J Korean Soc Spine Surg.  2005 Jun;12(2):91-100. 10.4184/jkss.2005.12.2.91.

Phenotypical Stability and Matrix Synthesis of Human Intervertebral Disc Cells in Response to Dexamethasone and Transforming Growth Factor-beta1

• Pyo NS
• Kwon UH
• Moon SH
• Kim H
• Lee KI
• Jun JA
• Kim KT
• Kim HS
• Lee HM

• KMID: 1583451
• DOI: http://doi.org/10.4184/jkss.2005.12.2.91

Abstract

STUDY DESIGN: An in vitro experiment.
OBJECTIVES
To evaluate the mRNA expressions of matrix components, and analyze the cellular proliferation and proteoglycan synthesis of human intervertebral disc cells in response to dexamethasone and TGF-beta1 SUMMARY OF LITERATURE REVIEW: Corticosteroids are responsible for the regulation of a diverse range of biological processes through modulation of the expression of target genes. The direct injection of methylprednisolone to the intervertebral disc (IVD) has been shown to cause degeneration and calcification of the disc in rabbits. Systemic administration of hydrocortisone induced degeneration of notochordal cells, which accelerated the aging process of the disc in mice. Transforming growth factor beta-1 (TGF-beta1) is known as a potent agent for the proliferation, differentiation and matrix synthesis of IVD.
MATERIALS AND METHODS
IVD cells were isolated from ten patients, and subsequently cultured. Various doses of dexamethasone (DEX) and/or TGF-beta1 were administered to the IVD cultures. DNA and proteoglycan syntheses were measured by the incorporation of [3H]-thymidine and [35S]-sulfate, respectively. RT-PCRs were performed for the expressions of aggrecan, collagen types I and II, and osteocalcin mRNA.
RESULTS
Cultures with DEX showed increased cellular proliferation and decreased proteoglycan synthesis (p<0.05). TGF-beta1 potentiated the proliferative effect of DEX, but failed to stimulate proteoglycan synthesis in the cultures containing DEX. There were no recognizable changes in the mRNA expressions of aggrecan, collagen types I and II, and osteocalcin in response to DEX and TGF-beta1.
CONCLUSIONS
DEX demonstrated a proliferative effect on human IVD cells, with the combination of DEX and TGF-beta1 showing potentiation of the proliferative effect, while at high doses(100 and 1000nM, the DEX was shown to down-regulate the proteoglycan synthesis. Caution should be exercised in the use of corticosteroid in the therapeutic approaches for the treatment of disc disease or in the regenerative matrix of the IVD.

Keyword

Dexamethasone; Intervertebral disc (IVD); TGF-beta1; Proteoglycan

MeSH Terms

Adrenal Cortex Hormones
Aggrecans
Aging
Animals
Biological Processes
Cell Proliferation
Collagen
Dexamethasone*
DNA
Humans*
Hydrocortisone
Intervertebral Disc*
Methylprednisolone
Mice
Notochord
Osteocalcin
Proteoglycans
Rabbits
RNA, Messenger
Transforming Growth Factor beta1
Transforming Growth Factors
Adrenal Cortex Hormones
Aggrecans
Collagen
DNA
Dexamethasone
Hydrocortisone
Methylprednisolone
Osteocalcin
Proteoglycans
RNA, Messenger
Transforming Growth Factor beta1
Transforming Growth Factors

Figure

• 

  Fig. 1. Survival of intervertebral disc cells with various concentration of dexamethasone(0.1, 1, 10, 100, 1000nM). There is no significant cytotoxicity of dexamethasone with dexamethasone compared to control.

• 

  Fig. 2. [3H]-thymidine incorporation with various dose of dexamethasone and TGF-β1(10 ng/ml). White bar denotes culture with TGF- β1. Dexamethasone with a concentration of 10 nM, 100 nM, and 1000 nM renders increased DNA synthesis compared to control(p<0.05). Culture with dexamethasone and TGF-β1(10 ng/ml) showed increased DNA synthesis from the 1nM of dexamethasone and also demonstrated synergistic effect in DNA synthesis. ∗ p<0.05

• 

  Fig. 3. [35S]-sulfate incorporation with various dose of dexamethasone and TGF-β1(10 ng/ml). White bar denotes culture with TGF-β1. Dexamethasone with a concentration of 10 nM, 100 nM, and 1000 nM renders decreased proteoglycan synthesis compared to control (p<0.05). Culture with dexamethasone and TGF- β1(10 ng/ml) showed further decrease in proteoglycan synthesis at 1nM of dexamethasone. ∗ p<0.05

• 

  Fig. 4. Expression of aggrecan, type I collagen, and type II collagen mRNA measured by reverse transcriptase poly-merase chain reaction. Cultures with various dose of dexamethasone showed no significant changes in the expression of aggrecan, type I collagen and type II collagen mRNA expression. Densitometric date was nor-malized by β-actin.

• 

  Fig. 5. Expression of aggrecan, type I collagen, and type II collagen mRNA measured by reverse transcriptase poly-merase chain reaction. Cultures with various dose of dexamethasone and with TGF-β1(10 ng/ml) showed down-regulation of aggrecan mRNA expression while unchanged pattern in type I collagen and type II collagen mRNA expression. Densitometric date was normal-ized by β-actin.

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