Biochemistry of the intervertebral disc

In comparison to other tissues, understanding the disc biochemistry is only in the very early stage. The few facts that are known would point to strategies for interventions to alter disc biology and maintain disc biomechanics.     

Aging and degeneration in the lumbar intervertebral disc

1. The maintenance of the nucleus pulposus matrix in the adult human disc is dependent on the functional integrity of the cartilage end plate cells. 2. Cartilage end plate senescence is followed by compensatory cartilaginous metaplasia of annulus fibrosus cells. 3. It is proposed that disc narrowing and collapse are related to metabolic failure of matrix production by end plate and annulus fibrosus cells. 4. Calcium pyrophosphate dihydrate crystal deposits, a visible manifestation of a metabolic abnormality, are found frequently in the annulus fibrosus and cartilage end plates of elderly patients with degenerative disc disease.     

DNA minor groove recognition by a tetrahydropyrimidinium analogue of hoechst 33258: NMR and molecular dynamics studies of the complex with d(GGTAATTACC)2

Hoechst 43254 (H43254), a 2,3,4,5-tetrahydropyrimidin-1-ium analogue of the bis-benzimidazole minor groove binding agent Hoechst 33258 (H33258), has been studied by NMR and restrained molecular dynamics in its complex with d(GGTAATTACC)2. We investigate the origin of the enhanced complex stability afforded by the replacement of the N-methylpiperazine ring of H33258 with the tetrahydropyrimidinium ring of H43254, the latter presenting the opportunity for specific minor groove-directed recognition through a pyrimidinium NH. A set of 25 drug-DNA NOEs define the binding site with some precision and are used as part of the structural analysis using restrained molecular dynamics simulations considering explicit solvation and the treatment of electrostatic interactions using the particle mesh Ewald method within AMBER 4.1. Starting with three different initial structures with the drug located at different sites in the groove (pairwise RMSD 4.3-12.6 A) we arrive at three very similar structures (pairwise RMSD 0.80-1.34 A) representing one converged binding site at the centre of the AATT tract. Two of the three structures show the tetrahydropyrimidinium ring to be suitably positioned for an -NH to adenine N3 hydrogen bond suggesting that electrostatic interactions may play an important role in the enhanced affinity as well as imparting additional A-T specificity. The NMR data show that the pyrimidinium NH interaction is dynamic since signal averaging from the two sides of the ring indicate rapid rotations in the bound form.     

MRI of the ageing and herniating intervertebral disc

OBJECTIVE: To evaluate the relationship between specific cleavage of aggrecan at the Glu373-Ala374 'aggrecanase' site and degradation and release of proteoglycan catabolites from cartilage in explant cultures. DESIGN: The monoclonal antibody, BC-3, which specifically recognizes the new N-terminus, ARGSVIL, generated by cleavage of aggrecan at the Glu373-Ala374 'aggrecanase' site, was used to follow the generation of fragments produced by cleavage at this site as compared to degradation of proteoglycan as assessed by glycosaminoglycan (GAG) release from cartilage in response to cytokines and the ability of inhibitors to block this cleavage. RESULTS: (1) There was a strong correlation between specific cleavage at the Glu373-Ala374 bond and the release of aggrecan catabolites in response to interleukin-1 (IL-1) or tumour necrosis factor (TNF) stimulation. (2) This cleavage in the interglobular domain of aggrecan was inhibited by the inclusion of cycloheximide, thus indicating a requirement for de novo protein synthesis in the induction of 'aggrecanase' activity. (3) The inhibitors, indomethacin, naproxen, tenidap, dexamethasone and doxycycline were ineffective in blocking either specific cleavage at the 'aggrecanase' site or aggrecan degradation as measured by GAG release from cartilage. (4) In contrast, compounds which act through two different mechanisms to inhibit MMPs were effective in blocking both specific cleavage at the 'aggrecanase' site and proteoglycan degradation. CONCLUSIONS: Our data suggest that 'aggrecanase' is primarily responsible for proteoglycan cleavage in these experimental systems and that this protease has properties in common with metalloproteases including members of the MMP and ADAM family. Inhibition of 'aggrecanase' may have utility in preventing cartilage loss in arthritis.     

Matrix metalloproteinases in the human intervertebral disc: role in disc degeneration and scoliosis

STUDY DESIGN: Biochemical study of human intervertebral discs collected at surgery from patients with low back pain associated with disc degeneration or scoliosis. Matrix metalloproteinases were studied by quantitative zymography. OBJECTIVE: To determine whether changes in the expression of matrix metalloproteinases will bring about tissue remodelling that contributes to the progressive nature and pathology of these diseases of the intervertebral disc. SUMMARY OF BACKGROUND DATA: The diseases of the intervertebral disc, degenerative disc disease and scoliosis, are both characterized by changes in the extracellular matrix components that will affect the mechanical function of the tissue. Matrix metalloproteinases are known to have the capability of degrading all the known extracellular matrix components of the disc. METHODS: Matrix metalloproteinases 2 and 9 were detected by gelatin-gel zymography and quantified by laser scanning densitometry. Both pro and active forms of the enzymes were measured. Thirty-four discs from patients with low back pain and 29 from patients with scoliosis were investigated. RESULTS: A correlation was found between the increasing levels of matrix metalloproteinases 2 and 9 and the grade of degenerative disc disease. In addition, the levels of these enzymes show a differential expression across the scoliotic disc with the highest levels in samples taken from the convexity of the curve. CONCLUSIONS: The difference between the concave and convex side of the scoliotic curve indicates that mechanical loads might influence the expression of these enzymes. The increased expression of these enzymes in both degenerative disc disease and scoliosis strongly suggests that they may affect the progressive nature of these diseases.     

A method for the determination of hyaluronate in the presence of other glycosaminoglycans and its application to human intervertebral disc

The intrapleural air of a pneumothorax may largely collect medial to the anterior aspect of the lung in supine infants. The mechanical basis for this phenomenon is presented with supporting experimental evidence. This distribution of intrapleural air poses problems in recognition of the pneumothorax radiographically, in differentiation of the pneumothorax from pneumomediastinum, in estimation of the true volume of intrapleural air, and in the proper placement of drainage tubes. The importance of horizontal-beam lateral radiographs and of careful attention to the position of intrapleural tubes is emphasized, especially in supine, immobile newborns.     

The chemical constitution of the proteoglycan of human intervertebral disc

Proteoglycan was prepared from three pools of normal human intervertebral discs by extraction with buffered 4M-guanidinium chloride followed by CsCl-density-gradient ultracentrifugation. Chromatography on agarose (Bio-Gel A-150m) and on DEAE-cellulose suggested a single polydisperse proteoglycan species. The intrinsic viscosities of three preparations were 166, 122 and 168 ml/g. After degradation with 0.5M-KOH containing 0.02M-NaBH4, the glycosaminoglycans were recovered quantitatively and their Ca2+ salts separated into a hexuronate-rich fraction (fraction 1), which was precipitated in 0-45% (v/v) ethanol, and a hexose-rich fraction (fraction2), which was precipitated in 45-70% (v/v) ethanol. Qualitative and quantitative analyses of the glycosaminoglycans revealed fraction 1 to be chondroitin sulphate, and fraction 2 to be keratan sulphate; the latter was contaminated with protein and possibly a small amount of another glycosaminoglycan. For both glycosaminoglycans, plots of log(mol.wt.) against weight fell close to a normal distribution. The mode for chondroitin sulphate was close to 20000; that for keratan sulphate, 10000. A threefold range of molecular weight included the central 16-84% [+/- 1 S.D. of log(mol.wt.)] of the weight of both fractions.     

Frozen storage affects the compressive creep behavior of the porcine intervertebral disc

STUDY DESIGN: A biomechanical study of the compressive creep behavior of the porcine intervertebral disc before and after frozen storage. OBJECTIVE: To determine whether frozen storage alters the creep response, hydration, and nuclear swelling pressure of the intact intervertebral disc. SUMMARY OF BACKGROUND DATA: The mechanical response of the disc is dominated by swelling and fluid flow, whose effects are time-dependent. Because fluid content, which may change during storage, plays a significant role in the disc's time-dependent behavior, changes in mechanical response due to freezing may have been missed in previous studies that focused on time-independent behavior only. METHODS: Porcine intervertebral discs were tested in repeated cycles of compressive creep either immediately postmortem or after 3 weeks of frozen storage. Swelling pressure and nuclear hydration were also measured in fresh and frozen discs. A fluid transport model was used to analyze the creep data. RESULTS: The creep behavior of the intact porcine intervertebral disc is dramatically affected by frozen storage. The apparent permeability of the frozen discs was 82% higher than that of the fresh discs, and the swelling pressure of frozen discs was 25% lower in frozen discs (P < 0.01). The behavior of fresh and frozen discs became more dissimilar with repeated cycles of creep. CONCLUSIONS: In vitro tests of frozen porcine intervertebral discs do not represent fresh behavior. Frozen storage appears to permanently alter disc behavior. The precise nature of any freezing-induced damage, and whether frozen storage similarly affects human discs, remains to be seen.     

Metabolism of the extracellular matrix formed by intervertebral disc cells cultured in alginate

STUDY DESIGN: Cells from normal rabbit nucleus pulposus (NP) and anulus fibrosus (AF) were cultured in alginate beads for as long as 14 days to allow them to reform a matrix made up of two compartments: the cell-associated matrix (CM) and further removed matrix (FRM). At different time points, the CM and FRM made by each cell population were analyzed using histologic, biochemical, and immunologic assays. OBJECTIVES: To study the metabolism of normal rabbit NP and AF cells in alginate by characterizing the CM and FRM formed by each cell population, and to identify metabolic properties that may shed light on mechanisms at play in disc degeneration. SUMMARY OF BACKGROUND DATA: Little is known about the metabolism of intervertebral disc cells, in part because of the lack of microculture systems appropriate for the study of these cells in vitro. In recent studies from our laboratories, it was suggested that articular chondrocytes cultured in alginate beads remain phenotypically stable and reform a matrix similar to the one they populate in vivo. This culture system appears ideally suited for the study of intervertebral cells available only in limited numbers. METHODS: Rabbit NP and AF cells released from the matrix by sequential enzyme digestion were encapsulated in alginate beads (20,000 cells/bead) and cultured for as long as 14 days. At selected time points, beads were solubilized with calcium chelating agents, and the CM and FRM were isolated. The rate of 35S-sulfate incorporation into proteoglycans, and the contents of various extracellular matrix molecules (total sulfated proteoglycans, antigenic keratan sulfate, hyaluronan, collagen, and pyridinium crosslinks) were measured. RESULTS: Both NP and AF cells remained phenotypically stable in the alginate gel throughout the culture period and reestablished a matrix composed of CM and FRM compartments. The two cell populations exhibited numerous differences in their metabolic activities in vitro. Nucleus pulposus cells synthesized fewer proteoglycan and collagen molecules and were less effective in incorporating these into the CM than AF cells. CONCLUSIONS: Intervertebral disc cells, especially NP cells, are extremely sluggish in reforming a CM, a protective shell rich in proteoglycans and collagen molecules. This may help explain why damage to the NP often is accompanied by progressive degeneration of the disc in vivo.     

A new culture system to study the metabolism of the intervertebral disc in vitro

STUDY DESIGN: This study determined whether entrapment of a rabbit intervertebral disc in alginate gel helped to promote the retention of normal metabolic activities by the nucleus pulposus and anulus fibrosus in tissue culture. OBJECTIVES: To establish an in vitro culture system to study the metabolism of the intervertebral disc as a whole integral organ. SUMMARY OF BACKGROUND DATA: In vitro studies of the metabolism of intervertebral discs have been scarce because of the difficulties involved in maintaining the integrity of the tissues, especially that of the nucleus pulposus, in culture medium. METHODS: Rabbit intervertebral discs were embedded in alginate gel and maintained in culture for as long as 1 month. At weekly intervals, experiments were performed to measure the rate of proteoglycan synthesis and to characterize proteoglycans newly synthesized by cells in the anulus fibrosus and nucleus pulposus. In addition, at these same time intervals, the contents of sulfated proteoglycans, antigenic keratan sulfate, hyaluronan, and collagen in these two intervertebral disc tissues were measured to evaluate tissue integrity. Intervertebral discs cultured in medium alone were used as controls and analyzed in parallel. RESULTS: The anulus fibrosus and nucleus pulposus of intervertebral discs cultured in alginate gel sustained a higher rate of proteoglycan synthesis and maintained a higher content of extracellular matrix components than the respective controls at all times. CONCLUSIONS: This new alginate tissue culture system should prove useful for studying the metabolism of whole intervertebral discs.     

Multimode interaction of Hoechst 33258 with eukaryotic DNA; quantitative analysis of the DNA conformational changes

The interaction of the minor groove binding ligand Hoechst 33258 (Hoe) with natural DNA was investigated by high resolution titration rotational viscometry. Analysis of the concomitant DNA conformational changes was performed with two DNA samples of sufficiently different molar mass M, at 4 degrees C, 22 degrees C and 40 degrees C, for Hoe/DNA-P ratios below r = 0.02. In this narrow r range several interaction modes could be resolved. The measured conformational changes were quantified in terms of relative changes of both apparent DNA persistence length, delta a/a, and hydrodynamically operative DNA contour length, deltaL/L. Delta a/a(r) primarily is a measure of ligand-induced DNA helix stiffening, but both, delta a/a(r) and deltaL/L(r), generally depend also on ligand binding induced DNA bending or DNA unbending. The essential difference obviously is that delta a/a(r) is influenced by the randomly distributed helix bends and deltaL/L(r) by phased ones. The measurements performed at different temperatures deliver informations about existence and temperature dependent abolition of intrinsic helix curvature. Both Hoe and netropsin (Nt) prefer binding to AT rich DNA segments, which are candidates for intrinsic DNA helix bends. But our data for Hoe interaction with calf thymus DNA (ctDNA) show characteristic differences to those for Nt-ctDNA interaction. Especially for Hoe, the mode of highest affinity is saturated already at a ligand concentration of roughly 1 nM (r approximately = 0.0015 Hoe/DNA-P). It exhibits an unusually strong temperature dependence of the conformational DNA response. A Hoe-Nt competition experiment shows that Hoe binding to the sites of the very first Hoe mode is almost unaffected by bound Nt. But Hoe binding to the sites of the following Hoe modes does not occur due to the competition with Nt. Thus this mode of strongest Hoe-DNA interaction reflects a unique mechanism, possibly of high relevance for gene regulatory systems.     

Monocyte chemoattractant protein-1 in the intervertebral disc. A histologic experimental model

STUDY DESIGN: Monocyte chemoattractant protein-1 was investigated in an experimental rat model using immunohistochemistry. OBJECTIVE: To ascertain the precise mechanism of macrophage recruitment in the early phase of disc resorption. SUMMARY OF BACKGROUND DATA: In previous studies, many investigators reported that disc herniation was resorbed by monocytic phagocytosis. However, how the recruitment of monocytes was triggered is still unknown. METHODS: The autologous intervertebral discs from tails of Wistar rats were subcutaneously implanted into the abdomen. These discs were obtained on days 2, 3, 7, and 14 after implantation and were used for immunohistochemical study and for quantitative analysis of monocyte chemoattractant protein-1 by sandwich enzyme-linked immunosorbent assay. RESULTS: Monocyte chemoattractant protein-1-positive granulocytes and macrophages were observed surrounding the intervertebral disc, and monocyte chemoattractant protein-1-positive disc chondrocytes were observed in the nucleus pulposus and the inner anulus fibrosus on day 3. By day 7, monocyte chemoattractant protein-1-positive and TRPM-3-positive macrophages appeared in the granulation tissue, and some of these cells invaded the nucleus pulposus and inner anulus fibrosus. The concentration of monocyte chemoattractant protein-1 was highest on day 3. CONCLUSION: Intervertebral disc chondrocytes have chemotactic properties and play an active role in the recruitment of monocytes involved in disc resorption.     

The location of the intervertebral lumbar disc on the posterior aspect of the spine

Lack of in vitro cultivation methods has inhibited the development of rapid, reliable diagnostic procedures for adenovirus-associated necrotizing bronchopneumonia in guinea pigs. Because polymerase chain reaction (PCR) techniques are well established for human adenoviruses, primers for the amplification of guinea pig adenovirus DNA were evaluated. The DNA for PCR was purified from the lung tissue of spontaneously infected and healthy guinea pigs. Adenovirus DNA could only be detected in the lungs of the infected animals. Subsequent sequence analysis of PCR products revealed that the guinea pig adenovirus is a distinct adenovirus.     

Intragenic polymorphisms of the vitamin D receptor gene associated with intervertebral disc degeneration

STUDY DESIGN: A study in genetic epidemiology of disc degeneration, based on lifetime exposure data, findings on magnetic resonance imaging, and genotyping of intragenic markers. OBJECTIVES: To pursue the potential correlation between common allelic variations in the vitamin D receptor locus and degeneration of the intervertebral disc. SUMMARY OF BACKGROUND DATA: Familial aggregation has been observed in intervertebral disc degeneration, but the relative significance of the genetic component and shared environmental influences is unknown. The identification of relevant candidate genes associated with disc degeneration would specify a genetic component and increase our understanding of the etiopathogenesis of disc degeneration. METHODS: From the population-based Finnish Twin cohort, 85 pairs of male monozygotic twins were selected based on exposure to suspected risk factors for disc degeneration. Interview data were gathered on relevant lifetime exposures, and thoracic and lumbar disc degeneration was determined through quantitative and qualitative assessments of signal intensity on magnetic resonance imaging, and qualitative assessments of disc bulging and disc height narrowing. Possible associations were examined between disc degeneration measures and two polymorphisms of the coding region of the vitamin D receptor locus. RESULTS: Two intragenic polymorphisms of the vitamin D receptor gene revealed an association with disc degeneration. Quantitatively assessed signal intensities of thoracic and lumbar (T6-S1) discs were 12.9% worse in men with the Taql tt genotype and 4.5% worse in men with the Tt genotype, compared with signal intensity in men with the TT genotype (age adjusted P = 0.003). A similar pattern was found between disc signal intensity and Fokl genotypes; men with the ff and Ff genotypes had mean signal intensities that were 9.3% and 4.3% lower, respectively, than those in men with FF genotypes (age-adjusted P = 0.006). The summary scores of qualitatively assessed signal intensity, bulging, and disc height were 4.0% and 6.9% worse in men with Ff and ff genotypes, respectively, when compared with those in men with the FF genotype (age-adjusted P = 0.029). CONCLUSION: Specific vitamin D receptor alleles were associated with intervertebral disc degeneration as measured by T2-weighted signal intensity, demonstrating for the first time, the existence of genetic susceptibility to this progressive, age-related degenerative process.     

Isolation and characterization of metallic wear debris from a dynamic intervertebral disc prosthesis

A dynamic intervertebral disc prosthesis (DIDP) has been developed. It consists of a CoCrMo body and uses Ti6Al4V springs to replicate the mechanical function of the lumbar joint. Wear studies have been performed previously on the DIDP using two specialized simulators to test the wear properties of the moving parts of the disc prosthesis. A pin-in-slot simulator generates wear that would occur in the hinge-pin assembly of the prosthesis. A spring-in-pocket simulator approximates the conditions under which the springs would wear against the body of the prosthesis. The spring-pocket interface is responsible for the production of approximately 90% of the total wear occurring in the prosthesis, and is therefore the main focus of this study. Bovine serum with a preservative has been used as a lubricant in both simulators. The spring-in-pocket simulator compares the effects of two different manufacturing techniques of CoCrMo (HIPing and forging) on their wear characteristics against Ti6Al4V springs. Debris from the spring-in-pocket simulator has been isolated from the serum lubricant and characterized using scanning electron microscopy techniques. The morphology of the Ti6Al4V fragments is rough and irregularly shaped. The size of these fragments ranges from < 1 microns to > 30 microns. The forged CoCrMo alloy debris has an irregular polyhedral shape, with sizes in the same range as the spring fragments. The morphology of the HIPed CoCrMo debris is spherical with a size range < 5 microns to > 30 microns. Length and width measurements of micron-size particles were made with the particle measurements feature of the scanning electron microscope. Micron-size particles were found in all stations. This article provides a unique way to isolate and analyze debris from serum lubricants used in simulators.     

《内经》肾主骨理论与腰椎间盘突出症的相关性

肾主骨理论是中医理论体系中重要的组成部分,该理论首载于<内经>,并经历代医家的继承和发展,一直指导着临床实践.随着社会的进步,医学技术的发展,肾主骨理论的现代研究也取得了相当的进展.在此基础之上,该理论指导临床治疗疾病的范围也不断扩大.因此,肾主骨理论也越来越受到人们的重视.近年来,虽然人们生活水平提高,重物搬运皆有机械帮助,而且计算机的应用也减轻了人类工作的强度,但是患骨病特别是腰椎间盘突出症的患者却越来越多.因此,利用传统中医的肾主骨理论来探讨腰椎间盘突出症的发病原因并指导该病的治疗,显得十分必要.     

Body mass index and height in patients requiring surgery for lumbar intervertebral disc herniation

The immediate preoperative body mass index and standing body height of 1128 patients who underwent surgery for lumbar intervertebral disc herniation were compared in a cross-sectional study with the corresponding values obtained from a general population sample. The material was divided into sex- and age-specific subgroups. To delineate possible differences, the 99% confidence intervals for the anthropometric mean values were constructed instead of hypothesis testing. With the exception of the oldest age group, from 50 to 59 years, the patients who underwent surgery for a disc herniation were more obese and taller than the population on average in all other sex- and age-specific subgroups. The major contrast emerged in women aged 20-29 years, in whom the 99% confidence interval for the mean body mass index of the patients undergoing surgery on was 25.1-27.3 kg/m2 versus 22.3-23.1 kg/m2 in the general population. In patients aged 20-39 years the mean body mass index was increased also when the body height of the patients was less than the mean value of the general population samples. Both an increased body mass index and a tall stature seem to have a clear association with those severe lumbar intervertebral disc herniations that require operative treatment.