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Study design An investigation of mechanical destabilization of the lumbar ovine intervertebral disc (IVD) inducing IVD degeneration (IVDD) as determined by multiparameter outcome measures (magnetic resonance imaging [MRI], IVD composition, biomechanical testing, gene profiling, immunohistochemistry, and immunoblotting). Objective To assess the effect of IVD mechanical destabilization on matrix protein and metalloproteinase gene expression to investigate the pathophysiological mechanisms of lumbar IVDD. Summary of background data Several earlier studies have used annular transection to induce IVDD in sheep, but none have optimized or validated the most appropriate lesion size. Methods The annulus fibrosus (AF) incision inducing maximal change in IVD biomechanics was applied to L1-L2, L3-L4, and L5-L6 discs in vivo to compare with a sham procedure at 3 months post operation. IVDs were evaluated by MRI, biomechanics, histopathology, proteoglycan and collagen content, gene expression, and aggrecan proteolysis by Western blotting. Results Significant changes were observed in lesion (6 × 20 mm(2)) compared with sham IVDs at 3 months post operation: reduced disc height on MRI; increased neutral zone in biomechanical testing; depleted proteoglycan and collagen content in the nucleus pulposus (NP) and lesion half of the AF but not in the contralateral AF; increased messenger RNA for collagen I and II, aggrecan, versican, perlecan, matrix metalloproteinase (MMP)-1 & 13, and ADAMTS-5, in the lesion-site AF and NP but not in the contralateral AF. ADAMTS-4 messenger RNA was increased in the lesion-site AF but decreased in the NP. Despite an upregulation in MMPs, there was no change in MMP- or ADAMTS-generated aggrecan neoepitopes in any region of the IVD in lesion or sham discs. Conclusion Lumbar IVDD was reproducibly induced with a 6 × 20 mm(2) annular lesion, with focal dysregulation of MMP gene expression, cell cloning in the inner AF, loss of NP aggrecan, and disc height. Loss of aggrecan from the NP was not attributable to increased proteolysis in the interglobular domain by MMPs or ADAMTS.

Improved staging of cartilage degeneration is required, particularly during early stages when minimal surface damage is visible arthroscopically. Degradation of articular cartilage extracellular matrix, resulting from degenerative changes associated with osteoarthritis, can influence its functional properties. Cartilage mechanical properties therefore may provide a quantitative method for monitoring degenerative change in this tissue. We determined whether dynamic mechanical properties of cartilage (effective shear modulus and phase lag) measured with a handheld indenter correlated with histopathology scores, proteoglycan, and collagen content or expression of chondrocyte-specific (aggrecan, collagen II) or dedifferentiation (collagen I and III) genes in human osteoarthritic cartilage with International Cartilage Repair Society scores of 0 to 1. We observed an association between the histopathologic stage of cartilage disease and dynamic shear modulus and phase lag. In contrast, there generally was a poor relationship between cartilage biomechanical properties and biochemistry with the only noteworthy correlation being between shear modulus and collagen. Phase lag but not shear modulus correlated with gene expression. These data support the potential of dynamic indentation for assessing the stage of cartilage degeneration in tissue with minimal gross surface damage.

Study design The spatial and temporal localization of fibroblast growth factor-2, transforming growth factor-beta, osteonectin, and alpha-smooth muscle cell actin in the injured anulus fibrosus was investigated. Objective To assess the involvement of fibroblast growth factor-2, transforming growth factor-beta, osteonectin, and alpha-smooth muscle cell actin in anulus fibrosus repair. Summary of background data Fibroblast growth factor-2 and transforming growth factor-beta have been localized to disc herniation tissue, and alpha-smooth muscle cell actin has been identified in a number of mesenchymal cell types, but their roles have not been evaluated in repair processes in the experimentally injured anulus fibrosus. Methods For this study, 32 two adult merinos received a 4-mm deep standard annular incision in their L1L2 and L3L4 discs (lesion group). A similar number of sham-surgery animals served as control subjects. Osteonectin, fibroblast growth factor-2, transforming growth factor-beta, and alpha-smooth muscle cell actin were immunolocalized in sagittal disc sections 3, 6, 12, and 26 months after the operation. Selected specimens also were stained with hematoxylin and eosin, Masson-trichrome, toluidine blue, and picrosirius red. Results Early focal depletion of proteoglycan was evident in the anulus fibrosus and reorganization of outer annular lamellas 3 to 6 months after the operation. Blood vessel ingrowth and fibroblast infiltration from the outer anulus fibrosus along the plane of the annular defect were maximal 12 months after the operation. Focal upregulation in alpha-smooth muscle cell actin expression was evident with maximal staining in the 12-month lesion samples near infiltrating blood vessels at the lesion site, and also in cells well away from these vessels. Some of the anulus fibrosus cells of the sham sections also stained positively for alpha-smooth muscle cell actin, but this staining was significantly less than in the lesion samples. Staining for fibroblast growth factor-2, transforming growth factor-beta, and osteonectin was strongly localized to blood vessels and cells in the vicinity of the annular lesion. It was maximal 12 months after the operation and diminished by 26 months after the operation. Osteonectin expression also was significantly elevated in outer anulus fibrosus cells distant from the lesion site and its associated blood vessels. In the sham discs, immunoreactivity to fibroblast growth factor-2, transforming growth factor-beta, osteonectin, and alpha-smooth muscle cell actin was confined to sparsely distributed cells in the anulus fibrosus. No matrix staining was observed. Conclusions Immunoreactivity for the noted agents was strongly associated with regions of the annular lesions undergoing matrix reorganization consistent with an active repair response. This response extended as far as the middle third of the anulus fibrosus, which also demarcated the extent of blood vessel ingrowth and cellular infiltration in this model. The alpha-smooth muscle cell actin expression suggested an active involvement of myofibroblasts in the anulus fibrosus repair processes.

The objective of this study was to determine whether disc cells could be cultured under serum-free conditions and whether they synthesized bovine pancreatic trypsin inhibitor (BPTI)-like serine proteinase inhibitory proteins (SPIs) previously demonstrated for ovine chondrocytes. Intervertebral discs from 1- to 2-year-old merino wether sheep were dissected into the annulus fibrosus and nucleus pulposus, and the cells isolated by sequential enzymatic digestion. The cells were grown encapsulated in calcium alginate microspheres under serum-free conditions for 10 days. They remained more than 92% viable as assessed using the vital fluorescent dyes chloromethyl fluorescein diacetate and ethidium homodimer-1 to delineate live/dead cells, respectively. Western and affinity blotting identified a 12-16-kDa media SPI and an additional 34-36-kDa BPTI-like species in solubilized bead samples. This study has demonstrated ovine disc cells synthesized BPTI-like SPIs in serum-free alginate bead culture similar to chondrocyte SPIs; however, the 58-kDa precursor SPI form was not detected suggesting differences in the endogenous processing of these SPIs.

Study Design. The proteoglycan metabolism of ovine disc nucleus pulposus and anulus fibrosus cells was investigated in relation to age, spinal level, and intrinsic spinal biomechanical properties. Objective. To evaluate the hypothesis that with aging loss of proteoglycans from the lumbosacral disc exceeds that from upper lumbar discs because of its proximity to a rigid segment, the sacrum. of Background Data. The proteoglycan and associated water of the disc decreases with aging, Methods. Proteoglycans were extracted directly from the disc tissues using 4 M GuHCI and examined by composite agarose polyacrylamide gel electrophoresis. Disc cells were cultured in alginate beads, and their metabolic activity was assessed by 3 H-thymidine incorporation into DNA and by bloreduction of a cell proliferation dye. Newly synthesized proteoglycans were radiolabeled with 3 S, and their molecular weight distributions and ability to aggregate with hyaluronan were determined by Sephacryl S1000 gel chromatography. Resident proteoglycans extracted from disc tissues with 4 M GuHCl were similarly evaluated. A group of adult animals also were studied biomechanically to evaluate the range of spinal motion (L4/L5 to L7/S1). Results. In contrast to the neonatal proteoglycan samples, the biosynthesis of proteoglycans by nucleus pulposus cells of adult discs increased progressively toward the sacrum. This correlated with increased metabolic activity. Analysis of the resident proteoglycans by composite agarose polyacrylamide gel electrophoresis indicated that although the aggrecan-1 population was present almost exclusively in the neonatal group, it was the aggrecan-2 population that predominated in the adult discs, and it became progressively more heterogeneous with aging and proximity of the disc to the sacrum. Conclusions. The proteoglycans of the lumbosacral disc of adult animals turned over faster than proteoglycans of adjacent lumbar discs. The reduced proteoglycan content and ability to aggregate, particularly in the nucleus pulposus of lumbosacral discs, indicated that proteoglycan catabolism exceeded the rate of biosynthesis. These events in the lumbosacral disc are thought to be determined mechanically by its proximity to the sacrum.

Study Design. The current report describes a study in beagles in which the effects of intradiscal injection of three doses of chymopapain were evaluated with respect to the reduction of disc width and reconstitution of the nucleus pulposus. Objectives. To establish an intradiscal dose of chymopapain that would achieve optimal reduction in disc height followed by maximum reconstitution of the nucleus pulposus. Summary of Background Data. Earlier reports of the efficacy of high and low doses of chymopapain for chemonucleolysis have provided conflicting data, and a scientific basis for an appropriate dose is lacking. Methods. Four mature, female beagles were subjected to chemonucleolysis using three doses of chymopapain as Chymodiactin (31, 63 and 125 picokatals/disc) injected into the L2-L3, L1-L2, and L3-L4 discs. Disc widths were monitored radiographically over 32 weeks. Proteoglycans were radiolabeled by intravenous injection with Na235SO4 (1 mCi/kg) 24 hours before sacrifice, and their specific activities (disintegrations per minute/mg proteoglycan), hydrodynamic size, and ability to aggregate determined. Results. Sixty-three picokatals of Chymodiactin produced optimal disc reconstitution after chemonucleolysis. A reduction in disc height of approximately 35% was evident within 1 month and this slowly returned to approximately 90% of the preinjection value after 32 weeks. The nucleus pulposus contained approximately 75% of the proteoglycan content of control tissues, and most of these formed aggregates with hyaluronan. Disc collagen levels remained relatively unaffected by treatment. Conclusions. This study demonstrates that an effective reduction in disc width compatible with later reconstitution of the nucleus pulposus can be achieved experimentally with an appropriate dose of chymopapain. These data clearly indicate that an optimal dose of chymopapain for chemonucleolysis in humans needs to be established.

An established model of experimental disc degeneration (Osti et al., Spine 15:762, 1990; Melrose et al., J. Orthop Res 10:665, 1992) was used in this study. Four 2-year-old sheep received anterolateral incisions (4 x 10 mm) in the outer one-third of the annulus fibrosus of their L2-L3 and L4-L5 discs (lesion group). The annulus was not incised in another four sham-operated animals. After 6 months the sheep were killed, lumbar discs were dissected into lateral halves of the annulus fibrosus and the nucleus pulposus. Cells were isolated from disc tissues enzymatically and were grown in alginate bead culture to examine the proteoglycan metabolism of cells from lesion and control zones. The media of lesion zone cultures contained relatively high levels (compared with sham cultures) of catabolic fragments of the large, high-buoyant-density proteoglycans as demonstrated by Western blotting using monoclonal antibodies (5-D-4, 3-B-3, 1-C-6) and biotinylated hyaluronan and also by gel chromatography. Furthermore, cells from the vicinity of the lesion site also synthesized significantly lower levels (compared with sham cultures) of aggrecan that was retained within the alginate beads. Collectively, these data indicated that focal depletion of large, high-buoyant-density proteoglycans was evident within lesion sites in this model of experimental disc degeneration. The introduction of an annular lesion therefore significantly affected the proteoglycan metabolism of endogenous disc cell populations. The unique hydrodynamic and viscoelastic properties of the intervertebral disc are dependent to a large degree on the tissue levels of aggrecan. The focal depletion of aggrecan by annular lesions therefore may represent an important predisposing factor to the subsequent degeneration of these intervertebral discs.