Open Access, Peer-reviewed
pISSN 2093-4378
eISSN 2093-4386
    J Korean Soc Spine Surg. 2003 Mar;10(1):1-7. Korean.
    Published online Mar 31, 2003.
    https://doi.org/10.4184/jkss.2003.10.1.1
    Copyright © 2003 Korean Society of Spine Surgery
    Original Article

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

    Seong-Hwan Moon, M.D., Hyang Kim, M.Sc., Un-Hye Kwon, B.Sc., Keong-Hee Kim, B.Sc, Hong Ki Youn, M.D., Hak-Sun Kim, M.D., Soo-Bong Hahn, M.D. and Hwan-Mo Lee, M.D.
      • Department of Orthopedic Surgery, Yonsei University, College of Medicine, Seoul, Korea.
    • Address reprint requests to Hwan-Mo Lee, M.D. Department of Orthopaedic Surgery, Yonsei University College of Medicine, #134 Shinchon-dong, Soedaemun-gu, Seoul 120-752, Korea. Tel: 82-2-361-5649, Fax: 82-2-363-1139, Email: hwanlee@yumc.yonsei.ac.kr

    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.

    Keywords
    Ligamentum flavum; BMP-2; Adenovirus; Gene transfer

    Figures

    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.

    References

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