiolabel their proteoglycans. Lumbar discs were removed and dissected into nucleus pulposus and annulus fibrosus. Proteoglycans were extracted at 4 degrees C from these tissues with buffered 4.0 M Gdn-HCl containing proteinase inhibitors, and purified by CsCl density gradient ultracentrifugation. The average hydrodynamic size and ability of the purified proteoglycans to aggregate in the presence of excess hyaluronic acid was determined by Sepharose CL-2B chromatography. The galactosamine/glucosamine ratios of these proteoglycans as well as their non-aggregating fractions were also ascertained. The proteoglycan content of discs of old animals was significantly less than in the young. The proportion of 35S-labelled, or non-labelled proteoglycans which could aggregate in the presence of hyaluronic acid was also much lower in the preparations isolated from the older discs. In contrast, the average hydrodynamic size of the non-aggregating proteoglycans isolated from the annuli fibrosi of group 2 animals were larger than the corresponding population of group 1 animals. Aminosugar analysis of these same proteoglycan fractions from older animals afforded galactosamine/glucosamine ratios (mean 1.81 +/- 0.14) which were less than the younger age group (mean 2.63 +/- 0.40). These data suggest that with ageing and degeneration the proteoglycans of the beagle disc undergo increased degradation with the accumulation in the annulus fibrosus of a population which is of larger average hydrodynamic size and richer in keratan sulphate than proteoglycans present in younger tissues. 相似文献
7.
We have used the racemization of aspartic acid as a marker for the "molecular age" of aggrecan components of the human intervertebral disc matrix (aggregating and non-aggregating proteoglycans as well as the different buoyant density fractions of aggrecan). By measuring the D/L(Asp) ratio of the various aggrecan species as a function of age and using the values of the racemization constant, k(i), found earlier for aggrecan in articular cartilage, we were able to establish directly the relative residence time of these molecules in human intervertebral disc matrix. For A1 preparations taken from normal tissue, turnover rates of 0.059 +/- 0.01 and 0.063 +/- 0.01/year correspond to half-life values of 12 +/- 2.0 and 11.23 +/- 1.9 years for nucleus pulposus and annulus fibrosus, respectively; the turnover rates of 0.084 +/- 0.022 and 0.092 +/- 0.034/year for degenerate tissue correspond to half-life values of 8.77 +/- 2.2 and 8.41 +/- 2.8 years, suggesting increased rate of removal of small aggrecan fragments. For the large monomer, fraction A1D1, turnover is 0.13 +/- 0.04/year, corresponding to a half-life of 5.56 +/- 1.58 years, similar to 3.4 years in human articular cartilage. For the binding region (A1D6), turnover is 0.033 +/- 0.0012/year, corresponding to a half-life of 21.53 +/- 0.6 years, similar to 23.5 years in articular cartilage. A1 preparations from nucleus pulposus contain a lower proportion of aggregating proteoglycans as compared with annulus fibrosus, suggesting increased proteolytic modification in the nucleus pulposus. D/L(Asp) values in aggregating and non-aggregating proteoglycans of a 24-year-old individual show similar results, suggesting that the non-aggregating molecules are synthesized initially as aggregating proteoglycans, which thereafter undergo cleavage and detachment from hyaluronan. 相似文献
8.
We evaluated the immunohistochemical distribution of three major proteoglycans of cartilage, i.e., aggrecan, versican and
perlecan vis-a-vis collagens I and II in the developing human spine of first-trimester foetuses. Aggrecan and perlecan were
prominently immunolocalised in the cartilaginous vertebral body rudiments and to a lesser extent within the foetal intervertebral
disc. In contrast, versican was only expressed in the developing intervertebral disc interspace. Using domain-specific monoclonal
antibodies against the various modules of versican, we discovered the V0 isoform as the predominant form present. Versican
immunolocalisations conducted with antibodies directed to epitopes in its N and C termini and GAG-α and GAG-β core protein
domains provided evidence that versican in the nucleus pulposus was either synthesised devoid of a G3 domain or this domain
was proteolytically removed in situ. The V0 versican isoform was localised with prominent fibrillar components in the annular
lamellae of the outer annulus fibrosus. Perlecan was a notable pericellular proteoglycan in the annulus fibrosus and nucleus
pulposus but poorly immunolocalised in the marginal tissues of the developing intervertebral disc, apparently delineating
the intervertebral disc–vertebral body interface region destined to become the cartilaginous endplate in the mature intervertebral
disc. The distribution of collagens I and II in the foetal spine was mutually exclusive with type I present in the outer annulus
fibrosus, marginal tissues around the vertebral body rudiment and throughout the developing intervertebral disc, and type
II prominent in the vertebral rudiment, absent in the outer annulus fibrosus and diffusely distributed in the inner annulus
fibrosus and nucleus pulposus. Collectively, our findings suggest the existence of an intricate and finely balanced interplay
between various proteoglycans and collagens and the spinal cell populations which synthesise and assemble these components
during spinal development. 相似文献
9.
Potassium channels play a major role in intracellular homeostasis and regulation of cell volume. Intervertebral disc cells respond to mechanical loading in a complex manner. Mechanical loading may play a role in disc degeneration. Lumbar intervertebral disc samples from 5 patients (average age: 47 years, range: 25-64 years) were used for this study, investigating cells from the nucleus pulposus and the annulus fibrosus duplicate samples to determine RNA expression and protein expression. Analysis of mRNA expression by RT-PCR demonstrated that TREK 1 was expressed by nucleus pulposus (n=5) and annulus fibrosus (n=5) cells. Currently, TREK-1 is the only potassium channel known to be activated by intracellular acidosis, and responds to mechanical and chemical stimuli. Whilst the precise role of potassium channels in cellular homeostasis remains to be determined, TREK-1 may be important to protect disc cells against ischaemic damage, and subsequent disc degeneration, and may also play a role in effecting mechanotransduction. Further research is required to fully elucidate the role of the TREK-1 ion channel in intervertebral disc cells. 相似文献
10.
An axisymmetric finite element model of a body-disc-body unit has been developed and used to study the relative effects of two distinct direction-dependent material representations of the disc annulus on the predicted state of stresses in the disc. The annulus fibrosus is modelled either as nonhomogeneous fibre reinforced composite or alternatively as homogeneous orthotropic with transverse isotropy. In order to have identical states of displacements and hence strains, the unknown properties of the latter model are chosen to be equivalent with those of the former. The fibre slopes of 20 degrees, 30 degrees, and 40 degrees are considered in this study. The stresses in the annulus matrix in the circumferential planes parallel to the fibre layers are predicted to be significantly different depending on the annulus model used. In the nonhomogeneous model, the fibre membranes while under tensile forces, in turn, apply compression to the annulus matrix and, hence, decrease the annulus normal stresses in the above planes. Had the membranes carried compressive forces, a reverse trend would have resulted. The foregoing relative differences are dependent on the fibre orientation, and the magnitude of the tensile forces carried by the fibre layers. The latter also depends, amongst others, on the orientation of the fibres, decreasing as the fibre slope increases from 20 degrees to 30 degrees and 40 degrees. On the basis of the annulus micro-structure and the relative mechanical functions of its components, namely the annulus bulk and the collagenous fibre layers, it appears that nonhomogeneous fibre reinforced composite model of the disc annulus is more realistic resulting in a more accurate computation of stresses in the annulus fibrosus. 相似文献
11.
Chemokines are an important group of soluble molecules with specialized functions in inflammation. The roles of many specialized chemokines and their receptors remain poorly understood in the human intervertebral disc. We investigated CXCL16 and its receptor, CXCR6, to determine their immunolocalization in disc tissue and their presence following exposure of cultured human annulus fibrosus cells to proinflammatory cytokines. CXCL16 is a marker for inflammation; it also can induce hypoxia-inducible factor 1α (HIF-1α), which is a phenotypic marker of heathy nucleus pulposus tissue. We found CXCL16 and CXCR6 immunostaining in many cells of the annulus portion of the disc. Molecular studies showed that annulus fibrosus cells exposed to IL-1ß, but not TNF-α, exhibited significant up-regulation of CXCL16 expression vs. control cells. There was no significant difference in the percentage of annulus cells that exhibited immunolocalization of CXCL16 in grade I/II, grade III or grade IV/V specimens. The presence of CXCL16 and its receptor, CXCR6, in the annulus in vivo suggests the need for future research concerning the role of this chemokine in proinflammatory functions, HIF-1α expression and disc vascularization. 相似文献
12.
Assays of several proteases, incorporating guanidinium chloride extracts of human femoral head cartilage and intervertebral disc, demonstrated that both tissues contain inhibitors of certain serine proteases. Trypsin, chymotrypsin and a granule extract of human polymorphonuclear leukocytes containing elastase and cathepsin G activities, were inhibited by low molecular weight fractions prepared by Sephadex G-75 chromatography. Using a radioassay, it was further shown that these fractions inhibit proteolysis of cartilage proteoglycan. The inhibitor in intervertebral disc is concentrated in the nucleus pulposus, with a decreasing gradient to the periphery of the annulus fibrosus. It is proposed that these inhibitors confer at least partial protection against pathological proteolysis of the proteoglycans in human articular cartilage and nucleus pulposus. 相似文献
14.
Intervertebral disc degeneration results in disorganization of the laminate structure of the annulus that may arise from mechanical microfailure. Failure mechanisms in the annulus were investigated using composite lamination theory and other analyses to calculate stresses in annulus layers, interlaminar shear stress, and the region of stress concentration around a fiber break. Scanning electron microscopy (SEM) was used to evaluate failure patterns in the annulus and evaluate novel structural features of the disc tissue. Stress concentrations in the annulus due to an isolated fiber break were localized to approximately 5 microm away from the break, and only considered a likely cause of annulus fibrosus failure (i.e., radial tears in the annulus) under extreme loading conditions or when collagen damage occurs over a relatively large region. Interlaminar shear stresses were calculated to be relatively large, to increase with layer thickness (as reported with degeneration), and were considered to be associated with propagation of circumferential tears in the annulus. SEM analysis of intervertebral disc annulus fibrosus tissue demonstrated a clear laminate structure, delamination, matrix cracking, and fiber failure. Novel structural features noted with SEM also included the presence of small tubules that appear to run along the length of collagen fibers in the annulus and a distinct collagenous structure representative of a pericellular matrix in the nucleus region. 相似文献
15.
X-ray diffraction has been used to measure the orientation of the collagen fibres in the ventral annulus fibrosus of intact L1/2 rabbit intervertebral disc during in vitro bending and torsion. Fibres are tilted with respect to the axis of the spine. As predicted by theory, fibre tilt decreases in those regions of the annulus which are stretched by bending but increases in the slackened regions. Good agreement with the quantitative predictions of bending theory was obtained in three of the six series of experiments, the predicted trend being found in all six. Tilt direction alternates in successive lamellae of the annulus. When discs were subjected to both clockwise and anticlockwise torsion of 5°, the two families of titled fibres reoriented in the expected directions. 相似文献
16.
Assays of several proteases, incorporating guanidinium chloride extracts of human femoral head cartilage and intervertebral disc, demonstrated that both tissues contain inhibitors of certain serine proteases. Trypsin, chymotrypsin and a granule extract of human polymorphonuclear leukocyte containing elastase and cathepsin G activities, were inhibited by low molecular weight fractions prepared by Sephadex G-75 chromatography. Using a radioassay, it was further shown that these fractions inhibit proteolysis of cartilage proteoglycan. The inhibitor in intervertebral disc is concentrated in the nucleus pulposus, with a decreasing gradient to the periphery of the annulus fibrosus.It is proposed that these inhibitors confer at least partial protection against pathological proteolysis of the proteoglycans in human articular cartilage and nucleus pulposus. 相似文献
17.
Low back pain is a significant socioeconomic burden in the United States and lumbar intervertebral disc degeneration is frequently implicated as a cause. The discs play an important mechanical role in the spine, yet the relationship between disc function and back pain is poorly defined. The objective of this work was to develop a technique using magnetic resonance imaging (MRI) and three-dimensional modeling to measure in vivo disc deformations. Using this method, we found that disc geometry was measurable with precision less than the in-plane dimensions of a voxel (≈100 µm, 10% of the MRI pixel size). Furthermore, there was excellent agreement between mean disc height, disc perimeter, disc volume and regional disc height measurements for multiple trials from an individual rater (standard deviation <3.1% across all measurements) and between mean height, perimeter, and volume measurements made by two independent raters (error <1.5% across all measurements). We then used this measurement system to track diurnal deformations in the L5-S1 disc in a young, healthy population (n = 8; age 24.1 ± 3.3 yrs; 2 M/6F). We measured decreases in the mean disc height (−8%) and volume (−9%) with no changes in perimeter over an eight-hour workday. We found that the largest height losses occurred in the posterior (−13%) and posterior-lateral (−14%) regions adjacent to the outer annulus fibrosus. Diurnal annulus fibrosus (AF) strains induced by posterior and posterior-lateral height loss may increase the risk for posterior disc herniation or posterior AF tears. These preliminary findings lay a foundation for determining how deviations from normal deformations may contribute to back pain. 相似文献
18.
The functional role of versican in influencing intervertebral disc cell adhesion and proliferation was analyzed in bovine intervertebral disc. We have previously demonstrated the C-terminal globular G3 (or selectin-like) domain of versican to influence mesenchymal chondrogenesis and fibroblast proliferation in vitro. For this study, a versican G3 expression construct was generated to examine the role of the G3 domain of versican. Nucleus pulposus and annulus fibrosus cells were isolated from adult bovine caudal discs using sequential enzymatic digestion and versican expression characterized by RT-PCR. In cell proliferation assays, we observed that there was greater cellular proliferation in the presence of versican G3 for both disc cell types. The higher proliferation rate of annulus fibrosus cells when compared to nucleus pulposus cells seeded in monolayer supports heterogeneity of intervertebral disc cell populations. The presence of versican G3 construct enhanced the adhesion of isolated nucleus pulposus and annulus fibrosus cells approximately 4 to 6 fold, respectively. Cellular adhesion was greater in the presence of versican G3 in a dose dependent manner. G3 product was purified using affinity columns, and the purified G3 also enhanced cell adhesion. 相似文献
19.
Degenerate intervertebral discs exhibit both material and structural changes. Structural defects (lesions) develop in the anulus fibrosus with age. While degeneration has been simulated in numerous previous studies, the effects of structural lesions on disc mechanics are not well known. In this study, a finite element model (FEM) of the L4/5 intervertebral disc was developed in order to study the effects of anular lesions and loss of hydrostatic pressure in the nucleus pulposus on the disc mechanics. Models were developed to simulate both healthy and degenerate discs. Degeneration was simulated with either rim, radial or circumferential anular lesions and by equating nucleus pressure to zero. The anulus fibrosus ground substance was represented as a nonlinear incompressible material using a second-order polynomial, hyperelastic strain energy equation. Hyperelastic material parameters were derived from experimentation on sheep discs. Endplates were assumed to be rigid, and annulus lamellae were assumed to be vertical in the unloaded state. Loading conditions corresponding to physiological ranges of rotational motion were applied to the models and peak rotation moments compared between models. Loss of nucleus pulposus pressure had a much greater effect on the disc mechanics than the presence of anular lesions. This indicated that the development of anular lesions alone (prior to degeneration of the nucleus) has minimal effect on disc mechanics, but that disc stiffness is significantly reduced by the loss of hydrostatic pressure in the nucleus. With the degeneration of the nucleus, the outer innervated anulus or surrounding osteo-ligamentous anatomy may therefore experience increased strains. 相似文献
20.
Lipopolysaccharide (LPS) induces matrix degradation and markedly stimulates the production of several cytokines, i.e., interleukin-1β, −6, and −10, by disc cells and chondrocytes. We performed a series of experiments to compare cellular responses of cells from the bovine intervertebral disc (nucleus pulposus and annulus fibrosus) and from bovine articular cartilage to LPS. Alginate beads containing cells isolated from bovine intervertebral discs and articular cartilage were cultured with or without LPS in the presence of 10% fetal bovine serum. The DNA content and the rate of proteoglycan synthesis and degradation were determined. In articular chondrocytes, LPS strongly suppressed cell proliferation and proteoglycan synthesis in a dose-dependent manner and stimulated proteoglycan degradation. Compared with articular chondrocytes, nucleus pulposus cells responded in a similar, although less pronounced manner. However, treatment of annulus fibrosus cells with LPS showed no significant effects on proteoglycan synthesis or degradation. A slight, but statistically significant, inhibition of cell proliferation was observed at high concentrations of LPS in annulus fibrosus cells. Thus, LPS suppressed proteoglycan synthesis and stimulated proteoglycan degradation by articular chondrocytes and nucleus pulposus cells. The effects of LPS on annulus fibrosus cells were minor compared with those on the other two cell types. The dissimilar effects of LPS on the various cell types suggest metabolic differences between these cells and may further indicate a divergence in pathways of LPS signaling and a differential sensitivity to exogenous stimuli such as LPS.This work was supported in part by NIH grants 2-P50-AR39239 and 1-P01-AR48152. 相似文献
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