Bone Morphogenetic Protein-2 Facilitates Expression of Chondrogenic, not Osteogenic, Phenotype of Human Intervertebral Disc Cells

Dong Jun Kim, Seong Hwan Moon, Hyang Kim, Un Hye Kwon, Moon Soo Park, Keong Jin Han, Soo Bong Hahn, Hwan Mo Lee

Research output: Contribution to journalArticle

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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 upregulated 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.

Original languageEnglish
Pages (from-to)2679-2684
Number of pages6
JournalSpine
Volume28
Issue number24
DOIs
Publication statusPublished - 2003 Dec 15

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Bone Morphogenetic Protein 2
Intervertebral Disc
Phenotype
Proteoglycans
Coloring Agents
Aggrecans
Collagen Type II
Osteocalcin
Messenger RNA
Collagen Type I
Osteogenesis
Alkaline Phosphatase
Osteoblasts
Cell Culture Techniques
Ilium
Chondrogenesis
Anabolic Agents
Chondrocytes
Cricetulus
Sulfates

All Science Journal Classification (ASJC) codes

  • Orthopedics and Sports Medicine
  • Clinical Neurology

Cite this

Kim, Dong Jun ; Moon, Seong Hwan ; Kim, Hyang ; Kwon, Un Hye ; Park, Moon Soo ; Han, Keong Jin ; Hahn, Soo Bong ; Lee, Hwan Mo. / Bone Morphogenetic Protein-2 Facilitates Expression of Chondrogenic, not Osteogenic, Phenotype of Human Intervertebral Disc Cells. In: Spine. 2003 ; Vol. 28, No. 24. pp. 2679-2684.
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title = "Bone Morphogenetic Protein-2 Facilitates Expression of Chondrogenic, not Osteogenic, Phenotype of Human Intervertebral Disc Cells",
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 upregulated 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.",
author = "Kim, {Dong Jun} and Moon, {Seong Hwan} and Hyang Kim and Kwon, {Un Hye} and Park, {Moon Soo} and Han, {Keong Jin} and Hahn, {Soo Bong} and Lee, {Hwan Mo}",
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journal = "Spine",
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Bone Morphogenetic Protein-2 Facilitates Expression of Chondrogenic, not Osteogenic, Phenotype of Human Intervertebral Disc Cells. / Kim, Dong Jun; Moon, Seong Hwan; Kim, Hyang; Kwon, Un Hye; Park, Moon Soo; Han, Keong Jin; Hahn, Soo Bong; Lee, Hwan Mo.

In: Spine, Vol. 28, No. 24, 15.12.2003, p. 2679-2684.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Bone Morphogenetic Protein-2 Facilitates Expression of Chondrogenic, not Osteogenic, Phenotype of Human Intervertebral Disc Cells

AU - Kim, Dong Jun

AU - Moon, Seong Hwan

AU - Kim, Hyang

AU - Kwon, Un Hye

AU - Park, Moon Soo

AU - Han, Keong Jin

AU - Hahn, Soo Bong

AU - Lee, Hwan Mo

PY - 2003/12/15

Y1 - 2003/12/15

N2 - 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 upregulated 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.

AB - 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 upregulated 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.

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