Osteogenesis induced in goat bone marrow progenitor cells by recombinant adenovirus coexpressing bone morphogenetic protein 2 and basic fibroblast growth factor

Braz J Med Biol Res. 2013 Sep;46(9):809-14. doi: 10.1590/1414-431X20132929. Epub 2013 Sep 6.

Abstract

Bone morphogenetic protein 2 (BMP2) and basic fibroblast growth factor (bFGF) have been shown to exhibit a synergistic effect to promote bone repair and healing. In this study, we constructed a novel adenovirus with high coexpression of BMP2 and bFGF and evaluated its effect on osteogenic differentiation of goat bone marrow progenitor cells (BMPCs). Recombinant adenovirus Ad-BMP2-bFGF was constructed by using the T2A sequence. BMPCs were isolated from goats by density gradient centrifugation and adherent cell culture, and were then infected with Ad-BMP2-bFGF or Ad-BMP2. Expression of BMP2 and bFGF was detected by ELISA, and alkaline phosphatase (ALP) activity was detected by an ALP assay kit. In addition, von Kossa staining and immunocytochemical staining of collagen II were performed on BMPCs 21 days after infection. There was a high coexpression of BMP2 and bFGF in BMPCs infected with Ad-BMP2-bFGF. Twenty-one days after infection, ALP activity was significantly higher in BMPCs infected with Ad-BMP2-bFGF than in those infected with Ad-BMP2. Larger and more mineralized calcium nodules, as well as stronger collagen II staining, were observed in BMPCs infected with Ad-BMP2-bFGF than in those infected with Ad-BMP2. In summary, we developed a novel adenovirus vector Ad-BMP2-bFGF for simultaneous high coexpression of BMP2 and bFGF, which could induce BMPCs to differentiate efficiently into osteoblasts.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenoviridae / genetics
  • Adenoviridae / metabolism*
  • Alkaline Phosphatase / metabolism
  • Analysis of Variance
  • Animals
  • Base Sequence
  • Bone Marrow Cells / cytology*
  • Bone Marrow Cells / virology
  • Bone Morphogenetic Protein 2 / genetics
  • Bone Morphogenetic Protein 2 / metabolism*
  • Cell Differentiation / physiology*
  • Centrifugation, Density Gradient
  • Enzyme-Linked Immunosorbent Assay
  • Fibroblast Growth Factor 2 / genetics
  • Fibroblast Growth Factor 2 / metabolism*
  • Gene Transfer Techniques
  • Genetic Vectors / metabolism
  • Goats
  • Immunohistochemistry
  • Osteoblasts / cytology
  • Osteogenesis / physiology*
  • Primary Cell Culture
  • Recombinant Proteins / genetics
  • Stem Cells / cytology*
  • Stem Cells / virology

Substances

  • Bone Morphogenetic Protein 2
  • Recombinant Proteins
  • Fibroblast Growth Factor 2
  • Alkaline Phosphatase