Biologic modification of ligamentum flavum cells by marker gene transfer and recombinant human bone morphogenetic protein-2.
METHODS
The study involves an in vitro experiment using human ligamentum flavum (LF), adenovirus lacZ construct (Ad/lacZ), and recombinant human bone morphogenetic protein-2 (BMP-2).


OBJECTIVE
To demonstrate the feasibility of marker gene transfer to human LF cells and the effect of BMP-2 on the osteogenic differentiation of human LF cells.


BACKGROUND
BMP-2 is a widely known pivotal osteoinductive agent. Clinically and experimentally, BMP-2 has proven to be an effective in spinal fusion. Degenerated LF has only been implicated to be of pathophysiological significance in spinal stenosis. However, biologic modifications of LF to enhance osteogenesis have not been attempted previously.


METHODS
Human LF and cancellous bone from the ilium were harvested from patients with lumbar spinal stenosis. LF cells and osteoblasts were isolated and cultured, and adenovirus lacZ construct (Ad/ lacZ), luciferase construct (Ad/luciferase), and BMP-2 were designed and produced. LF cell cultures were then exposed to various concentrations of Ad/lacZ (25, 50, 75, 100, 150 multiplicity of infection) and BMP-2 (50, 100, 500, 1,000, and 1,500 ng/mL). Osteoblast cultures were used as a positive control for LF culture. LF cell cultures with Ad/luciferase served as viral controls for culture with Ad/ lacZ. The transgene expression of lacZ was assessed by X-gal stain and beta-galactosidase assay. Alkaline phosphatase, Von Kossa, and Alizarin red-S stains were used to confirm osteogenic differentiation and bone nodule formation. Immunocytochemical staining was also performed to detect osteocalcin expression.


RESULTS
LF cell cultures transduced with Ad/lacZ showed extensive X-gal expression and increased beta-galactosidase activity compared to viral (Ad/luciferase) and saline controls. In LF cultures treated with BMP-2, robust alkaline phosphatase expression, and bone nodule formations were observed as evidenced by positive Von Kossa and Alizarin red-S staining, and the strong expression of osteocalcin. The osteogenic response of LF cells to BMP-2 was dose dependent.


CONCLUSIONS
Human LF cells were found to be susceptible to adenovirus-mediated marker gene transfer, which offers the possibility of a new range of possible genetic modifications. In human LF cells, BMP-2 was found to markedly up-regulate the expression of osteogenic phenotypes and to induce bone nodule formation. The results of this study support the notion that biologically modified LF cells, i.e., LF cells treated with BMP-2, or with adenovirus-mediated BMP-2 cDNA gene transfer, may facilitate spinal fusion.
Moon
Seong-Hwan
SH
Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, Korea.
Park
Seung-Rim
SR
Kim
Hyang
H
Kwon
Un-Hye
UH
Kim
Keong-Hee
KH
Kim
Hak-Sun
HS
Lee
Hwan-Mo
HM
D000256:Adenoviridae / Q000235:genetics; D000368:Aged; D000469:Alkaline Phosphatase / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D005240:Feasibility Studies; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D006801:Humans; D017843:Ligamentum Flavum / Q000166:cytology / Q000378:metabolism*; D008875:Middle Aged; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D015203:Reproducibility of Results; D013194:Staining and Labeling; D029961:Zebrafish Proteins; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D011994:Recombinant Proteins; D029961:Zebrafish Proteins; C405936:bmp2b protein, zebrafish; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase