Howard S. An

Recombinant human osteogenic protein–1 (RHOP-1) upregulates extracellular matrix metabolism by human annulus fibrosus and nucleus pulposus cells

Abstract 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.

Chondroitinase ABC-induced intervertebral disc degeneration is minimized when the enzyme is co-injected with osteogenic protein-1

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<.001) and was no longer significantly different from the control group at 12 weeks (control, 86% ± 6%; C-ABC + OP-1; 92% ± 12%). Biochemical and histological analyses supported those findings (Fig. 1). Relationship between findings and existing knowledge: Importantly, the addition of OP-1 to the intradiscally injected solution did not interfere with C-ABC–induced chemonucleolysis. Furthermore, measurement of DHI showed that OP-1 was very effective in subsequently stimulating matrix repair. Overall significance of findings: These in vivo findings illustrate the potential of OP-1 to promote nucleus pulposus regeneration in humans after chemonucleolysis. Disclosures: Device or drug: recombinant human osteogenic protein-1. Status: investigational. Conflict of interest: Howard An, and Koichi Masuda, grant research support: Stryker Biotech.