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Proteoglycan aggregates free of non-aggregating proteoglycan have been prepared from the annuli fibrosi and nuclei pulposi of intervertebral discs of three human lumbar spines by extraction with 4M-guanidinium chloride, associative density gradient centrifugation, and chromatography on Sepharose CL-2B. The aggregate (A1-2B.V0) was subjected to dissociative density-gradient ultracentrifugation. Three proteins of Mr 38 900, 44 200 and 50 100 found in the fraction of low buoyant density (A1-2B.V0-D4) reacted with antibodies to link protein from newborn human articular cartilage. After reduction with mercaptoethanol, two proteins of Mr 43 000 and two of Mr 20 000 and 14 000 were seen. The A1-2B.V0-D4 fraction, labelled with 125I, coeluted with both hyaluronate and a hyaluronate oligosaccharide (HA14) on a Sepharose CL-2B column. HA10 and HA14 reduced the viscosity of A1 fractions; HA4, HA6 and HA8 did not. HA14 decreased the viscosity of disc proteoglycans less than it did that of bovine cartilage proteoglycans. Thus, although a link protein was present in human intervertebral disc, it stabilized proteoglycan aggregates less well than did the link protein from bovine nasal cartilage.
STUDY DESIGN: Herniated lumbar disc specimens were analyzed using reverse transcriptase-polymerase chain reaction to determine the profile of chemokine expression. OBJECTIVE: To investigate the mechanism underlying the recruitment of inflammatory cells into herniated discs during the process of spontaneous regression. SUMMARY OF BACKGROUND DATA: Spontaneous regression of herniated intervertebral discs has been increasingly reported. Although macrophages are suggested to play a central role in this process, it remains unclear how these macrophages accumulate in the herniated discs. METHODS: RNA was extracted from 36 surgical specimens of the herniated lumbar disc, a disc specimen of idiopathic scoliosis and pyogenic spondylitis, and activated peripheral blood mononuclear cells of a normal donor. The RNA was reverse transcribed, and the resultant cDNA was amplified by PCR using primer pairs specific to the CXC chemokines (IL-8, MGSA-alpha, IP-10, MIG), the CC chemokines (MCP-1, MCP-2, MCP-3, MCP-4, MIP-1alpha, MIP-3alpha, RANTES, STCP-1), the C chemokine (lymphotactin), and the glyceraldehyde phosphate housekeeping gene. Thin cryostat sections also were made from the disc specimens and stained with hematoxylin and eosin. RESULTS: All the chemokines examined except MCP-4 were expressed by activated peripheral blood mononuclear cells. Glyceraldehyde phosphate was detected in 8 of 36 herniated discs and in 1 disc specimen each of idiopathic scoliosis and pyogenic spondylitis. Chemokine expression was examined for these 10 disc specimens. From among the 13 chemokines examined, MCP-3, MCP-4, RANTES, and IP-10 were detected in the disc from the idiopathic scoliosis, and MCP-3, MCP-4, RANTES, IP-10, MIG, and MGSA-alpha were detected in the infected or herniated discs. Histologic analysis showed infiltration of inflammatory cells in the infected disc and all 8 herniated discs. CONCLUSIONS: The findings suggest that chemoattractive properties exist in a selected population of human intervertebral discs, and that unique sets of chemokinesplay a role in spontaneous regression of these herniated disc tissues.
STUDY DESIGN: The histochemical changes in the multifidus muscle in 29 patients with L4-L5 lumbar intervertebral disc herniation were studied. OBJECTIVES: To clarify how nerve root impairment affects the histochemical properties of the lumbar multifidus muscle in patients with lumbar intervertebral disk herniation. SUMMARY OF BACKGROUND DATA: There have been several studies on histochemical changes in lumbar muscles in patients with nerve root impairment, but the findings concerning changes in muscle fiber sizes vary among investigators. METHODS: Biopsy specimens were obtained intraoperatively from the L4 and L5 bands of the multifidus muscle on the affected and nonaffected sides. The specimens were stained with ATPase to evaluate the size of the fibers and structural changes. RESULTS: In the L5 muscle band, the mean sizes of Type 1 and Type 2 fibers on the affected side were significantly smaller than those on the nonaffected side (Type 1: P < 0.01, Type 2: P < 0.001). The decrease in size was 6.4% for Type 1 and 9.8% for Type 2. Increased percentages of Type 1 fibers and a high incidence of small angular fibers and fiber type grouping were also shown on the affected side. In contrast, in the L4 muscle band, no side-to-side differences in the histologic findings were observed. There was no significant level-to-level difference in the mean size of Type 1 or Type 2 fibers on either the affected or the nonaffected side. CONCLUSIONS: These results suggest that nerve root impairment leads to atrophy of Type 1 and Type 2 fibers, with structural changes in the multifidus muscle only at the involved level.
OBJECTIVE: To compare therapeutic effects of catgut implantation at acupoint and routine acupuncture on prolapse of lumbar intervertebral disc. METHODS: One hundred and forty cases were randomly divided into a treatment group and a control group, 70 cases in each group. The treatment group were treated with catgut implantation at acupoint, once each week, 3 sessions constituting one course, and the control group with routine acupuncture, once every other day, 10 sessions constituting one course. RESULTS: The effective rate was 95.6% after treatment of one course and 88.2% 3 months later in the treatment group, which were better than 84.6% and 72.3% in the control group, respectively (Both P < 0.05). CONCLUSION: Catgut implantation at acupoint has a better therapeutic effect on prolapse of lumbar intervertebral disc, with lower cost.
The synthesis and turnover in vivo of 35S-labelled proteoglycans in mouse cervical, thoracic and lumbar intervertebral discs, and in costal cartilage, was investigated after intraperitoneal injection of [35S]sulphate. Intervertebral discs and costal cartilage synthesize similar amounts of 35S-labelled proteoglycans per microgram of DNA. Discs and cartilage all synthesize a major proteoglycan species (approx. 85%) of large hydrodynamic size and a minor species (approx. 15%) of small size. Both proteoglycans carry chondroitin sulphate chains. Keratan sulphate was not found associated with either species. The total 35S-labelled proteoglycan pool had a metabolic half-life (t1/2) of 10-12 days in discs, and 17 days in cartilage. The extractable major and minor species turned over at similar rates. Those proteoglycans left in the tissue after 29 days turn over very slowly. Approx. 50% of the major 35S-labelled proteoglycan species formed mixed aggregates with hyaluronic acid and rat chondrosarcoma proteoglycan. The ability to form aggregates did not decrease up to 45 days after synthesis. Of the heterogeneous population of proteoglycans comprising the major species, those remaining in the tissue 9 days after synthesis were of smaller average hydrodynamic size and had shorter chondroitin sulphate side chains than the average size at the time of synthesis. With increasing time after synthesis, proteoglycans were less readily extracted from the tissue by 4.0 M-guanidinium chloride than at the time of synthesis.
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An anatomically accurate, three-dimensional, nonlinear finite element model of the human cervical spine was developed using computed tomography images and cryomicrotome sections. The detailed model included the cortical bone, cancellous core, endplate, lamina, pedicle, transverse processes and spinous processes of the vertebrae; the annulus fibrosus and nucleus pulposus of the intervertebral discs; the uncovertebral joints; the articular cartilage, the synovial fluid and synovial membrane of the facet joints; and the anterior and posterior longitudinal ligaments, interspinous ligaments, capsular ligaments and ligamentum flavum. The finite element model was validated with experimental results: force-displacement and localized strain responses of the vertebral body and lateral masses under pure compression, and varying eccentric anterior-compression and posterior-compression loading modes. This experimentally validated finite element model was used to study the biomechanics of the cervical spine intervertebral disc by quantifying the internal axial and shear forces resisted by the ventral, middle, and dorsal regions of the disc under the above axial and eccentric loading modes. Results indicated that higher axial forces (compared to shear forces) were transmitted through different regions of the disc under all loading modes. While the ventral region of the disc resisted higher variations in axial force, the dorsal region transmitted higher shear forces under all loading modes. These findings may offer an insight to better understand the biomechanical role of the human cervical spine intervertebral disc.
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The nucleolytic action of chondroitinase ABC was studied by the use of rabbit lumbar intervertebral discs. The injection of one unit of enzyme results in necrosis of the nucleus pulposus. Radiologic, histologic, and biochemical changes were evident in discs studied 2 days after injection, and consistent nucleolysis was seen in those studied after 6 days. These results show that chondroitinase ABC deserves further evaluation as a possible chemonucleolytic enzyme.
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.
The present study investigates whether changes in barometric pressure influence low back pain in patients with vacuum phenomenon within lumbar intervertebral discs. Twenty-four patients with low back pain were examined: 12 with vacuum phenomenon within the intervertebral discs (VP [+] group) and 12 without the vacuum phenomenon (VP [-] group). All patients consented to an evaluation of low back pain in a hyperbaric oxygen chamber. In the VP (+) group, low back pain became more severe in one patient when atmospheric pressure was elevated, and in eight patients when atmospheric pressure was decreased. In the VP (-) group, two patients experienced more severe low back pain when atmospheric pressure was decreased. Our study demonstrated that low back pain might be aggravated by atmosphere depression in patients with lumbar disease associated with the vacuum phenomenon.
If patient-specific finite element models of the spine could be developed, they would offer enormous opportunities in the diagnosis and management of back problems. Several generic models have been developed in the past, but there has been very little detailed examination of the sensitivity of these models' characteristics to the input parameters. This relationship must be thoroughly understood if representative patient-specific models are to be realized and used with confidence. In particular, the performance of the intervertebral discs are central to any spine model and need detailed investigation first. A generic non-linear model of an intervertebral disc was developed and subjected to compressive, flexion and torsional loading regimes. The effects of both material and geometric non-linearities were investigated for the three loading schemes and the results compared with experimental data. The basic material properties of the fibres, annulus and nucleus were then varied and the effects on the stiffness, annulus bulge and annulus stresses analysed. The results showed that the non-linear geometry assumption had a significant effect on the compression characteristics, whereas the non-linear material option did not. In contrast, the material non-linearity was more important for the flexural and torsional loading schemes. Thus, the inclusion of non-linear material and geometry analysis options in finite element models of intervertebral discs is necessary to predict in vivo load-deflection characteristics accurately. When the influence of the material properties was examined in detail, it was found that the fibre properties did not have a significant effect on the compressive stiffness of the disc but did affect the flexural and torsional stiffnesses by up to +/-20 per cent. All loading modes were sensitive to the annulus properties with stiffnesses varying by up to +/-16 per cent. The model also revealed that for a particular compressive deformation or flexural or torsional rotation, the disc bulge was not sensitive to any of the material properties over the range of properties considered. The annulus stresses did differ significantly as the material properties were varied (up to 70 per cent under a compressive load and 60 per cent during disc flexion).
The purpose of this research was to explore the in situ anatomic and mechanical environment of disc cells. Laser scanning confocal microscopy was used to characterize three-dimensional morphology of intervertebral disc cells, micromechanical deformation and interaction with extracellular matrix, and functional intercellular communication. Bovine coccygeal discs were used for both the anatomic and micromechanical investigations. Anulus fibrosus cells had a complex morphology with sinuous processes woven into the extracellular matrix, particularly in the outer aspect of the anulus where they were also interconnected via functional gap junctions. They were also found in an extensive pericellular matrix of type-VI collagen, joining as many as ten cells into linear cell arrays that could be extracted from the matrix as functional units. Mechanically, collagen fibril sliding was demonstrated to govern cell mechanics and strain transfer in the anulus fibrosus during loading activities. Lamellar cells were largely protected from direct tensile strains in the matrix, with minimal intercellular strains. However, intercellular strains between lamellar cells in adjacent arrays were large, illustrating shearing between linear cell arrays. Appreciable shear was observed across the lamellar cell bodies as well as to the cellular processes woven into the matrix. These findings demonstrated the morphologic and micromechanical complexity of anulus fibrosus cells. The knowledge of the in situ environment of disc cells will provide a base to investigate the mechanical implications of disc degeneration on the cellular environment and to better understand how mechanical and genetic risk factors can impact the cells that are essential to maintaining the intervertebral disc.
A new radiographic grading system for a more objective assessment of lumbar intervertebral disc degeneration has been described and tested in Part I of this study. The aim of the present Part II of the study was to adapt this system to the cervical spine, and to test it for validity and interobserver agreement. Some modifications of the grading system described in Part I were necessary to make it applicable to the cervical spine. Its basic structure, however, stayed untouched. The three variables "Height Loss", "Osteophyte Formation" and "Diffuse Sclerosis" first have to be graded individually. Then, the "Overall Degree of Degeneration" is assigned on a four-point scale from 0 (no degeneration) to 3 (severe degeneration). For validation, the radiographic degrees of degeneration of 28 cervical discs were compared to the respective macroscopic ones, which were defined as "real" degrees of degeneration. The interobserver agreement was determined between one experienced and one unexperienced observer using the radiographs of 57 cervical discs. Quadratic weighted Kappa coefficients (kappa) with 95% confidence limits (95% CL) were used for statistical evaluation. The validation of the new version of the radiographic grading system showed a moderate agreement with the "real", macroscopic overall degree of degeneration (kappa=0.599, 95% CL 0.421-0.786). In 64% of all discs the "real" overall degree of degeneration was underestimated but never overestimated. This underestimation, however, was much less pronounced and the Kappa coefficients were significantly higher for the three variables: Height Loss, Osteophyte Formation, and Diffuse Sclerosis separately. The agreement between the radiographic ratings of the experienced and the unexperienced observer was substantial for the overall degree of degeneration (kappa=0.688, 95% CL 0.580-0.796), almost perfect for the variable, Height Loss, moderate for Osteophyte Formation and fair for Diffuse Sclerosis. In conclusion, we believe that the new version of the radiographic grading system is a sufficiently valid and reliable tool to quantify the degree of degeneration of individual cervical intervertebral discs. In comparison to the version for the lumbar spine described in Part I, however, a slightly higher tendency to underestimate the "real" overall degree of degeneration and somewhat higher interobserver differences have to be expected.
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OBJECTIVE: To determine how partial removal of the nucleus changes the response of the annulus to compression. DESIGN: The deformation of the annulus in the mid-sagittal plane, during compression, was determined from digital video images. BACKGROUND: Several studies have shown that removal of the nucleus changes the external behaviour of the intervertebral disc, but few studies have investigated changes to internal behaviour. METHODS: Six frozen human lumbar discs were bisected in the sagittal plane to produce 12 specimens. The cut surfaces were marked with seven dots of Alcian blue stain. The specimens were sealed, enabling their internal structure to be viewed directly by a digital video recording system, and thawed. The video system recorded the response of each specimen as it was compressed by up to 1.8 mm at a rate of 0.2 mm s(-1). The displacements of the Alcian blue marks were measured using an image analysis program. Magnetic resonance imaging was used to investigate the validity of this technique. RESULTS: Partial removal of the nucleus changed the way that the disc deformed under compression. A highly significant change in direction of movement was seen in the inner posterior region of the annulus. CONCLUSIONS: Partial removal of the nucleus changes the response of the annulus to compression. RELEVANCE: Partial denucleation of the human intervertebral disc is shown to change the direction of bulging of the inner annulus when the disc is compressed. Increases in shear stress, arising from these changes, may lead to further disc degeneration in the form of circumferential tears.