Bone morphogenetic protein-2 facilitates expression of chondrogenic, not osteogenic, phenotype of human intervertebral disc cells.
METHODS
In vitro experiment using bone morphogenetic protein-2 (BMP-2) and human intervertebral disc (IVD) cells.


OBJECTIVE
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.


BACKGROUND
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.


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.
Kim
Dong-Jun
DJ
Ewha Women University, Seoul, Korea.
Moon
Seong-Hwan
SH
Kim
Hyang
H
Kwon
Un-Hye
UH
Park
Moon-Soo
MS
Han
Keong-Jin
KJ
Hahn
Soo-Bong
SB
Lee
Hwan-Mo
HM
D000328:Adult; D053674:Aggrecans; D000469:Alkaline Phosphatase / Q000032:analysis; D000880:Anthraquinones; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D024042:Collagen Type I / Q000096:biosynthesis / Q000235:genetics; D024043:Collagen Type II / Q000096:biosynthesis / Q000235:genetics; D004396:Coloring Agents; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D037181:Lectins, C-Type; D008875:Middle Aged; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D015675:Osteocalcin / Q000096:biosynthesis / Q000235:genetics; D010641:Phenotype; D011509:Proteoglycans / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta
D053674:Aggrecans; D000880:Anthraquinones; C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D024042:Collagen Type I; D024043:Collagen Type II; D004396:Coloring Agents; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D011509:Proteoglycans; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2; D015675:Osteocalcin; C004468:Alizarin Red S; D000469:Alkaline Phosphatase