title: Synergistic effect of Indian hedgehog and bone morphogenetic protein-2 gene transfer to increase the osteogenic potential of human mesenchymal stem cells.
abstract: BACKGROUND
  To stimulate healing of large bone defects research has concentrated on the application of mesenchymal stem cells (MSCs).
  
  
  METHODS
  In the present study, we induced the overexpression of the growth factors bone morphogenetic protein 2 (BMP-2) and/or Indian hedgehog (IHH) in human MSCs by adenoviral transduction to increase their osteogenic potential. GFP and nontransduced MSCs served as controls. The influence of the respective genetic modification on cell metabolic activity, proliferation, alkaline phosphatase (ALP) activity, mineralization in cell culture, and osteogenic marker gene expression was investigated.
  
  
  RESULTS
  Transduction had no negative influence on cell metabolic activity or proliferation. ALP activity showed a typical rise-and-fall pattern with a maximal activity at day 14 and 21 after osteogenic induction. Enzyme activity was significantly higher in groups cultured with osteogenic media. The overexpression of BMP-2 and especially IHH + BMP-2 resulted in a significantly higher mineralization after 28 days. This was in line with obtained quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analyses, which showed a significant increase in osteopontin and osteocalcin expression for osteogenically induced BMP-2 and IHH + BMP-2 transduced cells when compared with the other groups. Moreover, an increase in runx2 expression was observed in all osteogenic groups toward day 21. It was again more pronounced for BMP-2 and IHH + BMP-2 transduced cells cultured in osteogenic media.
  
  
  CONCLUSIONS
  In summary, viral transduction did not negatively influence cell metabolic activity and proliferation. The overexpression of BMP-2 in combination with or without IHH resulted in an increased deposition of mineralized extracellular matrix, and expression of osteogenic marker genes. Viral transduction therefore represents a promising means to increase the osteogenic potential of MSCs and the combination of different transgenes may result in synergistic effects.
authors.forename: Johannes C
authors.initials: JC
authors.lastname: Reichert
authors.forename: Jonas
authors.initials: J
authors.lastname: Schmalzl
authors.forename: Patrick
authors.initials: P
authors.lastname: Prager
authors.forename: Fabian
authors.initials: F
authors.lastname: Gilbert
authors.forename: Verena M C
authors.initials: VM
authors.lastname: Quent
authors.forename: Andre F
authors.initials: AF
authors.lastname: Steinert
authors.forename: Maximilian
authors.initials: M
authors.lastname: Rudert
authors.forename: Ulrich
authors.initials: U
authors.lastname: Nöth
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D001854:Bone Marrow Cells / Q000166:cytology; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D053823:Hedgehog Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D010012:Osteogenesis; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: D055396:Bone Morphogenetic Protein 2; D050659:Core Binding Factor Alpha 1 Subunit; D053823:Hedgehog Proteins; D015675:Osteocalcin; D053495:Osteopontin