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Abstract
Study Design
A n in-vitro experiment using human ligamentum flavum (LF) and the adnovirus- BMP- 2 construct, A d/ 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. A n adenovirus construct, containing BMP- 2 cDNA (A d/BMP- 2), was also produced. The LF cell cultures were exposed to the adenoviral construct. The Osteocalcin expression was analysed by W estern 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 A d/BMP- 2, showed transgene expression in the Western blot analysis. A lso, the cultures exhibited the mR NA expressions of both osteocalcin and BMP- 2, in a dosedependent manner. The LF cultures, with A d/BMP- 2, demonstrated alkaline phosphatase expression and bone nodule formations from the Von Kossa staining.
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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.
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