title: Percutaneous gene therapy heals cranial defects.
abstract: Nonhealing bone defects are difficult to treat. As the bone morphogenic protein and transforming growth factor beta pathways have been implicated in bone healing, we hypothesized that percutaneous Smad7 silencing would enhance signaling through both pathways and improve bone formation. Critical sized parietal trephine defects were created and animals received percutaneous injection of: agarose alone or agarose containing nonsense or Smad7 small interfering RNA (siRNA). At 12 weeks, SMADs1, 2, 3, 5, 7 and 8 levels were assessed. Smad1/5/8 osteogenic target, Dlx5, and SMAD2/3 angiogenic target, plasminogen activator inhibitor-1 (Pai1), transcription levels were measured. Noncanonical signaling through TGFβ activated kinase-1 (Tak1) and target, runt-related transcription factor 2 (Runx2) and collagen1α1 (Col1α1), transcription were also measured. Micro-computed tomography and Gomori trichome staining were used to assess healing. Percutaneous injection of Smad7 siRNA significantly knocked down Smad7 mRNA (86.3 ± 2.5%) and protein levels (46.3 ± 3.1%). The SMAD7 knockdown resulted in a significant increase in receptor-regulated SMADs (R-SMAD) (Smad 1/5/8 and Smad2/3) nuclear translocation. R-SMAD nuclear translocation increased Dlx5 and Pai1 transcription. Additionally, noncanonical signaling through Tak1 increased Runx2 and Col1α1 target transcription. Compared with animals treated with agarose alone (33.9 ± 2.8% healing) and nonsense siRNA (31.5 ± 11.8% healing), animals treated Smad7 siRNA had significantly great (91.2 ± 3.8%) healing. Percutaneous Smad7 silencing increases signal transduction through canonical and noncanonical pathways resulting in significant bone formation. Minimally invasive gene therapies may prove effective in the treatment of nonhealing bone defects.
authors.affiliation: The Department of Plastic Surgery, Institute of Reconstructive Plastic Surgery Laboratories, New York University Medical Center, New York, NY, USA.
authors.forename: J
authors.initials: J
authors.lastname: Layliev
authors.forename: F
authors.initials: F
authors.lastname: Sagebin
authors.forename: A
authors.initials: A
authors.lastname: Weinstein
authors.forename: A
authors.initials: A
authors.lastname: Marchac
authors.forename: C
authors.initials: C
authors.lastname: Szpalski
authors.forename: P B
authors.initials: PB
authors.lastname: Saadeh
authors.forename: S M
authors.initials: SM
authors.lastname: Warren
mesh_terms: D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D050723:Fractures, Bone / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D055785:Gene Knockdown Techniques; D015316:Genetic Therapy; D006801:Humans; D008297:Male; D051379:Mice; D010012:Osteogenesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering; D015398:Signal Transduction; D012886:Skull / Q000378:metabolism; D051896:Smad Proteins, Receptor-Regulated / Q000235:genetics / Q000378:metabolism; D051906:Smad7 Protein / Q000235:genetics* / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism
chemical_list: D019485:Bone Morphogenetic Proteins; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051896:Smad Proteins, Receptor-Regulated; D051906:Smad7 Protein; D016212:Transforming Growth Factor beta