J Korean Soc Spine Surg.  2003 Mar;10(1):1-7. 10.4184/jkss.2003.10.1.1.

Dual Roles of Ligamentum Flavum for Spinal Fusion: As an Osteoinductive Agent and Carrier for Ex-vivo Gene Transfer

• Moon SH
• Kim H
• Kwon UH
• Kim KH
• Youn HK
• Kim HS
• Hahn SB
• Lee HM

• KMID: 2209698
• DOI: http://doi.org/10.4184/jkss.2003.10.1.1

Abstract

STUDY DESIGN: An in-vitro experiment using human ligamentum flavum (LF) and the adnovirus-BMP-2 construct, Ad/BMP-2.
OBJECTIVES
To determine the dual roles of LF as an osteoinductive agent and carrier for ex-vivo gene transfer. SUMMARY OF LITERATURE REVIEW: LF is known to have osteogenic potential. Pathologically, ossified LF may cause myelopathy and radiculopathy. BMP-2 is known as an important factor in the differentiation, and maintenance, of osteoblast phenotypes. Ex-vivo gene transfer, using human LF for spinal fusion, has never been attempted before. MATERIALS AND METHODS: The LF cells were cultured from the degenerated LF of spinal stenosis patients. An adenovirus construct, containing BMP-2 cDNA (Ad/BMP-2), was also produced. The LF cell cultures were exposed to the adenoviral construct. The Osteocalcin expression was analysed by Western blot analysis. The osteocalcin and BMP-2 mRNA expressions were analysed by RT-PCR. Bone formation was assessed by alkaline phosphatase and Von Kossa stains.
RESULTS
The LF cell cultures, with Ad/BMP-2, showed transgene expression in the Western blot analysis. Also, the cultures exhibited the mRNA expressions of both osteocalcin and BMP-2, in a dose-dependent manner. The LF cultures, with Ad/BMP-2, demonstrated alkaline phosphatase expression and bone nodule formations from the Von Kossa staining.
CONCLUSION
The genetically modified LF strongly induced osteogenesis, which can be used during a spinal fusion, as an osteoinductive agent and carrier, for ex-vivo gene transfer.

Keyword

Ligamentum flavum; BMP-2; Adenovirus; Gene transfer

MeSH Terms

Adenoviridae
Alkaline Phosphatase
Blotting, Western
Cell Culture Techniques
Coloring Agents
DNA, Complementary
Humans
Ligamentum Flavum*
Osteoblasts
Osteocalcin
Osteogenesis
Phenotype
Radiculopathy
RNA, Messenger
Spinal Cord Diseases
Spinal Fusion*
Spinal Stenosis
Transgenes
Alkaline Phosphatase
Coloring Agents
DNA, Complementary
Osteocalcin
RNA, Messenger

Figure

• 

  Fig. 1. Transgene expression (BMP-2 mRNA) and expression of osteogenic phenotype (osteocalcin mRNA) in cell culture of ligamentum flavum with an Ad/BMP-2 (MOI of 50, 100, 150). Expression was detected by reverse transcriptase polymerase chain reaction.

• 

  Fig. 2. Expression of osteocalcin protein in supernatant of ligamentum flavum cell culture with an Ad/BMP-2 (MOI of 50, 100, 150). Expression was detected by Western Blot analysis. Negative control denotes CHO cell without Ad/BMP-2 and positive control denotes saline with osteocalcin protein.

• 

  Fig. 3. Ligamentum flavum cell culture with various dose of Ad/BMP-2 (MOI of 50, 100, 150) showed dose dependent increase of reactivity with (A) alkaline phosphatase stain and (B) Von Kossa stain, while culture with saline exhibited negative stain.

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