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One of the advantages of chemonucleolysis for the treatment of a herniated intervertebral disc is the potential for the disc to self-repair. It has been suggested that the enzymes used for chemonucleolysis differentially affect the potential of the disc cells to promote repair.To test the ability of nucleus pulposus and anulus fibrosus cells to repair the extracellular matrix degraded in vitro by either chondroitinase ABC or chymopapain.An alginate cell culture system was used to monitor the progress of matrix repair after chemonucleolysis in vitro.Rabbit nucleus pulposus or anulus fibrosus cells precultured for 10 days in alginate gel were briefly exposed to low concentrations of chondroitinase ABC or chymopapain and then returned to normal culture conditions for up to 4 weeks. At each time point, the contents of DNA and matrix macromolecules and proteoglycan synthesis were measured.The DNA content of enzyme-treated alginate beads during the following 4 weeks of culture was higher in the chondroitinase ABC group than in the chymopapain group (NP, p.01, and AF, p.05). The content of proteoglycan in beads containing nucleus pulposus and anulus fibrosus cells in the chondroitinase ABC group was higher than that in the chymopapain group (NP and AF, p.001). The rate of proteoglycan synthesis and the content of collagen did not, however, differ between those two groups.Intervertebral disc cells exposed to chondroitinase ABC reestablish a matrix richer in proteoglycan than cells exposed to chymopapain. This may be because of differences in the substrate spectrum of each enzyme. Although these results cannot be translated directly to the in vivo situation, they suggest the possibility that cells in discs subjected to chondroitinase ABC-induced chemonucleolysis retain a greater ability to replenish their extracellular matrix with proteoglycans than cells in discs exposed to chymopapain.

Despite the significant impairment associated with degenerative disc disease, a clear understanding of its pathogenesis is still lacking. Currently, no particular model parallels the complex nature of human disc degeneration. Naturally occurring animal models have the drawback that the basis for the high rate of disc degeneration is not known. Although the interventions in artificial animal models that create disc degeneration are known, the relationship of those to the events leading to disc degeneration in humans is not. With the recent progress in biomechanics, cell biology and molecular biology, an easily reproducible and valid animal model may help unlock the complex cascade of events surrounding human disc degeneration.

Purpose of study: Poor vascularity to the intervertebral discs (IVD) and sclerosis of the end plate may result in the decrease of nutrient diffusion and subsequent disc deterioration. The purpose of this study was to determine the effect of nutrition on disc metabolism using our recently developed in vitro organ culture model with end plate, and to see if osteogenic protein (OP)-1 can protect discs from a low-nutrient environment. Methods used: Twenty-four lumbar IVDs were obtained from adult New Zealand rabbits weighing 3 kg. Specimens were cultured over 21 days in DMEM/F12 with different concentrations of FBS (5%, 10% and 20%). Four of the specimens cultured in 5% FBS were injected with 0.2 mg OP-1 at 7 days. At 21 days, discs were dissected from end plate and annulus fibrosus (AF) and nucleus pulposus (NP) were separated. After measuring dry weight, the AF and NP tissue were analyzed for DNA content and proteoglycan content (PG) by Hoechst dye method and DMB, respectively. The results were statistically analyzed using analysis of variance with a Fisher's PLSD test as a post hoc test. of findings: The DNA content of the NP decreased as the concentration of FBS decreased (5%=1.28 ug/mg, 10%=2.67 ug/mg, 20%=3.92 ug/mg; p=.03). The PG content in NP showed a positive correlation with the concentration of FBS (5%=25.4 ug/mg, 10%=95.5 ug/mg, 20%=152.7 ug/mg; p=.02). The concentration of FBS did not, however, have an effect on the DNA or PG content in the AF. Discs cultured in 5% FBS plus OP-1 demonstrated increased DNA content in NP (1.52 ug/mg, p=.04) and PG content in NP (36.1 ug/mg, p=.05) when compared with discs cultured in 5% FBS without OP-1. The DNA content and PG content in the AF were not affected. Relationship between findings and existing knowledge: Numerous studies have demonstrated that decreased vascularity to the IVD is linked to disc degeneration. This is presumably because of decreased nutrition to the disc cells. However, no studies have been done on the direct relationship between nutrient supply and disc growth in an in vitro organ culture. Moreover, no studies are available on the effect of OP-1 on the IVD in the nutrient-poor environment. Overall significance of findings: In the in vitro organ culture system with end plate, the NP is very sensitive to nutrient supply. AF tissue was not dependent on the nutrition. These results suggest that the diminished vascularity and/or sclerotic end plate changes may contribute to the progress of disc degeneration. Administration of OP-1 appears to protect NP tissue from undergoing degeneration from poor disc nutrition. Disclosures: Device or drug: OP-1. Status: investigational. Conflict of interest: No conflicts.

Purpose of study: Chondroitinase-ABC (C-ABC) is commonly used to degrade the chondroitin sulfate and dermatan sulfate chains of proteoglycans. In some countries, C-ABC, injected intradiscally, shows promise as a chemonucleolytic agent. However, the removal of glycosaminoglycans may lead, over the long-term, to significant degradation of the injected intervertebral disc (IVD). Osteogenic protein-1 (OP-1) has been shown to stimulate IVD matrix synthesis in vitro and, thus, may be useful in inhibiting and/or slowing down IVD matrix degradation in vivo. The purpose of this study was to monitor the effect of recombinant human OP-1 (rhOP-1) on the in vivo repair of IVDs co-injected with C-ABC. Methods used: Twenty-four New Zealand White rabbits (3 kg) were divided equally into four groups (control vehicle; C-ABC [10 mU] alone; rhOP-1 [100 μg] alone; or C-ABC + OP-1 [10 mU+100 μg, respectively] co-injected). The assigned agents (10 μl/disc) were injected into three levels of IVDs for each rabbit in each group. Radiographs of the lumbar spine were taken every 2 weeks after in the injections. At 4 or 12 weeks after the injections, three rabbits in each group were euthanized and the IVDs assessed biochemically and histologically. of findings: Significant disc space narrowing was observed 2 weeks after the injection of either C-ABC or C-ABC + OP-1, but not after injection of the control vehicle. In the C-ABC group, this narrowing was sustained for up to 12 weeks (4 weeks, 77 ± 11%; 12 weeks, 75 ± 8%). In the C-ABC + OP-1 group, the disc height index (DHI) had begun to return toward normal after 4 weeks (C-ABC vs. C-ABC + OP-1, p

Purpose of study: Recombinant human osteogenic protein–1 (rhOP-1) was recently shown to be a potent stimulator of cartilage matrix repair. It was also found to stimulate proteoglycan (PG) and collagen synthesis by rabbit nucleus pulposus (NP) and annulus fibrosus (AF) cells [1], suggesting that it may be applied intradiscally for the treatment of disc degeneration. At this time, however, it is not known if human adult NP and AF cells can be similarly stimulated by OP-1. The purpose of this study was to examine the effect of OP-1 on cell proliferation and PG synthesis by human NP and AF cells cultured in alginate gel. Methods used: Human lumbar intervertebral discs were obtained from an adult patient who underwent anterior lumbar discectomy and fusion for scoliosis. The NP and AF were separated by blunt dissection and separately pooled. Cells were enzymatically released from each tissue by digestion with 0.4% pronase for 1 hour and then with 0.025% collagenase P and 0.004% deoxyribonuclease II for 16 hours. The isolated cells were encapsulated in 1.2% low-viscosity alginate at 2 million cells /ml, as previously described [2]. After overnight culture in DMEM/F12 plus 10% FBS, the cells were cultured for another 14 days in this medium in the absence or presence of rhOP-1 (a gift from Stryker Biotech) at 20 or 200 ng/ml. The medium was changed daily. The content of DNA was measured using the Hoechst 33258 dye method and fluorometry [2]. The rate of PG synthesis was assessed by incubating the beads for the last 4 hours of culture in medium containing 35S-sulfate (20 uCi/ml) on day 14. The radiolabeled PGs were measured by a rapid filtration assay [3]. Total PG content in the beads was also assessed using the dimethylmethylene blue staining method [2] after papain digestion. of findings: Recombinant human OP-1 did not have a significant effect on cell proliferation in either cell type. The DNA content on day 14 was similar in beads cultured with or without rhOP-1. Recombinant human OP-1 significantly stimulated PG synthesis on day 14 by both AF and NP cells ( Fig. 1 Download : Download high-res image (82KB) Download : Download full-size image Fig. 1 . Effects of rhOP-1 on PG synthesis. ; NP, p Relationship between findings and existing knowledge: The results of this study show that rhOP-1 enhanced the production of PG by human NP and AF cells (Fig. 1) . Overall significance of findings: The results presented here suggest that rhOP-1 could prove most useful as a therapeutic agent in promoting synthesis and repair of matrix of both the AF and NP elements of degenerating human intervertebral discs. Disclosures: Device or drug: recombinant human osteogenic protein-1. Status: investigational. Conflict of interest: No conflicts.