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Tissue engineering of intervertebral discs (IVD) using mesenchymal stem cells (MSCs) induced to differentiate into a disc-cell phenotype has been considered as an alternative treatment for disc degeneration. However, since there is no unique marker characteristic of discs and since hyaline cartilage and immature nucleus pulposus (NP) possess similar macromolecules in their extracellular matrix, it is currently difficult to recognize MSC conversion to a disc cell. This study was performed to compare the proteoglycan to collagen ratio (measured as GAG to hydroxyproline ratio) in the NP of normal disc to that of the hyaline cartilage of the endplate within the same group of individuals and test the hypothesis that this ratio can be used for in vivo studies to distinguish between a normal NP and hyaline cartilage phenotype. Whole human lumbar spine specimens from fresh cadavers, ranging in age from 12 weeks to 79 years, were used to harvest the IVDs and adjacent endplates. The GAG to hydroxyproline ratio within the NP of young adults is approximately 27:1, whereas the ratio within the hyaline cartilage endplate of the same aged individuals is about 2:1. The production of an extracellular matrix with a high proteoglycan to collagen ratio can be used in vivo to distinguish NP cells from chondrocytes, and could help in identifying a NP-like phenotype in vivo as opposed to a chondrocyte when MSCs are induced to differentiate for tissue engineering of a disc.

Study Design: The contents of DNA, proteoglycan, type II collagen, and denatured type II collagen in the bovine coccygeal intervertebral discs were examined in situ in relation to disc level, age, and tissue region., Objective: To determine whether bovine coccygeal discs are a suitable model to study human lumbar discs., Summary of Background Data: Bovine coccygeal discs have been suggested as a suitable alternative model because they are readily available, in contrast to human discs, and represent a common source of tissue in the disc field. However, it is not known whether the changes in matrix contents in bovine coccygeal discs are similar to those found in the human lumbar spine., Methods: Intervertebral discs from bovine tails were dissected into the nucleus pulposus (NP) and anulus fibrosus (AF). Tissues were weighed and analyzed for matrix contents using specific assays., Results: Similar to water content, the proteoglycan content was higher in the NP than in the AF. Water content of the bovine NP did not change with age, unlike the proteoglycan content, which decreased. type II collagen content was higher in the NP than in the AF, and both did not change overall significantly with age. The percent of denatured type II collagen decreased with age only in the NP. The DNA content did not vary with age in the AF and in the NP., Conclusion: Differences in matrix contents exist between the bovine coccygeal discs and the human lumbar spine. Thus, caution must be exercised when using the bovine tail as a model for the human lumbar spine in biochemical studies.

The effect of tissue porosity on ion (sodium, potassium, and chloride) diffusivity in agarose gels and porcine intervertebral disc tissues was investigated using an electrical conductivity method. An empirical, constitutive model for diffusivity (D) of solutes in porous fibrous media was proposed: D/Do = exp[-alpha(r(s)/k(1/2))beta] where r(s) is the Stokes radius of a solute, kappa is the Darcy permeability of the porous medium, Do is the diffusivity in free solution, alpha and beta are two positive parameters whose values depend on material structure. It is found that alpha = 1.25 +/- 0.138, beta = 0.681 +/- 0.059 (95% confidence interval, R2 = 0.92, n = 72) for agarose gels and alpha = 1.29 +/- 0.171 and beta = 0.372 +/- 0.088 (95% confidence interval, R2 = 0.88, n = 86) for porcine annulus fibrosus. The functional relationship between solute diffusivity and tissue deformation was derived. Comparisons of our model prediction with experimental data on diffusion coefficients of macromolecules (proteins, dextrans, polymer beads) in agarose gels in the literature were made. Our results were also compared to the data on ion diffusivity in charged gels and in cartilaginous tissues reported in the literature. There was a good agreement between our model prediction and the data in the literature. The present study provides additional information on solute diffusivity in uncharged gels and charged tissues, and is important for understanding nutritional transport in avascular cartilaginous tissues under different mechanical loading conditions.

Study Design: Distribution and appearance of tumor necrosis factor-alpha (TNF-alpha) in the dorsal root ganglion (DRG) exposed to experimental disc herniation were investigated using an immunohistochemical method in rats., Objectives: To study the distribution and appearance of TNF-alpha in the DRG following experimental disc herniation in rats., Summary of Background Data: Nucleus pulposus in the epidural space induces spinal nerve root injury not only by mechanical but also chemical mechanisms. Cytokines may play a key role in the chemical damage. There is, however, no report on the distribution and appearance of TNF-alpha in the DRG exposed to nucleus pulposus., Methods: Nucleus pulposus from the discs was smeared on the glass slides and processed for immunohistochemistry by the avidin-biotinylated peroxidase complex technique using rabbit antisera to TNF-alpha in rats. A herniation of the nucleus pulposus was made by incision of the L4-L5 disc in rats. The L4 and L5 DRGs were resected 1, 3, 7, 14, and 21 days after surgery. The specimens were processed for immunohistochemistry using rabbit antisera to TNF-alpha. The TNF-alpha-positive cells were observed and counted using light microscopy. Distribution of the TNF-alpha products was compared on each day after surgery., Results: A positive staining was seen in the cell bodies and in the matrix between the cells in the smeared nucleus pulposus. In the L4 DRG sections, the number of positive cells was significantly higher in the disc incision group than in the sham group at 1, 3, 7, and 14 days after surgery (P < 0.05). The positive cells showed a decrease in number day by day after surgery. On the contrary, in the L5 DRG, only a few positive cells were observed in the disc incision group after surgery. There was no statistically significant difference between disc incision and the sham groups at each day after surgery for the L5 DRGs., Conclusions: The immunoreactivity of TNF-alpha in the DRG directly exposed to nucleus pulposus increases during 2 weeks. A collapse of the positive cells was seen in the DRG directly exposed to the nucleus pulposus.

Study Design: Human intervertebral disc anulus tissue was obtained in a prospective study of immunolocalization of SPARC (secreted protein, acidic and rich in cysteine) (osteonectin). Experimental studies were approved by the authors' Human Subjects Institutional Review Board. Discs were obtained from surgical specimens and from control donors., Objectives: To determine whether SPARC could be detected in the disc with immunohistochemistry and to determine the incidence of SPARC-positive cells., Summary of the Background Data: SPARC is a glycoprotein that has an important role in modulating interactions between cells and matrix. It influences remodeling, collagen fibrillogenesis, metalloproteinase expression, and cytokine expression. Little is known about SPARC in the disc, and one previous study reported the absence of its immunolocalization in fetal and adult disc tissue., Methods: Eight normal human discs from subjects aged newborn to 10 years, and 11 disc specimens from control donors or surgical patients aged 15to 76 years were examined for immunolocalization of SPARC. Anulus cells were also tested for the presence of SPARC in vitro in monolayer or three-dimensional agarose culture., Results: In discs of subjects aged newborn to 0.19 years, SPARC was present in all cells in the outer anulus, in 76.4% of inner anulus cells, and 76.0% of nucleus cells. Localization was significantly lower in anulus cells of study participants aged 4.7 to 76 years (66.7%, P = 0.04). Anulus cells cultured in agarose or monolayer showed positive localization in all cells., Conclusions: Findings show decreased presence of SPARC in disc cells of older subjects with disc degeneration and point to the importance of future studies designed to elucidate the unrecognized role of SPARC in disc remodeling, aging, and degeneration.

Study Design: Experimental scoliosis was created in goats and then treated using anterior thoracic stapling., Objective: To correct, without fusion, a progressive idiopathic-type scoliotic deformity in an immature goat model using a shape memory alloy staple., Summary of Background Data: Fusionless scoliosis treatment techniques, using minimally invasive approaches to the anterior thoracic spine, provide theoretical advantages over currently available forms of treatment., Methods: Experimental scoliosis was created in 40 goats using a posterior asymmetric tether with convex rib resection and concave rib tethering for a period of up to 15 weeks. Twenty-seven goats with progressive deformities were used for subsequent study and randomized into 4 treatment groups: group I, anterior thoracic stapling with removal of the posterior tether; group II, removal of the posterior tether only; group III, anterior thoracic stapling with persistent posterior tethering; and group IV, persistent posterior tethering with no treatment. The treatment period lasted an additional 6 to 14 weeks. Staple backout was graded radiographically. After killing the goats, histology and disc biochemistry analyses were conducted., Results: The goats in group I corrected from an initial 57 degrees of curvature to 43 degrees over the duration of the treatment period. Group II goats, which served as a control for group I, corrected from 67 degrees to 60 degrees during the treatment period. Group III goats demonstrated a modest correction from 65 degrees to 63 degrees with the stapling procedure, whereas group IV goats (controls for group III) progressed from 55 degrees to 67 degrees with a persistent posterior tether during the treatment period. The difference between the correction in group III and progression in group IV was statistically significant (P = 0.002). Complications were limited to partial staple backout in 27% of 56 staples., Conclusions: The results of this study support the efficacy of an anterior thoracic staple in correcting moderately severe scoliosis and halting the progression of more malignant scoliosis without fusion in a goat model.

While tremendous work has been performed to characterize degenerative disc disease through gross morphologic, biochemical, and histologic grading schemes, the development of an accurate and noninvasive diagnostic tool is required to objectively detect changes in the matrix with aging and disc degeneration. In the present study, quantitative magnetic resonance was used to determine if the quality of the nutritional supply to the intervertebral disc at various ages and levels of degeneration could be assessed through measurement of the apparent diffusion coefficients (ADCs). Modifications of the nucleus pulposus matrix content, specifically of water and glycosaminoglycan contents, with age and disc degeneration, were reflected in correlating changes in the ADCs. From unforced stepwise linear regression analyses, relations were established showing that decreases in glycosaminoglycan or water contents in the nucleus pulposus resulted in direct decreases in the ADCs. Relations obtained for the ADCs of the nucleus pulposus were direction dependent, in conformity with the anisotropic diffusion in the intervertebral discs. Changes in matrix integrity, as evidenced by the percentage of denatured collagen, were also detected in the nucleus pulposus with a low positive correlation to the ADC along the height of the disc and an inverse statistically significant regression to the ADC along the anterior to posterior axis of the disc. Correlations between the matrix content and integrity of the annulus fibrosus and its ADCs were not as evident, with only the ADC in the lateral direction of the disc of the anterior annulus fibrosus able to reflect changes in matrix content. The information obtained by the ADCs, particularly of the nucleus pulposus, can potentially be used in combination with quantitative T1, T2, and MT parameters to noninvasively obtain a quantitative assessment of the disc matrix composition and structural integrity.

Study Design: Cerebrospinal fluid biomarkers were evaluated in a setup using established pig models to mimic clinical disc herniation., Objectives: To investigate biomarkers for nerve tissue injury, inflammation, and pain in cerebrospinal fluid after mechanical compression and/or nucleus pulposus application to spinal nerve roots., Summary of Background Data: The association between mechanical compression, biochemical effects of nucleus pulposus, and nerve root injury in degenerative disc disorders is incompletely investigated., Methods: The unilateral S1 nerve root was exposed in 20 pigs. The animals were divided into four groups (n = 5 each): 1) slow-onset mechanical compression with an ameroid constrictor; 2) autologous nucleus pulposus application; 3) mechanical compression plus nucleus pulposus; and 4) sham operation. After 1 week, 6 mL of cerebrospinal fluid was collected, and four structural nerve proteins, neurofilaments, S-100, glial fibrillary acidic protein, neuron-specific enolase, the proinflammatory cytokine interleukin-8, the neurotransmitter nociceptin, and substance P endopeptidase activity were analyzed using immunoassays., Results: The concentration of neurofilament was increased in the mechanical compression group (17.0 microg/L +/- 5.0) and in the mechanical compression plus nucleus pulposus group (19.8 +/- 12.1 microg/L) compared with the sham group (0.9 +/- 0.9 microg/L) and the nucleus pulposus group (0.4 +/- 0.1 microg/L) (P < 0.01 for both). The concentration of nociceptin was increased significantly in the mechanical compression group (24.0 +/- 8.6 fm/mL) and in the mechanical compression plus nucleus pulposus group (31.2 +/- 6.6 fm/mL) compared with the sham group (7.0 +/- 1.3 fm/mL) (P < 0.05 and P < 0.01, respectively). A correlation was found between concentrations of neurofilament and nociceptin (r = 0.50, P < 0.05). There were no intergroup differences regarding glial fibrillary acidic protein, neuron-specific enolase, S-100, interleukin-8, or substance P endopeptidase activity., Conclusions: The present study demonstrates increased concentrations of neurofilament and nociceptin in cerebrospinal fluid after nerve root compression. A simultaneous application of nucleus pulposus did not increase the response.

Objective: To study the pathogenesis of the pain of discography and the discogenic low back pain., Methods: 19 specimens of lumbar intervertebral discs from 17 patients with discogenic low back pain during posterior lumbar interbody fusion, and 12 physiologically aging discs and 10 normal control discs were collected to investigate the morphologic features and innervation containing neuropeptides substance P (SP), neural filament (NF), and vasoactive-intestinal peptide (VIP)., Results: The distinct morphologic characteristic of the disc from the patient with discogenic low back pain was the formation of the strip zone of vascularized granulation tissue from the nucleus pulposus to the outer part of the annulus fibrosus in which there was one or several fissures. The structure of annulus fibrosus beyond the strip zone of granulation tissue was basically normal. The structures of the aging discs and the control discs showed the age-related changes. The innervation of SP, NF and VIP immunoreactive nerve fibers in the painful discs was more extensive compared with the aging discs and the control discs. The nerve in growth deep into annulus fibrosus and nucleus pulposus was observed mainly along the strip zone of granulation tissue in the painful discs., Conclusions: Findings indicate that the strip zone of granulation tissue with extensive innervation in the posterior part of the painful disc is the original site of the pain of discography and the discogenic low back pain. The strip zone of granulation tissue might originate from the injury and subsequent reparation of the margin of annulus fibrosus. The difference of the aging disc and painful disc which can not be differed each other on MRI is the formation of the strip zone of granulation tissue along tear histologically in posterior part of the painful disc.

Study Design: This study describes the formation of nucleus pulposus tissue using a novel tissue engineering approach., Objectives: To determine if a construct composed of nucleus pulposus tissue on the surface of a calcium polyphosphate substrate could be formed in vitro with properties similar to native nucleus pulposus tissue., Summary of Background Data: There is no optimal treatment for the persistent pain associated with intervertebral disc degeneration. Disc replacement using artificial intervertebral discs has met with some success, and biologic transplantation is limited by the availability of donor tissues., Methods: Nucleus pulposus cells were isolated from bovine caudal intervertebral discs. Cells were seeded at high density on the upper surface of a porous bone substitute material (calcium polyphosphate) and maintained up to 6 weeks in culture. In vitro formed tissue was compared to native nucleus pulposus for histologic appearance, biochemical composition (tissue cellularity, proteoglycan and collagen accumulation), and compressive mechanical properties., Results: When maintained on the surface of a three-dimensional substrate, nucleus pulposus cells formed a continuous layer of tissue with a proteoglycan content equivalent to the native tissue. Although collagen accumulation attained only 26% than that of the native tissue, there was no difference in tissue stiffness, viscosity, or weight-bearing capacity of the in vitro formed tissue when compared with the native tissue., Conclusion: Nucleus pulposus-like tissue formed in vitro on the surface of a calcium polyphosphate substrate resembles the native tissue in terms of proteoglycan content and compressive mechanical properties. These studies are the first step toward developing a functional spinal unit in vitro.

Study Design: By the technique of tissue engineering, composite intervertebral disc implants were fabricated as novel materials for disc replacement, implanted into athymic mice, and removed at times up to 12 weeks., Objectives: The goal of this study was to construct composite intervertebral disc structures consisting of anulus fibrosus cells and nucleus pulposus cells seeded on polyglycolic acid and calcium alginate matrices, respectively., Summary of Background Data: Previous work has documented the growth of anulus fibrosus cells on collagen matrices and nucleus pulposus cells cultured on multiple matrices, but there is no documentation of composite disc implants., Methods: Lumbar intervertebral discs were harvested from sheep spine, and the nucleus pulposus was separated from surrounding anulus fibrosus. Each tissue was digested in collagenase type II. After 3 weeks in culture, cells were seeded into implants. The shape of the anulus fibrosus scaffold was fabricated from polyglycolic acid and polylactic acid, and anulus fibrosus cells were pipetted onto the scaffold and allowed to attach for 1 day. Nucleus pulposus cells were suspended in 2% alginate and injected into the center of the anulus fibrosus. The disc implants were placed in the subcutaneous space of the dorsum of athymic mice and harvested at 4, 8, and 12 weeks. At each time point, 4 samples were stored in -70 degrees C for collagen typing and analysis of proteoglycan, hydroxyproline, and DNA. Other samples were fixed in 10% formalin for Safranin-O staining., Results: The gross morphology and histology of engineered discs strongly resembled those of native intervertebral discs. Biochemical markers of matrix synthesis were present, increasing with time, and were similar to native tissue at 12 weeks. Tissue-engineered anulus fibrosus was rich in type I collagen but nucleus pulposus contained type II collagen, similar to the native disc., Conclusion: These results demonstrate the feasibility of creating a composite intervertebral disc with both anulusfibrosus and nucleus pulposus for clinical applications.

In this study, we investigated the profile of integrin expression in human and porcine intervertebral disc tissue. Differences in extracellular matrix composition between anulus fibrosus (AF) and nucleus pulposus (NP) regions of the disc, as well as differences in cellular responses to environmental stimuli, suggest a role for integrins in presenting matrix signals that may mediate these responses. Human disc tissue and porcine AF and NP tissue were stained with antibodies to alpha integrin subunits 1-6, V and IIb, and beta integrin subunits 1-6 and graded for evidence of positive staining on a scale from 0 (no staining) to 3 (high incidence of staining). Human tissue expressed alpha and beta integrin subunits shown to be present in articular cartilage, including alpha(1), alpha(5) and alpha(V). Porcine AF tissue expressed similar integrin subunits to human disc, with both expressing alpha(1), alpha(5), beta(1), beta(3) and beta(5) subunits, whereas porcine NP tissue expressed higher levels of alpha(6), beta(1) and beta(4) than AF tissue. The expressed subunits are known to interact with proteins including collagens, fibronectin and laminin; however, additional studies will be required to characterize the interactions of the integrin subunits with specific matrix constituents, as well as their specific involvement in regulating environmental stimuli.

Study Design: Mechanically test lumbar discs of transgenic mice in compression-tension and torsion., Objectives: Determine if a reduction in type I collagen results in decreased disc mechanics., Summary of Background Data: Quantitative relationships between disc structure and function would improve the understanding of disc generation and are essential relationships for functional tissue engineering. The reduced type I collagen transgenic mouse has been used in structure-function studies of bone and tendon, but not intervertebral discs. Methods for testing mouse discs have recently been developed, making disc structure-function studies possible., Methods: Microradiographed and mechanically tested lumbar discs from control and collagen-reduced mice in both compression-tension and torsion were used. Disc area and polar moment of inertia were determined from radiographic data, stiffness from mechanical data, and apparent modulus from geometric and mechanical data., Results: Collagen-reduced discs had a larger area and polar moment of inertia compared to controls. The linear and torsional stiffness of collagen-reduced and control discs were not significantly different. Finally, the apparent modulus of collagen-reduced discs was significantly less than controls in compression (73% of control) and torsion (50%)., Conclusions: Compared to controls, collagen-reduced discs had reduced apparent modulus in both loading directions, suggesting that the transgenic disc tissue was mechanically inferior to controls. These results are consistent with the widely accepted functional role of type I collagen in disc mechanics, and therefore supports the use of transgenic mice to study structure-function relationships of the disc. Future work will focus on quantifying structure-function relationships related to degeneration, as well as those relevant to the design of tissue-engineered disc replacements.

Study Design: An intact bovine caudal disc model was used to investigate how combinations of biomechanical parameters influence the severity of disruption during compressive loading., Objectives: To quantify the combined influence of flex-ion, hydration level, and compressive loading rate on nuclear disruption., Summary of Background Data: The risk of disc pro-lapse is known to increase when the disc is loaded flexed. However, there are few experimental data available quantifying the extent to which loading parameters might interact to produce disruption in the healthy disc., Methods: Reproducible states of full and partial hydration were established for 96 isolated caudal discs. These discs were then subjected to compression under combined conditions of high or low hydration, zero or full flexion, and moderate or low loading rate. The extent of disc disruption was assessed macroscopically using a damage weighting procedure., Results: Maximum disruption of the intact, healthy disc occurred under combined conditions of full hydration and flexion. Loading rate, whether at 0.004 MPa/sec or 4 MPa/sec, had little influence when applied without flexion, but there was an increased risk of disruption when the moderate rate was combined with full flexion., Conclusions: The investigation demonstrates that significant levels of disruption can be induced by mechanical loading in intact discs free of degenerative change. Our findings support the hypothesis that mechanical injury to a healthy disc might initiate a process of degeneration.

The intervertebral disc is implicated as the source of low-back pain in a substantial number of patients. Because thermal therapy has been thought to have a therapeutic effect on collagenous tissues, this technique has recently been incorporated into several minimally invasive back pain treatments. However, patient selection criteria and precise definition of optimum dose are hindered by uncertainty of treatment mechanisms. The purpose of this study was to quantify acute changes in annulus fibrosus biomechanics after a range of thermal exposures, and to correlate these results with tissue denaturation. Intact annulus fibrosus (attached to adjacent vertebrae) from porcine lumbar spines was tested ex vivo. Biomechanical behavior, microstructure, peak of denaturation endotherm, and enthalpy of denaturation (mDSC) were determined before and after hydrothermal heat treatment at 37 degrees C, 50 degrees C, 60 degrees C, 65 degrees C, 70 degrees C, 75 degrees C, 80 degrees C, and 85 degrees C. Shrinkage of excised annular tissue (removed from adjacent vertebrae) was also measured after treatment at 85 degrees C. Significant differences in intact annulus biomechanics were observed after treatment, but the effects were much smaller in magnitude than those observed in excised annulus and those reported previously for other tissues. Consistent with this, intact tissue was only minimally denatured by treatment at 85 degrees C for 15 min, whereas excised tissue was completely denatured by this protocol. Our data suggest that in situ constraint imposed by the joint structure significantly retards annular thermal denaturation. These findings should aid the interpretation of clinical outcomes and provide a basis for the future design of optimum dosing regimens.

Previous experimental and analytical studies of solute transport in the intervertebral disc have demonstrated that for small molecules diffusive transport alone fulfils the nutritional needs of disc cells. It has been often suggested that fluid flow into and within the disc may enhance the transport of larger molecules. The goal of the study was to predict the influence of load-induced interstitial fluid flow on mass transport in the intervertebral disc. An iterative procedure was used to predict the convective transport of physiologically relevant molecules within the disc. An axisymmetric, poroelastic finite-element structural model of the disc was developed. The diurnal loading was divided into discrete time steps. At each time step, the fluid flow within the disc due to compression or swelling was calculated. A sequentially coupled diffusion/convection model was then employed to calculate solute transport, with a constant concentration of solute being provided at the vascularised endplates and outer annulus. Loading was simulated for a complete diurnal cycle, and the relative convective and diffusive transport was compared for solutes with molecular weights ranging from 400 Da to 40 kDa. Consistent with previous studies, fluid flow did not enhance the transport of low-weight solutes. During swelling, interstitial fluid flow increased the unidirectional penetration of large solutes by approximately 100%. Due to the bi-directional temporal nature of disc loading, however, the net effect of convective transport over a full diurnal cycle was more limited (30% increase). Further study is required to determine the significance of large solutes and the timing of their delivery for disc physiology.

Research in biologic methods of treating disc degeneration is still in its infancy. Many different strategies are being evaluated, but the gene therapy strategy stands out because of its potential for long-term efficacy. Choosing the correct gene for use in gene therapy is critically important. Of the many different classes of potentially therapeutic genes, the regulatory genes hold the most promise. Of the different gene therapy delivery methods, the most work has been performed with viral vectors, either ex vivo or in vivo. Current research now is turning toward in vivo experiments in rabbits. Efficacy and safety will be demonstrated first with smaller animal models. Beyond that, nonhuman primate experiments demonstrating efficacy and safety will be the penultimate step before initiation of human studies.

Hydrogels of poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP) blends may provide a material suitable for replacement of the nucleus pulposus of the intervertebral disc. This research examined the stability of these hydrogels under simulated physiological conditions. Polymer dissolution and stability were characterized over 120 days immersion, chemical surface analysis over 56 days immersion, and tensile mechanical behavior over 56 days immersion. Rubber elasticity theory was used by combining mechanical results with swelling data to calculate network characteristics such as the molecular weight between physical crosslinks and density of crosslinks. Properties were examined as a function of PVA/PVP composition as well as PVA molecular weight and PVP molecular weight. Results indicated that PVA/PVP blends prepared with moderate amounts of PVP (0.5-5%) resulted in a polymer network stabilized through interchain hydrogen bonding between hydroxyl groups on PVA chains and carbonyl groups on PVP chains. Most notably, a significant decrease in percentage of polymer mass loss was seen for blends prepared with 143K molecular weight PVA. Surface chemical analysis revealed that PVP unincorporated in the network structure suffered significant dissolution out of the polymer network and into solution. The molecular weight of PVA and PVP were shown to have a significant influence on the blends' network properties. Gels prepared with lower molecular weight PVA resulted in a more stable blend containing a higher density of crosslinks. However, blends prepared with a higher molecular weight PVA showed superior polymer network stability in dissolution studies. The blend that had the best combination of network stability under physiological conditions and a relatively tight, stable, and crosslinked network was prepared with 99% PVA (143K) and 1% PVP (40K). This material is proposed as an implant material for replacement of the degenerated nucleus pulposus., (Copyright 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 1329-1337, 2003)

Objective: Experimental immunity to the G1 domain of the cartilage proteoglycan (PG) aggrecan (AG1) leads to the development of spondylitis as well as polyarthritis in BALB/c mice. The PG versican contains a structurally similar G1 domain (VG1). This study was conducted to determine whether immunity to VG1 would elicit similar pathology in these mice., Methods: Recombinant natively folded VG1 and AG1 were prepared. BALB/c mice received either a series of 5 injections of human VG1 or AG1, or no protein. Polyarthritis was determined clinically, and spondylitis and sacroiliitis histologically. Immunohistochemistry of rat tissues was used to study the localization of versican. Enzyme-linked immunosorbent assays were employed to study humoral immunity to the recombinant proteins as well as to overlapping synthetic peptides covering all these human G1 domains and mouse homologs. Affinity-purified antibodies to human AG1 and VG1 were isolated from sera of hyperimmunized mice. T lymphocyte proliferation assays were performed using recombinant human proteins. T cell lines reactive with specific immunodominant T cell epitopes in human AG1 and VG1 were isolated. Synthetic peptides encoding sequences in these human proteins and in corresponding mouse proteins were used in these analyses. Guanidinium chloride extracts of mouse spines were also used in Western blots to study antibody cross-reactivity., Results: Immunity to recombinant VG1 did not result in clinical polyarthritis. There was, however, clear evidence that VG1, like AG1, could induce spondylitis in the lumbar spine and sacroiliitis. Accumulation of mononuclear cells was observed in spinal ligaments adjacent to the intervertebral disc, in the intervertebral disc, and in the sacroiliac joints, the same sites where versican is localized. In contrast to AG1-immunized mice, in which T cells reactive with human AG1 cross-reacted with mouse AG1, there was no evidence in VG1-immunized mice that T cell immunity to human VG1 was cross-reactive with a mouse synthetic peptide that contained the sequence corresponding to the single immunodominant T cell sequence recognized in human VG1. Antibodies to specific sequences in human VG1 did, however, cross-react with human AG1 and with corresponding peptide sequences in mouse versican and aggrecan and with mouse proteins containing VG1 and AG1, present in mouse spine extracts. Similarly, antibodies to human AG1 cross-reacted with human VG1 and with extracted mouse VG1 and AG1 and synthetic peptides containing mouse sequences that corresponded to the reactive human epitopes in AG1 and VG1., Conclusion: These observations suggest that humoral immunity to human VG1 is involved in the induction of experimental spondylitis and sacroiliitis in BALB/c mice. This humoral immunity is cross-reactive with mouse versican and aggrecan but is not associated with polyarthritis, probably because of the lack of cross-reactive T cell immunity and the absence of detectable versican in articular cartilage limbs. Induction of polyarthritis by bovine or human aggrecan requires the involvement of immunity mediated by T lymphocytes that are cross-reactive to a mouse aggrecan epitope. Together these observations suggest that humoral immunity to versican as well as immunity to aggrecan may be of importance in the development of the spinal pathology characteristic of spondylarthropathies.

Study Design: Immunohistochemical study of the angiogenesis and inflammatory cell invasion in lumbar disc herniation. OBJECTIVES To observe the blood vessel formation within the extracellular matrix in lumbar disc herniation, and to elucidate the role of angiogenesis in the natural shrinking of hernias., Summary of Background Data: There have been few reports of detailed observation of blood vessel formation within the extracellular matrix, and the role that angiogenesis plays in the natural shrinking of hernias has not been elucidated., Methods: Twenty tissue samples surgically removed from 17 patients with herniated discs were studied (9 men, 8 women, 23-58 years old, 11 extrusion type, 9 sequestration type). In the immunohistochemical study, an anti-CD34 antibody for vascular endothelial cells, an anti-CD68 for macrophages, and an anti-vascular endothelial growth factor antibody was used for vascular endothelial growth factor., Results: Many spindle-shaped cells expressing vascular endothelial growth factor were seen inside granulation tissue infiltrating the cartilage matrix, and the number of vascular endothelial growth factor-positive cells and the number of CD34+ cells were positively correlated (R = 0.73, P < 0.001). In the area surrounding CD34+ cells that had formed a lumen, many CD68+ cells were observed, and the number of CD34+ cells and the number of CD68+ cells were positively correlated (R = 0.66, P < 0.001)., Conclusions: The results suggest that the vascular endothelial growth factor produced by the spindle-shaped cells acts to promote angiogenesis inside granulation tissue infiltrating the cartilage matrix, and that new blood vessels play an important role as a passage for macrophages into the degenerated matrix.

The volume of the intrafibrillar water space--i.e. the water contained inside the collagen fibres--is a key parameter that is relevant to concepts of connective tissue structure and function. Confined compression and swelling experiments on annulus fibrosus samples are interpreted in terms of a dual porosity model that distinguishes between a non-ionised intrafibrillar porosity and an ionised extrafibrillar porosity. Both porosities intercommunicate and are saturated with a monovalent ionic solution, i.c. NaCl. The extrafibrillar fixed charge density of the samples is assessed using radiotracer techniques and the collagen content is evaluated by measurement of hydroxyproline concentration. The interpretation of the experimental data yields values for the intrafibrillar water content, the average activity coefficient of the ions, the Donnan osmotic coefficient, the fraction of intrafibrillar water, the stress-free deformation state, and an effective stress-strain relationship as a function of the radial position in the disc. A linear fit between the second Piola-Kirchhoff effective stress and Green-Lagrange strain yielded an effective stiffness: H(e)=1.087 +/- 0.657 MPa. The average fraction of intrafibrillar water was 1.16 g/g collagen. The results were sensitive to changes in the activity and osmotic coefficients and the fraction of intrafibrillar water. The fixed charge density increased with distance from the outer edge of the annulus, whereas the hydroxyproline decreased.

Study Design: Human cadaveric lumbar spines were used to assess the temperature and thermal dose distribution during intradiscal electrothermal therapy in vitro., Objectives: To determine whether intradiscal electrothermal therapy produces adequate tissue temperatures to denature annular collagen or ablate nerve cells., Summary of Background Data: Several hypothesized mechanisms for the effect of intradiscal electrothermal therapy have been suggested and include: 1) shrinkage of the nucleus and/or the annulus fibrosus by contraction of collagen fibers; and 2) thermal ablation of sensitive nerve fibers in the outer annulus., Methods: Intradiscal electrothermal therapy was performed using the standard clinical protocol on 12 lumbar specimens in a 37.0 degrees C water bath using the SpineCath by Oratec. Temperatures were recorded simultaneously at 40 different locations in the disc. Thermal dose (Equivalent Minutes 43.0 degrees C) was calculated at each temperature point., Results: The highest temperature measured (out of 520 points) was 64.0 degrees C and was within 1 mm of the heating coil. Temperatures in excess of 60 degrees C were all within 1 to 2 mm of the intradiscal electrothermal therapy catheter surface, the 50 to 60 degrees C range extended approximately 6 mm, above 48 degrees C extended approximately 7 mm, and above 45 degrees C extended to approximately 10 mm. Less than 2% of points achieved temperatures sufficient for collagen denaturation (>60 degrees C). On average, 42.5% of points accumulated >250 Equivalent Minutes 43.0 degrees C, a conservative common dose threshold for thermal necrosis of cells. The time history of thermal measurements demonstrated that the disc temperature had not reached steady state by the end of the heating protocol (16.5 minutes)., Conclusions: Except for a very limited margin (1-2 mm) around the catheter, the temperature necessary to induce collagen shrinkage was not observed within the disc. Temperatures sufficient to ablate nerves were developed in some areas but were not reliably produced in clinically relevant regions, such as the posterior annulus. These results suggest that beneficial clinical outcomes may be critically dependent on probe placement or other factors unknown.

Nucleus pulposus, the central zone of the intervertebral disc, is gel-like and has a similar collagen phenotype to that of hyaline cartilage. Amino-terminal protein sequence analysis of the alpha1(IX)COL3 domain purified from bovine nucleus pulposus gave a different sequence to that of the long alpha1(IX) transcript expressed in hyaline cartilage and matched the predicted sequence of short alpha1(IX). The findings indicate that the matrix of bovine nucleus pulposus contains only the short form of alpha1(IX) that lacks the NC4 domain. The sequence encoded by exon 7, predicted from human COL9A1, is absent from both short and long forms of alpha1(IX) from bovine nucleus pulposus and articular cartilage. A structural analysis of the cross-linking sites occupied in type IX collagen from nucleus pulposus showed that usage of the short alpha1(IX) transcript in disc tissue had no apparent effect on cross-linking behavior. As in cartilage, type IX collagen of nucleus pulposus was heavily cross-linked to type II collagen and to other molecules of type IX collagen with a similar site occupancy.

The characteristics of the nucleus pulposus cells from adult rabbits maintained in in vitro cultures were described in another study. Herein, the authors provide a parallel profile of adult rabbit nucleus pulposus in situ, therefore allowing direct comparisons between in vitro and in situ investigations. Nucleus pulposus specimens from adult rabbits were evaluated using biochemical and immunohistochemical morphologic techniques. The nucleus pulposus from adult rabbits contained cell clusters embedded in proteoglycan-collagen matrix. The cells exhibited a well-defined Golgi system, an extensive endoplasmic reticulum, and a complex vesicular system filled with beaded structures (proteoglycans). Neither necrotic nor apoptotic cells were evident. There was a lack of mitochondria. The extensive extracellular matrix contained amorphous, beaded, and fibrillar components. The fibrillar banding was indicative of Type VI collagen. The nucleus pulposus of adult rabbits expressed aggrecan, collagen Type I and Type II, and CD44, but not collagen Type X and displayed low alkaline phosphatase activity.

Human thoracic discs were analyzed for collagen and collagen cross-links to determine the distribution due to segmental, age, and gender influences. Thoracic discs from 26 cadaveric spines (1 to 90 years old) were graded macroscopically, then separated into anular and nuclear samples. Only grade I (i.e., normal) disc samples were selected (n=209). Pyridinoline and deoxypyridinoline cross-links were initially separated by column chromatography and analyzed by reverse-phase high-pressure liquid chromatography. The collagen content was lower and the extent of pyridinoline and deoxypyridinoline were significantly higher in the nucleus compared with the anulus (p <.001). The collagen content and extent of pyridinoline were significantly lower with increasing age in the anulus and nucleus (p <.001). Young male discs had a significantly higher extent of pyridinoline compared with females (p <.001). Age, gender, and disc region differences were found to have a significant influence on the biochemical composition of the normal disc extracellular matrix.

Study Design: An in vivo experimental study to examine the possibility of using fresh frozen intervertebral disc allograft in disc transplantation., Objectives: To investigate the long-term radiographic, pathologic, biochemical, and biomechanical changes of fresh frozen disc allograft in a bipedal animal model., Summary of Background Data: It has been shown that intervertebral disc autograft is able to survive and maintain some degree of tissue metabolism and segmental mobility after transplantation in a bipedal animal model. However, the long-term results of disc allografting and the associated problems of graft rejection are unknown., Methods: Seventeen rhesus monkeys (15 male, 2 female) between 5 and 8 years of age and weighing between 6.7 and 11.8 kg were used in this study. Of these 17 subjects, two were used as intervertebral disc donors and three were used as controls for the biomechanical testing. The remaining 12 monkeys were randomly divided into a short-term group (n = 4, followed up for 2, 4, 6, and 8 weeks, respectively), a midterm group (n = 6, 6 months), and a long-term group (n = 2, 24 months). Radiologic, histologic, biochemical, and biomechanical changes were investigated., Results: Radiography and macro- and microhistologic examination showed severe disc degeneration at 24 months of follow-up. Disc height decreased mainly in the early postoperative stage. Decreased water, proteoglycan, and hydroxyproline contents of the allograft were observed at 6 and 24 months of follow-up. The biomechanical properties of the transplanted allograft were similar to those of control., Conclusion: Fresh frozen disc allografts can survive and maintain some degree of cell metabolism and segmental mobility at 24 months after transplantation. However, severe disc degeneration is also observed at this stage.

A 3-dimensional formulation for a poroelastic and chemical electric (PEACE) model is presented and applied to an intervertebral disc slice in a 1-dimensional validation problem and a 2-dimensional plane stress problem. The model was used to investigate the influence of fixed charge density magnitude and distribution on this slice of disc material. Results indicated that the mechanical, chemical, and electrical behaviors were all strongly influenced by the amount as well as the distribution of fixed charges in the matrix. Without any other changes in material properties, alterations in the fixed charge density (proteoglycan content) from a healthy to a degenerated distribution will cause an increase in solid matrix stresses and can affect whether the tissue imbibes or exudes fluid under different loading conditions. Disc tissue with a degenerated fixed charge density distribution exhibited greater solid matrix stresses and decreased streaming potential, all of which have implications for disc nutrition, disc biomechanics, and tissue remodeling. It was also seen that application of an electrical potential across the disc can induce fluid transport.

Study Design: Complement membrane attack complexes were located in lumbar spine disc tissues by immunohistochemistry. Their occurrence was compared in control discs obtained from organ donors (CD), discs showing a normal macroscopic anatomy, samples of intervertebral disc herniations (DH), and intervertebral discs found to be degenerated by discography, but not herniated (DD)., Objective: To look for a possible role of complement activation, specifically complement membrane attack complexes, an end product of the classic immune complex-mediated complement activation pathway, in disc pathophysiology., Summary of Background Data: Recent immunohistochemical and biochemical studies suggest a possible role for immune complexes, as observed by immunohistochemical location and biochemical assay of immunoglobulins M and G in intervertebral disc pathophysiology. Immune complexes may trigger complement activation and ultimately cell lysis. There are, however, currently no reports on complement activation in disc tissues, although immune (antigen-antibody) complexes have been demonstrated. Such immune complexes have been reported to occur on or near to disc cells in DH tissues., Methods: Thin frozen sections of disc tissue from CD (n = 9 discs), DH (n = 58 discs), and DD (n = 11 discs) were cut and then immunostained with a monoclonal antibody to the complement membrane attack complex (C5b-9) using avidin-biotin complex (ABC) immunostaining. The presence or absence of complement membrane attack complex immunoreactivity was compared in the various subtypes of DH and also with preoperative duration of radicular pain., Results: Complement membrane attack complexes could be observed in none of the CDs studied. In contrast, in more than one third of both the DH (21/58, 36.2%) and the DD (4/11, 36.4%), immunoreactivity to complement membrane attack complexes could be observed in disc cells. In DD discs, immunoreactivity to complement membrane attack complexes was most often present in anulus fibrosus samples (5/13, 38.5%). With respect to subtype of DH, complement membrane attack complexes were observed in 19 of 36 sequestrated discs (52.8%), 1 of 16 extrusions (6.3%), and 1 of 6 protrusions (16.7%). Complement membrane attack complexes were more often present with shorter pain duration (P= 0.03), but showed no relation to age. Disc cells often showed a heavy staining pattern for complement membrane attack complexes, suggesting an abundance of these complexes lodged in the membrane of the cells., Conclusions: The predominant presence of complement membrane attack complexes in sequestrated disc tissue could suggest a role in DH tissue-induced sciatica. Possibly immune (antigen-antibody) complexes, reported in previous studies, trigger the classic pathway of complement activation, with complement membrane attack complexes as the final product. Complement membrane attack complexes also appear to have some as yet undefined role in degenerated nonherniated disc tissue, with a predominant presence in the anulus fibrosus cells of such discs.

Elastic fibres have been revealed by both elastin immunostaining and conventional histological orcein-staining in the intervertebral discs of the bovine tail. These fibres are distributed in all regions of the disc but their organization varies from region to region. In the centre of the nucleus, long (> 150 microm) elastic fibres are orientated radially. In the transitional region between nucleus and annulus, the orientation of the elastic fibres changes, producing a criss-cross pattern. In the annulus itself, elastic fibres appear densely distributed in the region between the lamellae and also in 'bridges' across the lamellae, particularly in the adult. Elastic fibres are apparent within the lamellae, orientated parallel to the collagen fibres of each lamella, particularly in the young (12-day-old) discs. In the region between the disc and the cartilaginous endplate, elastic fibres appear to anchor into the plate and terminate there. The results of this study suggest that elastic fibres contribute to the mechanical functioning of the intervertebral disc. The varying organization of the elastic fibres in the different regions of the disc is likely to relate to the different regional loading patterns.

Study Design: Experimental investigation of the electrical conductivity of normal and trypsin-treated lumbar anulus fibrosis specimens., Objectives: To measure the electrical conductivity of intervertebral disc tissues and to study the effects of tissue porosity (volume fraction of water) and fixed charge density on the electrical conductivity of anulus fibrosis in physiologic saline., Summary of Background Data: Specific electrical conductivity is one of the material properties of intervertebral discs. Their value depends on ion concentrations and ion diffusivities within the tissue, which in turn are functions of tissue composition and structure. To our knowledge, the electrical conductivity of intervertebral discs has not been studied. Investigation of the electrical conductivity of intervertebral discs and understanding of their relationship to tissue porosity and fixed charge density will provide insights into electromechanical phenomena (e.g., streaming potential) and ion transport in intervertebral discs., Methods: A total of 35 porcine lumbar anulus fibrosis specimens were divided into two groups: one control group (n = 10) and one trypsin-treated group (n = 25). The specimens in the control group were subjected to one-dimensional free swelling in phosphate-buffered saline (pH 7.4), and electrical conductivity and porosity (water content) were measured over a period of about 45 minutes. The specimens in the treated group were immersed in a trypsin solution (372 U/mL phosphate-buffered saline) for 45 minutes at room temperature, and the electrical conductivity and porosity were measured after treatment. The electrical conductivity was correlated to tissue porosity for the control and treated specimens. The influences of porosity and fixed charge density were studied., Results: The average value for control specimens was 5.60 +/- 0.89 mS/cm (mean +/- SD; n = 10) before swelling and 9.11 +/- 0.90 mS/cm (mean +/- SD; n = 10) after swelling. Tissue porosity increased from 0.74 +/- 0.03 (mean +/- SD; n = 10) before swelling to 0.83 +/- 0.02 (mean +/- SD; n = 10) after swelling. The trypsin treatment reduced anulus fibrosis porosity by 3.6% (P < 0.05) and conductivity by 13% (P < 0.05) compared to those for control specimens after swelling. No significant changes werefound in wet and dry tissue densities between control and treated groups. There was a significant, linear correlation between conductivity and porosity for control anulus fibrosis specimens (R2 = 0.87; 86 measurements)., Conclusions: Measured electrical conductivity was sensitive to tissue porosity, but not to fixed charged density for anulus fibrosis specimens in phosphate-buffered saline.

Study Design: Intervertebral disc tissue was analyzed during or removed at routine surgery for correction of scoliosis. Tissue was analyzed for glucose, lactate, oxygen, glycosaminoglycan, collagen concentrations, and cell viability., Objectives: To investigate the cell viability of the scoliotic disc on the concave and convex sides and in relation to curve apex, and to relate cell viability to concentrations of nutrients, metabolites, and extracellular matrix components., Summary of Background Data: Compositional differences have been measured in relation to the deformation of scoliotic discs. However, the causes of these in relation to cellular activity or viability are unknown., Methods: Oxygen concentration was measured at surgery using a microelectrode. A segment of disc then was removed and sections at defined locations measured for cell viability and glucose, lactate, glycosaminoglycan, and collagen concentrations. RESULTS Cell viability was lower toward the convex side of the curve, with the greatest difference between the sides in the apical disc. The apical disc had the lowest oxygen and highest lactate concentrations, and lowest total number of cells. Glucose concentration correlated with the number of live cells. Concentrations of glycosaminoglycans and collagen per dry weight of tissue were similar on both sides of the disc., Conclusions: Differences in cell viability correlated with changes in nutrient and metabolite levels, and also with disc deformity (convex concave and distance from curve apex). Thus asymmetrical loads, tissue deformation, and nutrient supply may work separately or in combination to cause cell death. A loss of matrix macromolecules was not seen, possibly because the period between cell death and surgery was too short, as compared with long matrix turnover times. Cell death is expected eventually to have a deleterious effect on cell matrix and disc function.

Study Design: This study correlates the mRNA and protein levels of large and small proteoglycans with the morphologic grade of degeneration., Objectives: To investigate changes in mRNA and protein levels of aggrecan, versican, biglycan, decorin and fibromodulin in the anulus fibrosus and the nucleus pulposus at different stages of tissue degeneration., Summary of Background Data: Proteoglycans are found in both the anulus fibrosus and nucleus pulposus and contribute to the hydration of the tissue (aggrecan) and the regulation of matrix assembly (small proteoglycans). Changes in their abundance may influence the biochemical and biomechanical properties of the disc and may have an effect on disc function., Methods: Thirty-four human lumbar intervertebral discs were graded using Thompson's morphologic scale. The mRNA and protein content of aggrecan, versican, biglycan, decorin and fibromodulin was measured in extracts of anulus fibrosus and nucleus pulposus tissues dissected from these specimens. Protein levels were analyzed by Western blotting, and mRNA levels were semiquantified by reverse transcription polymerase chain reaction., Results: In the anulus fibrosus, the mRNA and protein content of all proteoglycans was significantly elevated at early stages of degeneration then declined in severely degenerated tissues. In contrast, matrix components in the nucleus pulposus showed a continuous decrease in content with increasing grade of degeneration., Conclusions: Cells of the anulus fibrosus and nucleus pulposus react to tissue degeneration differently. Decreased mRNA expression by nucleus pulposus cells and declining protein content of the matrix make the nucleus more vulnerable to degeneration than the anulus. The cells in the anulus fibrosus respond to early degeneration by upregulating biosynthetic processes. However, in heavily degenerated tissues, the decline in the synthesis of aggrecan and the increase in the concentrations of small proteoglycans may be responsible for the failure of the repair processes.

Objective: To assess the effects of human intervertebral disc aggrecan on nerve growth and guidance, using in vitro techniques., Methods: Aggrecan extracted from human lumbar intervertebral discs was incorporated into tissue culture substrata for the culture of the human neuronal cell line, SH-SY5Y, or explants of chick dorsal root ganglia. The effects on nerve growth of different concentrations of aggrecan extracted from the anulus fibrosus and nucleus pulposus, and of these aggrecan preparations following enzymic deglycosylation, were compared., Results: Disc aggrecan inhibited the growth of neurites from SH-SY5Y cells and induced growth cone turning of chick sensory neurites in a concentration-dependent manner. Aggrecan isolated from the anulus fibrosus was more inhibitory than that isolated from the nucleus pulposus, but enzymic pretreatments to reduce the glycosylation of both types of disc aggrecan partially abrogated their inhibitory effects., Conclusion: Nerve growth into degenerate intervertebral discs has been linked with the development of low back pain, but little is known about factors affecting disc innervation. The finding that disc aggrecan inhibits nerve growth in vitro, and that this inhibitory activity depends on aggrecan glycosylation, has important implications for our understanding of mechanisms that may regulate disc innervation in health and disease.

Study Design: A study was conducted to investigate the expression of cytokines related to the immune reaction in herniated lumbar disc tissues., Objective: To investigate the immunologic status of lumbar disc tissue and the type of immune reaction that occurs in response to lumbar disc herniation., Summary of Background Data: It has been proposed that herniated lumbar disc tissue causes an immune reaction. Various inflammatory cells, proinflammatory cytokines, antibodies, and immunoglobulins have been identified in and around herniated lumbar disc tissue. Recently, it has been reported that lumbar disc tissue may be another potential immune-privileged site in the human body., Methods: This study included 40 herniated lumbar disc tissues: 20 contained and 20 noncontained discs. The concentrations of interleukin-12, T-helper Type 1, interferon-gamma (IFN-gamma), and Th2 (IL-4) cytokines were determined by enzyme-linked immunosorbent assay, respectively. The results were compared between the two groups., Results: The concentrations of interleukin-12 and interferon-gamma were higher in the noncontained discs than in the contained discs: 28.3 +/- 10.7 pg/mL vs 9.2 +/- 4.2 pg/mL (P = 0.001) and 4.7 +/- 5.0 pg/mL vs 2.3 +/- 3.8 pg/mL (P = 0.029), respectively. On the contrary, the concentration of IL-4 was higher in the contained discs than in the noncontained discs: 24.3 +/- 20.1 pg/mL vs 1.9 +/- 4.5 pg/mL; P= 0.001. The degrees of interleukin-12 and interferon-gamma expression were negatively correlated with that of IL-4 (n = 40): correlation coefficient, -0.671 (P = 0.001) and correlation coefficient, -0.344 (P = 0.03), respectively., Conclusions: The findings suggest that preferential expression of Th2 cytokines by disc cells, as shown in contained discs, is another factor contributing to the immune privileged status of lumbar disc tissue. The exposure of lumbar disc tissue to the epidural space may increase the concentration of interleukin-12 in herniated lumbar disc tissue, changing the pattern of T-helper Types 1 and 2 cytokine expression.

The three-dimensional architecture of cells in the annulus fibrosus was studied by a systematic, histological examination using antibodies to cytoskeletal components, in conjunction with confocal microscopy. Variations in cell shape, arrangement of cellular processes and cytoskeletal architecture were found both within and between the defined zones of the outer and inner annulus. The morphology of three, novel annulus fibrosus cells is described: extended cordlike cells that form an interconnected network at the periphery of the disc; cells with extensive, sinuous processes in the inner region of the annulus fibrosus; and cells with broad, branching processes specific to the interlamellar septae of the outer annulus. The complex, yet seemingly deliberate arrangement of various cell shapes and their processes suggests multiple functional roles. Regional variations in the organization of the actin and vimentin cytoskeletal networks is reported across all regions of the annulus. Most notable is the continuous, strand arrangement of the actin label at the disc's periphery in contrast to its punctate appearance in all other regions. The gap junction protein connexin 43 was found within cells from all regions of the annulus, including those which did not form physical connections with surrounding cells. These observations of the cellular matrix in the healthy intervertebral disc should contribute to a better understanding of site-specific changes in tissue architecture, biochemistry and mechanical properties during degeneration, injury and healing.

Study Design: To analyze the effects of tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma on cauda equina function and to define if any of these cytokines could induce nerve root dysfunction comparable with the situation with application of nucleus pulposus., Summary of Background Data: Proinflammatory cytokines derived from the intervertebral disc have been suggested to mediate the nucleus pulposus-induced nerve root injury following local application of nucleus pulposus. However, it is not known if such cytokines may induce similar injury if applied separately., Methods: A total of 29 pigs were used. Nucleus pulposus was harvested from lumbar discs and applied to the sacrococcygeal cauda equina following laminectomy of the first coccygeal vertebra in seven pigs. Five pigs received 1.66 microg of tumor necrosis factor-alpha, five pigs received 0.85 microg of interleukin-1beta, and five pigs received 1.66 microg of interferon-gamma. Seven pigs received autologous fat for control. Nerve conduction velocity was studied by local electrical stimulation and recordings in the tail muscles 7 days after the application., Results: Application of nucleus pulposus and fat induced similar effects as seen in previous studies, with normal nerve conduction velocity for fat and a significant reduction for nucleus pulposus. Application of both interleukin-1beta and IFN-gamma induced slight reductions of nerve conduction velocity compared with fat, but they were not statistically significant. Tumor necrosis factor-alpha, however, induced a reduction of the velocity that was even more pronounced than for nucleus pulposus., Conclusion: Based on previous observations and the data of the present study, one may conclude that tumor necrosis factor-alpha from nucleus pulposus cells seems to be intimately involved with the basic pathophysiologic events leading to both nerve root dysfunction and pain after local, epidural application of nucleus pulposus. One may therefore also suspect that pharmacologic inhibition of tumor necrosis factor-alpha may at least theoretically be considered in the clinical situation with disc herniation and sciatica.

Objective: To determine the hydration-over-time behaviour of ovine intervertebral discs and intact joints in a saline bath at body temperature and the effect this has on their stiffness compared to air at ambient temperature., Design: The hydration-over-time behaviour and stiffness of the ovine functional spinal unit and disc were quantified., Background: The fluid content of an intervertebral disc is not constant but varies with external load and load history. The stiffness of ovine functional spinal units in a hydrated environment and how this compares to testing in air have not been quantified., Methods: Intervertebral discs and functional spinal units were weighed and soaked in a saline water bath at 37 degrees C and reweighed each hour for 6 h. They were then allowed to stand in air at room temperature while the time to return to initial weight was recorded. Functional spinal units were randomly assigned to two groups. Axial compression, flexion, extension, lateral bending and axial torsion tests were performed on both the intact functional spinal unit and isolated disc. Group 1 was tested in air then in a saline water bath at 37 degrees C with the testing order reversed for Group 2., Results: Hydration of the disc reached a plateau after an average 3-4 h of soaking with the largest increase seen in the first hour. Four hours, standing in air at room temperature, was required to return specimens to their initial weight. The functional spinal unit stiffness was significantly lower for those specimens tested in the bath compared to air., Conclusions: Ovine intervertebral discs show similar hydration-over-time behaviour when compared to human discs. Stiffnesses in different modes of loading were significantly different when tested in a hydrated environment compared with the standard method of testing in air., Relevance: It has been shown that there are biomechanical and biochemical similarities between sheep and human intervertebral discs. Despite these similarities, no studies have looked at how ovine intervertebral discs behave over time in a hydrated environment. In humans, hydration levels are an important aspect of intervertebral disc degeneration. There is also a relationship between decreased hydration levels and increased stiffness. This study demonstrates the similarities between human and ovine hydration-over-time behaviour. The importance of intervertebral disc hydration and its effects on stiffness under different modes of loading were also demonstrated and have not been previously shown using the ovine model. In this context, the results from this study provide further support for the use of the ovine model.

Study Design: Scoliotic and herniated human intervertebral disc tissue obtained intraoperatively was cultured, and the medium was analyzed for the production of monocyte chemoattractant protein-1 and interleukin-8., Objectives: This study was conducted to determine whether the human intervertebral disc is capable of spontaneous production of the chemokines monocyte chemoattractant protein-1 and interleukin-8., Summary of Background Data: Lumbar disc herniations undergo spontaneous regression with time. This is believed to occur via macrophage-mediated phagocytosis of herniated disc material. Monocyte chemoattractant protein-1, a chemotactic agent for macrophages, has recently been identified in rat intervertebral disc tissue., Methods: Disc material obtained from patients undergoing surgery for scoliosis and sciatica was cultured using a serumless technique, and the medium was subsequently analyzed for levels of monocyte chemoattractant protein-1 and interleukin-8., Results: Monocyte chemoattractant protein-1 and IL-8 were detected in both control and herniated disc specimens. Noncontained herniations produced higher levels of chemokines than those with an intact anulus., Conclusions: Human intervertebral disc tissue is capable of spontaneously producing the proinflammatory chemokines monocyte chemoattractant protein-1 and interleukin-8. These are chemotactic for macrophages and capillaries and may explain the ingrowth of granulation tissue seen in spontaneous disc herniation resorption.

Rationale and Objectives: The authors performed this study to evaluate whether a semiquantitative method of in vivo sodium imaging of the human intervertebral disk could provide diagnostic quality images in a reasonable time., Materials and Methods: In vivo sodium imaging of the human spine was performed with a 4-T whole-body magnetic resonance (MR) unit by using custom-built hardware and software. Short-echo-time images were obtained with a modified three-dimensional gradient-echo sequence, a custom-built surface coil, and receiver modifications to allow for nonproton data acquisition. Corrections for surface coil image intensity were performed with phantom image data. An estimation of the fixed charge density within the intervertebral disk was made with the surface coil-corrected images by using cerebrospinal fluid as an internal reference standard., Results: In vivo sodium images of the spine with high signal-to-noise ratio and resolution were obtained in approximately 10 minutes. Correction of in vivo images for surface coil effects demonstrated that the sodium content of the cerebrospinal fluid in the spinal canal may be used as an internal standard to estimate the proteoglycan content of the intervertebral disk., Conclusion: Diagnostic quality in vivo sodium images of the intervertebral disk are technically feasible and may be useful for assessing the proteoglycan content of the intervertebral disk. This would provide a method to detect early degenerative changes in the disk and evaluate the effects of any subsequent therapy.

Attempts to study the biology of the nucleus pulposus have been limited in scope due to the low rates of cell proliferation, difficulties in maintaining viable disc cells in culture and the absence of a clearly defined phenotype. The major objective of this communication is to construct a phenotypic signature for cells of the nucleus pulposus that is based on the hypothesis that in response to restriction on oxygen and nutrient flux, there is expression of HIF-1, GLUT-1 and MMP-2. Nucleus pulposus, as well as annulus fibrosus and cartilage of the vertebral end plates, was collected from rat spinal units. Western blot analysis and immunohistochemistry clearly showed that there was a significant level of expression of the HIF-1 beta isoform in the nucleus pulposus; HIF-1 beta was present at lower levels in cells of the annulus and the end plate. In contrast to HIF-1 beta, HIF-1 alpha was expressed only in the nucleus pulposus. This isoform was absent from both the cartilage end plate and annulus. We detected HIF-1 alpha immunohistochemically in the nucleus pulposus; however, the staining was light and diffuse. Cells of the nucleus pulposus expressed GLUT-1; in contrast, when probed by Western blot analysis the annulus and cartilage were negative for this protein. Western blot analysis also showed that in the nucleus pulposus the level of MMP-2 was high when compared to the adjacent tissues. We suggest that the differential expression of the two HIF isoforms, and GLUT-1 and MMP-2, provides a phenotypic signature that permits cells of the nucleus pulposus to be distinguished from neighboring tissues. Moreover, the presence of these isoforms provides evidence that cells of the disc respond to hypoxia and nutrient stress by upregulating stress-responsive genes.

Collagen possesses a strong second-order nonlinear susceptibility, a nonlinear optical property characterized by second harmonic generation in the presence of intense laser beams. We present a new technique involving polarization modulation of an ultra-short pulse laser beam that can simultaneously determine collagen fiber orientation and a parameter related to the second-order nonlinear susceptibility. We demonstrate the ability to discriminate among different patterns of fibrillar orientation, as exemplified by tendon, fascia, cornea, and successive lamellar rings in an intervertebral disc. Fiber orientation can be measured as a function of depth with an axial resolution of approximately 10 microm. The parameter related to the second-order nonlinear susceptibility is sensitive to fiber disorganization, oblique incidence of the beam on the sample, and birefringence of the tissue. This parameter represents an aggregate measure of tissue optical properties that could potentially be used for optical imaging in vivo.

Study Design: This study examined how the culture system and region of cellular origin affect disc cell morphology and extracellular matrix production., Objective: To determine the role of the cell populations in the different regions of the adult intervertebral disc in maintaining gradients in composition across the disc., Summary of Background Data: It is not known whether the steep profiles in composition across the intervertebral disc are maintained by distinct cell populations or whether differences in cell metabolism are determined by changes in the physical environment across the disc. Very little information exists on the matrix produced by cells from the mature, non-notochordal nucleus pulposus., Methods: Cells were extracted from articular cartilage, nucleus pulposus, and the inner and outer anulus fibrosus of caudal discs from 18- to 24-month-old steers cultured in alginate or collagen gels or in monolayer. The effect of culture system and cell origin on cell morphology and matrix synthesis was measured using 35S-sulphate labeling and indirect immunolocalization., Results: Distinct morphologic differences between cells from different regions cultured in monolayer were retained through two passages. The rate of sulfate incorporation varied with cell type. Immediately after isolation, it was two- to threefold greater for nucleus cells than for cells from the disc inner anulus or articular cartilage. The rate was lowest for outer anulus cells. It also varied with culture system. For all cell types, the incorporation rate was highest in alginate and lowest in monolayer. Immunolocalization showed that nucleus cells stained strongly for all proteoglycan epitopes, whereas outer anulus cells stained least and in monolayer produced little proteoglycan., Conclusions: The disc has at least three distinct cell populations, which differ in morphology and in amount and type of matrix they produce. Cells from mature nucleus pulposus produced sulfated glycosaminoglycans at a high rate in contrast to reported results for notochordal nucleus cells. Alginate, although an appropriate culture system for inner anulus and nucleus cells, may not be a suitable medium for outer anulus cells.

Aims: To investigate the phenotype of cells in normal and degenerate intervertebral discs by studying the expression of molecules characteristic of chondrocytes in situ., Methods: Human intervertebral discs taken at surgery were graded histologically, and classified on this basis as normal or degenerate. Eighteen of each type were selected, and in situ hybridisation was performed for the chondrocytic markers Sox9 and collagen II using (35)S labelled cDNA probes. Aggrecan was located by immunohistochemistry, using the monoclonal antibody HAG7E1, and visualised with an avidin-biotin peroxidase system., Results: In the normal discs, strong signals for Sox9 and collagen II mRNA, and strong staining for the aggrecan protein were seen for the cells of the nucleus pulposus (NP), but reactions were weak or absent over the cells of the annulus fibrosus (AF). In degenerate discs, the Sox9 and collagen II mRNA signals remained visible over the cells of the NP and were again absent in the AF. Aggrecan staining was not visible in the NP cells, and was again absent in the AF., Conclusions: Cells of the normal NP showed expression of all three markers, clearly indicating a chondrocytic phenotype. In degeneration, there was evidence of a loss of aggrecan synthesis, which may contribute to the pathogenesis of disc degeneration. AF cells showed no evidence of a chondrocytic phenotype in either normal or degenerate discs.

Our study establishes a rabbit model of disc degeneration which requires neither a chemical nor physical injury to the disc. Disc degeneration similar to that seen in man was created at levels proximal (L4-L5) and caudal (L7-S1) to a simulated lumbar fusion and was studied for up to nine months after arthrodesis. Loss of the normal parallel arrangement of collagen bundles within the annular lamellae was observed in intervertebral discs adjacent to the fusion at three months. By six months there was further disorganisation as well as loss of distinction between the lamellae themselves. By nine months the structure of the disc had been replaced by disorganised fibrous tissue, and annular tears were seen. There was an initial cellular proliferative response followed by loss of chondrocytes and notochordal cells in the nucleus pulposus. Degeneration was accompanied by a decrease in the monomer size of proteoglycans. Narrowing of the disc space, endplate sclerosis and the formation of osteophytes at adjacent disc spaces were observed radiologically.

In order to be able to study the metabolism of nucleus pulposus (NP) tissue, we developed a cell culture system that resulted in the formation of NP-like tissue in vitro. NP cells were isolated from sheep lumbar spines and grown on filter inserts (Millicell CM). Histological examination showed that the cells accumulated extracellular matrix and formed a continuous layer of NP-like tissue. The accumulation of sulfated proteoglycans in the NP-like tissue continued up to 10 weeks and this was paralleled by an increase in tissue thickness and dry weight. DNA content remained stable during the first 4 weeks but then decreased over time. The amount of DNA, glycosaminoglycan (GAG) and collagen per mg dry weight of the tissue generated after 10 weeks in culture were 1.25+/-0.02, 301.6+/-27.7 and 411+/-65 microg, respectively, compared with 1.04+/-0.08, 320.6+/-21.2 and 399+/-4.4 microg (mean +/- SEM) for the in vivo tissue. There was no significant difference between in vitro and in vivo tissue. The cells in culture synthesized large proteoglycans (kav = 0.26+/-0.03, mean +/- S.D.) which were similar in size to those synthesized by cells in NP tissue in ex vivo culture (kav = 0.22+/-0.02, mean +/- S.D.) as determined by Sepharose CL-2B column chromatography. The in vitro generated tissue contained type II collagen as demonstrated by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and silver staining as well as Western blot analysis. NP cells grown on filters generate tissue similar in composition to the in vivo tissue, for the characteristics examined to date, and should be a suitable model to use to study NP metabolism and extracellular matrix turnover.

Stanniocalcin (STC) is a polypeptide hormone discovered first in fish and more recently in mammals. In mammals, the gene is widely expressed and the hormone is, so far, known to be involved in regulating the transport of calcium or phosphate across renal and gut epithelia, and into neuronal cells. Gene expression is also high during development, and in an earlier study we mapped the temporal and spatial pattern of gene expression in the mouse urogenital system. Our data suggested that STC probably acted as a signaling molecule that was produced in mesenchyme cells and targeted to epithelial cell layers in both kidney and testes. Here we have examined STC mRNA and protein distributions between developmental stages E10.5 and E18.5 in the axial and appendicular skeleton. In the axial skeleton, STC was transiently expressed in a rostral-caudal fashion during vertebral development; protein appeared to be made in intervertebral disc mesenchyme cells and targeted to vertebral hypertrophic and prehypertrophic chondrocytes. By stage E18.5, the STC gene was active only in vertebral perichondrocytes. The pattern of expression in the appendicular skeleton was equally striking. Early in development, STC gene expression defined the initial lengths of bone primordia. The gene was expressed in mesenchyme cells at either ends of precartilaginous condensations defining future long bones and the secreted protein was targeted to the chondroblasts. Later on during joint formation, STC was highly expressed in interzone cells that defined all future joints. After cavitation, STC gene expression was greatest in perichondrocytes lining the joints. Underlying resting, proliferative and prehypertrophic chondrocytes appeared to be the targets of STC both during and after cavitation. Therefore, its pattern of expression was indicative of a role in early skeletal patterning and joint formation. Moreover, as occurs during urogenital development, it appeared that STC is made in undifferentiated mesenchyme cells and sequestered by those destined to differentiate.

Study Design: The prevalence of inflammatory cells in 205 disc herniations (DHs) and nine macroscopically normal discs for comparison was studied immunohistochemically. Inflammatory cells were separately analyzed in subtypes of DH. Immunohistochemical data were related to clinical parameters, the straight leg raising test (SLR) in particular., Objectives: The objectives of the study were to compare the occurrence of inflammatory cells in various subtypes of DH and to determine the association between clinical data and inflammatory cell occurrence in a more extensive sample of DH, with separate analysis of DH subtypes., Summary of Background Data: Previous studies have suggested a common occurrence of inflammation and inflammatory cells, particularly macrophages, in DHs. No studies on any larger material comprising different subtypes of DH have been done., Methods: For immunohistochemistry the alkaline phosphatase antialkaline phosphatase method was used. Monoclonal antibodies to T cells in general (CD2), activated T cells (CD25), B cells (CD22), and macrophages (CD68) were used. Obtained immunostaining results were then compared with clinical data, e.g., duration of pain, SLR, and type of DH (sequesters 86, extrusions 103, protrusions 16). Associations were studied by the chi2 test or Fisher's exact test, as applicable (level of significance P < 0.05)., Results: Abundant T cells were seen in 17% of the 205 DHs, activated T cells in 17%, B cells in 16%, and macrophages in 37%. All cell types were 2-3 times more prevalent in sequestrated discs than in extrusions. In protrusions macrophages were abundantly seen in 25% (4 of 16) and no other inflammatory cells. In patients with positive SLR and a sequestrated disc abundant lymphocytes were seen three times more often than in extrusions. When patients with bilaterally negative SLR were compared with those with tight SLR (< or =30 degrees ) with respect to inflammatory cell occurrence, some significant differences were noted (CD68, P < 0.025; CD25, P = 0.04). A comparison between SLR bilaterally positive and bilaterally negative also showed associations for all four inflammatory cell types (P = 0.016 to P = 0.029). There was no correlation between inflammatory cells and duration of pain. Abundant inflammatory cells were never seen in control discs., Conclusions: When SLR was positive and the DH type was sequestered, inflammatory cells were most commonly seen. Our results showed some statistically significant associations between inflammatory cells and SLR, most clearly when comparing bilaterally positive and negative SLR. Interestingly, a bilaterally positive SLR showed an association with all four inflammatory cell types analyzed. Tight SLR also showed an association, particularly with macrophages. In addition to tissue resorption, they may participate in sciatic pain. Even though lymphocytes were less prevalent, they may have some role in sequestered discs and bilaterally positive SLR.

Chondroitin sulfate (CS) isomers, 6-sulfate (CS6) and 4-sulfate (CS4), change their ratio to each other in cartilaginous tissues with aging. In this study, a quantitative measurement method of CS6 and CS4 was developed, using capillary electrophoresis (CE). Various buffer solutions, pH, and digestion times were studied, and the use of 0.1 M Tris-HCl at pH of 8.0 allowed the isolation of CS6 and CS4 from CS most efficiently when combined with chondrotinase ABC at a concentration of 1 mU/microg of the substrate during a 3 hr digestion period. Amounts of newly synthesized CS6 and CS4 in the intervertebral disk chondrocyte three-dimensional culture were quantified by this method after the proteoglycans were extracted by equilibrium density centrifugation.

An evaluation of the regional variation of certain cellular features in the human intervertebral disc (IVD) could lead to a better understanding of site-specific properties relative to degradation, response to injury, and healing processes. The objective of this study was to determine how cell density, cell morphology, cell grouping, and expression of a specific actin isoform varied with location and degeneration in the human disc. A total of 41 human L4-L5 and L5-S1 discs removed postmortem from 21 individuals were analyzed. The discs were graded for degeneration based on the Thompson scale and processed for evaluation. Microtomed sections from paraffin-embedded specimens were stained with hematoxylin and eosin or a monoclonal antibody to alpha-smooth muscle actin (alpha-SMA), an actin isoform often associated with contraction. A significant regional dependence was found for most of the measured parameters. A fourfold increase in cell density was found in proceeding from the nucleus pulposus (NP) to the outer annulus (OA) of the IVD. Approximately 30% of the cells in the NP were present in groups. Virtually all of the cells in the NP and 40% of those in the OA were round. Moreover, notable percentages (12-15%) of the cells in the NP and inner annulus (IA) contained alpha-SMA. Only pair density was found to be correlated with Thompson grade, with more degenerated specimens having higher values. A greater effect was also observed on the percentage of cells in groups. These findings provide the basis for future work to investigate the importance of cells in groups, the role of alpha-SMA in the disc, and the changes in these cellular characteristics in pathological disc conditions.

Cancellous bone morphometry was investigated in the sagittal plane of lumbar vertebrae using histoquantitation. The aim of this study was to identify variations in cancellous bone architecture at increasing states of intervertebral disc (IVD) disorganization after age adjustment and to investigate regional variations within the whole vertebral body. Measurements were taken of the ratio of bone volume (BV) to total volume (TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N). Lumbar spines (T12-L5) of 19 men and 8 women were removed at autopsy from an adult sample with no clinical history of bone-related disease or histologically identifiable bone disease. It was found that degeneration of the IVD becomes more common with increasing age. After age-adjustment, significant increases in the proportion of BV/TV were observed in the presence of advancing IVD disorganization. Significant architectural changes were observed in the anterior regions of the vertebral body with increases in Tb.Th and Tb.N and decreases in Tb.Sp. Minimal alterations were found at posterior regions. Bone loss was observed in central regions (most distant from the cortex) as IVD disorganization increased through reduction in both Tb.N and Tb.Th. The BV/TV increase in anterior areas of the centrum may be a response to a redistribution of load to the vertebral body periphery as a result of IVD disorganization. It appears that trabecular morphology is related to the condition of the associated IVD, rather than being the sole consequence of a loss of BV/TV with age. This relationship could influence the occurrence of vertebral body crush fracture.