De novo osteogenesis from human ligamentum flavum by adenovirus-mediated bone morphogenetic protein-2 gene transfer.
METHODS
In vitro and in vivo experiment using degenerated human ligamentum flavum (LF) and Type 5 adenovirus construct with bone morphogenetic protein-2 (BMP-2) cDNA.


OBJECTIVE
To demonstrate in vitro and in vivo osteogenic effect of BMP-2 gene transfer to human LF and to propose genetically modified LF as a substitute for autogenous bone graft in spinal fusion.


BACKGROUND
Spinal fusion is still considered to be an important option for treating various spinal disorders. To induce solid spinal fusion, osteoinductive and/or osteoconductive agents have been widely adopted. Autogenous LF, however, has never been seriously considered as a carrier for ex vivo osteoinductive gene therapy for spinal fusion.


METHODS
In vitro experiment: Degenerated human LF was harvested and cultured. Type 5 adenovirus lacZ (Ad/lacZ) and BMP-2 construct (Ad/BMP-2) were produced. LF cell cultures were then exposed to Ad/BMP-2. Expressions of osteocalcin and BMP-2 mRNA were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Western blot analysis was performed to detect osteocalcin protein. Alkaline phosphatase and von Kossa stains were used to detect osteogenic markers and bone nodule formation, respectively. In vivo experiment: Human LF tissues treated with Ad/lacZ, Ad/BMP-2, and saline were implanted into the subcutaneous tissue of nude mice. After 4 weeks, nude mice were radiographed and killed. Implanted LF tissues were harvested and histologically stained.


RESULTS
LF cell cultures with Ad/BMP-2 revealed strong expression of BMP-2 and osteocalcin mRNA in RT-PCR and osteocalcin protein in western blot analysis. LF cell culture with saline showed baseline expression of BMP-2, osteocalcin mRNA, and osteocalcin protein, respectively. Furthermore, LF cell culture with Ad/BMP-2 demonstrated the expression of alkaline phosphatase and bone nodule formation in the aforementioned histochemical stain. LF tissues with Ad/BMP-2 revealed de novo osteogenesis in nude mice, whereas LF with Ad/lacZ or saline showed only remaining LF tissue without sign of bone formation.


CONCLUSIONS
Human LF cells transduced with Ad/BMP-2 exhibited the expression of osteogenic phenotype and bone nodule formation. Additionally, genetically modified human LF with BMP-2 cDNA clearly demonstrated de novo osteogenesis, which supports the concept that biologically modified LF can be a substitute for autogenous bone graft in spinal fusion surgery.
Yang
Ick-Hwan
IH
Department of Orthopaedic Surgery, Cancer Center, Seoul, Korea.
Kim
Hyang
H
Kwon
Un-Hye
UH
Lee
Kwang-Il
KI
Jun
Ji-Ae
JA
Kim
Joo-Hang
JH
Yun
Che-Ok
CO
Park
Si-Young
SY
Lee
Hwan-Mo
HM
Moon
Seong-Hwan
SH
D000256:Adenoviridae / Q000235:genetics*; D000368:Aged; D000818:Animals; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000008:administration & dosage / Q000096:biosynthesis* / Q000235:genetics*; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D017843:Ligamentum Flavum / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008875:Middle Aged; D010012:Osteogenesis / Q000187:drug effects / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000008:administration & dosage / Q000096:biosynthesis* / Q000235:genetics*
C525718:BMP2 protein, human; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D016212:Transforming Growth Factor beta