Bone morphogenetic protein-2 facilitates expression of chondrogenic, not osteogenic, phenotype of human intervertebral disc cells

Spine (Phila Pa 1976). 2003 Dec 15;28(24):2679-84. doi: 10.1097/01.BRS.0000101445.46487.16.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aggrecans
  • Alkaline Phosphatase / analysis
  • Anthraquinones
  • Bone Morphogenetic Protein 2
  • Bone Morphogenetic Proteins / pharmacology*
  • Cells, Cultured
  • Chondrocytes / cytology
  • Chondrocytes / metabolism*
  • Collagen Type I / biosynthesis
  • Collagen Type I / genetics
  • Collagen Type II / biosynthesis
  • Collagen Type II / genetics
  • Coloring Agents
  • Extracellular Matrix Proteins / biosynthesis
  • Extracellular Matrix Proteins / genetics
  • Gene Expression / drug effects
  • Humans
  • Intervertebral Disc / cytology*
  • Intervertebral Disc / drug effects
  • Intervertebral Disc / metabolism
  • Lectins, C-Type
  • Middle Aged
  • Osteoblasts / cytology
  • Osteoblasts / metabolism
  • Osteocalcin / biosynthesis
  • Osteocalcin / genetics
  • Phenotype
  • Proteoglycans / biosynthesis
  • Proteoglycans / genetics
  • RNA, Messenger / metabolism
  • Recombinant Proteins
  • Transforming Growth Factor beta*

Substances

  • Aggrecans
  • Anthraquinones
  • BMP2 protein, human
  • Bone Morphogenetic Protein 2
  • Bone Morphogenetic Proteins
  • Collagen Type I
  • Collagen Type II
  • Coloring Agents
  • Extracellular Matrix Proteins
  • Lectins, C-Type
  • Proteoglycans
  • RNA, Messenger
  • Recombinant Proteins
  • Transforming Growth Factor beta
  • recombinant human bone morphogenetic protein-2
  • Osteocalcin
  • Alizarin Red S
  • Alkaline Phosphatase