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 ; NP, p<.05; AF, p<.01). The addition of rhOP-1 at 200 ng/ml to the medium resulted in a marked increase in the content of PG in NP beads (p<.05). This increase was minor during the first week but dramatic during the second week of culture with rhOP-1. Results in AF beads cultured under identical conditions were less pronounced but similar.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.