STUDY DESIGN: In vitro experiment using bone morphogenetic protein-2 (BMP-2) and human intervertebral disc (IVD) cells. OBJECTIVES: To demonstrate the effect of BMP-2 on mRNAs expression (collagen type I, collagen type II, aggrecan, and osteocalcin), proteoglycan synthesis, expression of alkaline phosphatase, bone nodule formation in human IVD cells. SUMMARY OF BACKGROUND DATA: BMP-2 was widely known as a powerful agent for osteoinduction and a crucial growth factor for early chondrogenesis and maintenance of cartilaginous phenotype. BMP-2 proved to be effective in stimulating proteoglycan synthesis in articular chondrocytes and IVD cells. Nevertheless, the effect of BMP-2 on IVD cells, whether chondrogenic or osteogenic, was not thoroughly elucidated in transcriptional level and histochemical stains. MATERIALS AND METHODS: Human IVDs were harvested and enzymatically digested. Then IVD cells were cultured three-dimensionally in alginate beads. Osteoblasts were cultured from cancellous bone of ilium for histochemical stains. Recombinant human BMP-2 (rhBMP-2) was produced by Chinese hamster ovary cells after transduction of BMP-2 cDNA, then concentrated and purified. Then IVD cell cultures were exposed to various concentrations of rhBMP-2. Reverse transcription-polymerase chain reaction for mRNA expression of aggrecan, collagen type I, collagen type II, and osteocalcin was performed. Newly synthesized proteoglycan was measured by 35S-sulfate incorporation on Sephadex G-25 M in PD 10 columns. As a histochemical examination, alkaline phosphatase and Alizarin red-S stains were used to detect osteogenic marker and bone nodule formation, respectively. RESULTS: In the rhBMP-2 treated cultures, there was increased newly synthesized proteoglycan (67% in 300 ng/mL and 200% in 1,500 ng/mL of rhBMP-2) and up-regulated expression of aggrecan, collagen type I, and collagen type II mRNA over untreated control. However, rhBMP-2 did not up-regulate expression of osteocalcin mRNA in the given dose and culture period. IVD cell cultures with rhBMP-2 showed no evidence of bone formation in histochemical stains, i.e., alkaline phosphatase and Alizarin red-S, while osteoblast culture exhibited strong positive stains. CONCLUSIONS: The rhBMP-2 clearly up-regulated mRNA expression of chondrogenic components and also stimulated proteoglycan synthesis without expression of osteogenic phenotype. Taken together, this study raise the possibility of rhBMP-2 can be anabolic agent for regenerating matrix of intervertebral disc.
STUDY DESIGN: In vitro experiment using bone morphogenetic protein-2 (BMP-2) and human intervertebral disc (IVD) cells. OBJECTIVES: To demonstrate the effect of BMP-2 on mRNAs expression (collagen type I, collagen type II, aggrecan, and osteocalcin), proteoglycan synthesis, expression of alkaline phosphatase, bone nodule formation in human IVD cells. SUMMARY OF BACKGROUND DATA: BMP-2 was widely known as a powerful agent for osteoinduction and a crucial growth factor for early chondrogenesis and maintenance of cartilaginous phenotype. BMP-2 proved to be effective in stimulating proteoglycan synthesis in articular chondrocytes and IVD cells. Nevertheless, the effect of BMP-2 on IVD cells, whether chondrogenic or osteogenic, was not thoroughly elucidated in transcriptional level and histochemical stains. MATERIALS AND METHODS:Human IVDs were harvested and enzymatically digested. Then IVD cells were cultured three-dimensionally in alginate beads. Osteoblasts were cultured from cancellous bone of ilium for histochemical stains. Recombinant humanBMP-2 (rhBMP-2) was produced by Chinese hamster ovary cells after transduction of BMP-2 cDNA, then concentrated and purified. Then IVD cell cultures were exposed to various concentrations of rhBMP-2. Reverse transcription-polymerase chain reaction for mRNA expression of aggrecan, collagen type I, collagen type II, and osteocalcin was performed. Newly synthesized proteoglycan was measured by 35S-sulfate incorporation on Sephadex G-25 M in PD 10 columns. As a histochemical examination, alkaline phosphatase and Alizarin red-S stains were used to detect osteogenic marker and bone nodule formation, respectively. RESULTS: In the rhBMP-2 treated cultures, there was increased newly synthesized proteoglycan (67% in 300 ng/mL and 200% in 1,500 ng/mL of rhBMP-2) and up-regulated expression of aggrecan, collagen type I, and collagen type II mRNA over untreated control. However, rhBMP-2 did not up-regulate expression of osteocalcin mRNA in the given dose and culture period. IVD cell cultures with rhBMP-2 showed no evidence of bone formation in histochemical stains, i.e., alkaline phosphatase and Alizarin red-S, while osteoblast culture exhibited strong positive stains. CONCLUSIONS: The rhBMP-2 clearly up-regulated mRNA expression of chondrogenic components and also stimulated proteoglycan synthesis without expression of osteogenic phenotype. Taken together, this study raise the possibility of rhBMP-2 can be anabolic agent for regenerating matrix of intervertebral disc.
Authors: Cristiane Sampaio de Mara; A S S Duarte; A R Sartori-Cintra; A C M Luzo; S T O Saad; I B Coimbra Journal: Rheumatol Int Date: 2012-01-12 Impact factor: 2.631