Bone morphogenic protein–2 induces chondrogenic gene expression and enhances disc matrix production

Abstract

Purpose of study: This study determines the effectiveness of recombinant human bone morphogenic protein (rhBMP)-2 in inducing the expression of chondrocytic genes (aggrecan and collagen II) and increasing sulfated-glycosaminoglycans (s-GAG) in cultured intervertebral rat disc cells.Methods used: Cells were isolated from annulus fibrosus of rat lumbar discs. The cells were grown in monolayer and treated with rhBMP-2 (0, 10, 100, 1,000 ng/ml) in DMEM/F-12 with 1% FBS (day 0). On days 2, 4 and 7 after rhBMP-2 treatment, s-GAG content in media was quantified using dimethylmethylene blue (DMMB) staining. The results were normalized according to culture duration and cell number. On day 7, mRNA was extracted reverse transcriptase polymerase chain reaction (RT-PCR) and real-time PCR were used to quantitate aggrecan, collagen I and II mRNA. Cell number was determined with a hemocytometer.of findings: RhBMP-2 at 100 and 1,000 ng/ml yielded a 60% and 90% increase in cell number on day 4 and yielded a 90% and 110% on day 7, respectively. RhBMP-2 at 10 ng/ml had no effect on cell number. Media s-GAG increase was greatest at day 7, increasing by 30%, 100% and 260% with rhBMP-2 treatments of 10, 100 and 1,000 ng/ml, respectively. Obvious increases in mRNA levels of aggrecan and collagen II were observed with rhBMP-2 concentrations of 100 and 1,000 ng/ml by RT-PCR. No detectable increase in mRNA level of collagen I was observed with rhBMP-2 treatments. Real-time PCR showed the greatest effect at 1,000 ng/ml rhBMP-2, leading to a six-fold increase in aggrecan and three-fold increase in collagen II mRNA above untreated control at day 7.Relationship between findings and existing knowledge: RhBMP-2 increases cell number and s-GAG (proteoglycan) synthesis. It also increases mRNA of aggrecan and collagen II but not collagen I; this is consistent with a more chondrocytic phenotype.Overall significance of findings: This study indicates that rhBMP-2 may be useful in enhancing disc matrix production of intervertebral disc cells.Disclosures: No disclosures.Conflict of interest: S. Tim Yoon, grant research support, Medtronic Sofamor Danek.