J Korean Soc Spine Surg.  2002 Dec;9(4):263-269. 10.4184/jkss.2002.9.4.263.

Osteogenesis by Transfer of Bone Morphogenetic Protein-2 cDNA in Ligamentum Flavum Cells: Approach Toward Tissue Engineering

• Moon SH
• Kim H
• Kwon UH
• Won JH
• Kim HS
• Hahn SB
• Lee HM

• KMID: 2209707
• DOI: http://doi.org/10.4184/jkss.2002.9.4.263

Abstract

STUDY DESIGN: In-vitro experiment.
OBJECTIVES
To determine the effect of bone morphogenetic protein-2 in the osteogenesis of human ligamentum flavum cells and test the feasibility of gene transfer to these cells. SUMMARY OF LITERATURE REVIEW: Bone morphogenetic protein-2 (BMP-2) is known to be an important factor in the differentiation and maintenance of the osteoblastic phenotype. Tissue engineering for osteogenesis in ligamentum flavum by BMP-2 and gene transfer has not been previously studied.
MATERIALS AND METHODS
Ligmentum flavum cells were harvested and cultured from surgical patients with spinal stenosis. BMP-2 was produced by transfecting pcDNA3.1/Hygro/BMP-2 into CHO cells using Lipofectamine 2000. Adenovirus-lacZ (Ad/lacZ) was also produced, and administered with BMP-2 to cell culture. The expression of lacZ was analyzed by X-gal staining. Bone formation was assessed by alkaline phosphatase, von Kossa, and alizarin Red-S staining, and the expression of osteocalcin was determined immunocytochemically.
RESULTS
Ligamentum flavum cell cultures with Ad/lacZ showed marker gene expression. BMP-2 induced osteogenesis in ligamentum flavum cells as evidenced by alkaline phosphatase, von Kosa, and alizarin Red-S staining. Also, cell culture with BMP-2 showed strong positivity with osteocalcin by immunocytochemistry.
CONCLUSION
BMP-2 more strongly induced the osteogenesis of ligamentum flavum, and also its gene transfer to ligamentum flavum was found to be feasible. These results may open a new era of ligamentum flavum tissue engineering.

Keyword

BMP-2; adenovirus; osteogenesis; Tissue engineering

MeSH Terms

Adenoviridae
Alkaline Phosphatase
Animals
Cell Culture Techniques
CHO Cells
Cricetinae
DNA, Complementary*
Gene Expression
Humans
Immunohistochemistry
Ligamentum Flavum*
Osteoblasts
Osteocalcin
Osteogenesis*
Phenotype
Spinal Stenosis
Tissue Engineering*
Alkaline Phosphatase
DNA, Complementary
Osteocalcin

Figure

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  Fig. 1. Expression of marker gene (lacZ) in cell culture of ligamentum flavum with an MOI of 0.5, 1, 2, 5, 10.

• 

  Fig. 2. Ligamentum flavum cell culture with high dose of BMP-2 (2000 ng/ml) expresses osteocalcin in immunocytochemistry. A: control culture without BMP-2, B: culture with BMP-2.

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  Fig. 3. Ligamentum flavum cell culture with various dose of BMP-2 showed dose dependent increase of reactivity with (A) alkaline phosphatase stain, (B) von Kossa stain, (C) Alizarin Red-S stain.

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