De novo osteogenesis from human ligamentum flavum by adenovirus-mediated bone morphogenetic protein-2 gene transfer

Ick Hwan Yang, Hyang Kim, Un Hye Kwon, Kwang Il Lee, Ji Ae Jun, Joo Hang Kim, Che Ok Yun, Si Young Park, Hwan Mo Lee, Seong Hwan Moon

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Study Design. 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. Objectives. 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. Summary of Background Data. 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. Conclusion. 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.

Original languageEnglish
Pages (from-to)2749-2754
Number of pages6
JournalSpine
Volume30
Issue number24
DOIs
Publication statusPublished - 2005 Dec 1
Externally publishedYes

Fingerprint

Ligamentum Flavum
Bone Morphogenetic Protein 2
Adenoviridae
Osteogenesis
Genes
Osteocalcin
Spinal Fusion
Cell Culture Techniques
Nude Mice
Bone Substitutes
Reverse Transcriptase Polymerase Chain Reaction
Messenger RNA
Alkaline Phosphatase
Coloring Agents
Complementary DNA
Western Blotting
Transplants

Keywords

  • Adenovirus
  • Bone morphogenetic protein-2 (BMP-2)
  • de novo osteogenesis
  • Gene therapy
  • Ligamentum flavum
  • Spinal fusion

ASJC Scopus subject areas

  • Orthopedics and Sports Medicine
  • Clinical Neurology

Cite this

Yang, I. H., Kim, H., Kwon, U. H., Lee, K. I., Jun, J. A., Kim, J. H., ... Moon, S. H. (2005). De novo osteogenesis from human ligamentum flavum by adenovirus-mediated bone morphogenetic protein-2 gene transfer. Spine, 30(24), 2749-2754. https://doi.org/10.1097/01.brs.0000192200.57312.13

De novo osteogenesis from human ligamentum flavum by adenovirus-mediated bone morphogenetic protein-2 gene transfer. / Yang, Ick Hwan; Kim, Hyang; Kwon, Un Hye; Lee, Kwang Il; Jun, Ji Ae; Kim, Joo Hang; Yun, Che Ok; Park, Si Young; Lee, Hwan Mo; Moon, Seong Hwan.

In: Spine, Vol. 30, No. 24, 01.12.2005, p. 2749-2754.

Research output: Contribution to journalArticle

Yang, IH, Kim, H, Kwon, UH, Lee, KI, Jun, JA, Kim, JH, Yun, CO, Park, SY, Lee, HM & Moon, SH 2005, 'De novo osteogenesis from human ligamentum flavum by adenovirus-mediated bone morphogenetic protein-2 gene transfer', Spine, vol. 30, no. 24, pp. 2749-2754. https://doi.org/10.1097/01.brs.0000192200.57312.13
Yang, Ick Hwan ; Kim, Hyang ; Kwon, Un Hye ; Lee, Kwang Il ; Jun, Ji Ae ; Kim, Joo Hang ; Yun, Che Ok ; Park, Si Young ; Lee, Hwan Mo ; Moon, Seong Hwan. / De novo osteogenesis from human ligamentum flavum by adenovirus-mediated bone morphogenetic protein-2 gene transfer. In: Spine. 2005 ; Vol. 30, No. 24. pp. 2749-2754.
@article{c5531d64e1f94949969cbfd37d2d899f,
title = "De novo osteogenesis from human ligamentum flavum by adenovirus-mediated bone morphogenetic protein-2 gene transfer",
abstract = "Study Design. 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. Objectives. 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. Summary of Background Data. 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. Conclusion. 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.",
keywords = "Adenovirus, Bone morphogenetic protein-2 (BMP-2), de novo osteogenesis, Gene therapy, Ligamentum flavum, Spinal fusion",
author = "Yang, {Ick Hwan} and Hyang Kim and Kwon, {Un Hye} and Lee, {Kwang Il} and Jun, {Ji Ae} and Kim, {Joo Hang} and Yun, {Che Ok} and Park, {Si Young} and Lee, {Hwan Mo} and Moon, {Seong Hwan}",
year = "2005",
month = "12",
day = "1",
doi = "10.1097/01.brs.0000192200.57312.13",
language = "English",
volume = "30",
pages = "2749--2754",
journal = "Spine",
issn = "0362-2436",
publisher = "Lippincott Williams and Wilkins",
number = "24",

}

TY - JOUR

T1 - De novo osteogenesis from human ligamentum flavum by adenovirus-mediated bone morphogenetic protein-2 gene transfer

AU - Yang, Ick Hwan

AU - Kim, Hyang

AU - Kwon, Un Hye

AU - Lee, Kwang Il

AU - Jun, Ji Ae

AU - Kim, Joo Hang

AU - Yun, Che Ok

AU - Park, Si Young

AU - Lee, Hwan Mo

AU - Moon, Seong Hwan

PY - 2005/12/1

Y1 - 2005/12/1

N2 - Study Design. 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. Objectives. 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. Summary of Background Data. 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. Conclusion. 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.

AB - Study Design. 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. Objectives. 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. Summary of Background Data. 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. Conclusion. 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.

KW - Adenovirus

KW - Bone morphogenetic protein-2 (BMP-2)

KW - de novo osteogenesis

KW - Gene therapy

KW - Ligamentum flavum

KW - Spinal fusion

UR - http://www.scopus.com/inward/record.url?scp=29444439847&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=29444439847&partnerID=8YFLogxK

U2 - 10.1097/01.brs.0000192200.57312.13

DO - 10.1097/01.brs.0000192200.57312.13

M3 - Article

VL - 30

SP - 2749

EP - 2754

JO - Spine

JF - Spine

SN - 0362-2436

IS - 24

ER -