title: Cationic Nanogel-mediated Runx2 and Osterix siRNA Delivery Decreases Mineralization in MC3T3 Cells.
abstract: BACKGROUND
  Heterotopic ossification (HO) may occur after musculoskeletal trauma, traumatic brain injury, and total joint arthroplasty. As such, HO is a compelling clinical concern in both military and civilian medicine. A possible etiology of HO involves dysregulated signals in the bone morphogenetic protein osteogenic cascade. Contemporary treatment options for HO (ie, nonsteroidal antiinflammatory drugs and radiation therapy) have adverse effects associated with their use and are not biologically engineered to abrogate the molecular mechanisms that govern osteogenic differentiation.
  
  
  OBJECTIVE
  We hypothesized that (1) nanogel-mediated short interfering RNA (siRNA) delivery against Runt-related transcription factor 2 (Runx2) and osterix (Osx) genes will decrease messenger RNA expression; (2) inhibit activity of the osteogenic marker alkaline phosphatase (ALP); and (3) inhibit hydroxyapatite (HA) deposition in osteoblast cell cultures.
  
  
  METHODS
  Nanogel nanostructured polymers delivered siRNA in 48-hour treatment cycles against master osteogenic regulators, Runx2 and Osx, in murine calvarial preosteoblasts (MC3T3-E1.4) stimulated for osteogenic differentiation by recombinant human bone morphogenetic protein (rhBMP-2). The efficacy of RNA interference (RNAi) therapeutics was determined by quantitation of messenger RNA knockdown (by quantitative reverse transcription-polymerase chain reaction), downstream protein knockdown (determined ALP enzymatic activity assay), and HA deposition (determined by OsteoImage™ assay).
  
  
  RESULTS
  Gene expression assays demonstrated that nanogel-based RNAi treatments at 1:1 and 5:1 nanogel:short interfering RNA weight ratios reduced Runx2 expression by 48.59% ± 19.53% (p < 0.001) and 43.22% ± 18.01% (both p < 0.001). The same 1:1 and 5:1 treatments against both Runx2 and Osx reduced expression of Osx by 51.65% ± 10.85% and 47.65% ± 9.80% (both p < 0.001). Moreover, repeated 48-hour RNAi treatment cycles against Runx2 and Osx rhBMP-2 administration reduced ALP activity after 4 and 7 days. ALP reductions after 4 days in culture by nanogel 5:1 and 10:1 RNAi treatments were 32.4% ± 12.0% and 33.6% ± 13.8% (both p < 0.001). After 7 days in culture, nanogel 1:1 and 5:1 RNAi treatments produced 35.9% ± 14.0% and 47.7% ± 3.2% reductions in ALP activity. Osteoblast mineralization data after 21 days suggested that nanogel 1:1, 5:1, and 10:1 RNAi treatments decreased mineralization (ie, HA deposition) from cultures treated only with rhBMP-2 (p < 0.001). However, despite RNAi attack on Runx2 and Osx, HA deposition levels remained greater than non-rhBMP-2-treated cell cultures.
  
  
  CONCLUSIONS
  Although mRNA and protein knockdown were confirmed as a result of RNAi treatments against Runx2 and Osx, complete elimination of mineralization processes was not achieved. RNAi targeting mid- and late-stage osteoblast differentiation markers such as ALP, osteocalcin, osteopontin, and bone sialoprotein) may produce the desired RNAi-nanogel nanostructured polymer HO prophylaxis.
  
  
  CONCLUSIONS
  Successful HO prophylaxis should target and silence osteogenic markers critical for heterotopic bone formation processes. The identification of such markers, beyond RUNX2 and OSX, may enhance the effectiveness of RNAi prophylaxes for HO.
authors.affiliation: Department of Biomedical Engineering, Carnegie Mellon University, 700 Technology Drive, Pittsburgh, PA, 15219, USA.
authors.forename: Arun R
authors.initials: AR
authors.lastname: Shrivats
authors.forename: Eric
authors.initials: E
authors.lastname: Hsu
authors.forename: Saadyah
authors.initials: S
authors.lastname: Averick
authors.forename: Molly
authors.initials: M
authors.lastname: Klimak
authors.forename: April C S
authors.initials: AC
authors.lastname: Watt
authors.forename: Marlene
authors.initials: M
authors.lastname: DeMaio
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Matyjaszewski
authors.forename: Jeffrey O
authors.initials: JO
authors.lastname: Hollinger
mesh_terms: D016475:3T3 Cells; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology; D002113:Calcification, Physiologic / Q000187:drug effects; D002412:Cations; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D017886:Durapatite / Q000378:metabolism; D005782:Gels; D051379:Mice; D049329:Nanostructures; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D011109:Polymethacrylic Acids / Q000737:chemistry*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D000076182:Sp7 Transcription Factor; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
chemical_list: C525718:BMP2 protein, human; D015415:Biomarkers; D055396:Bone Morphogenetic Protein 2; D002412:Cations; D050659:Core Binding Factor Alpha 1 Subunit; D005782:Gels; D011109:Polymethacrylic Acids; D034741:RNA, Small Interfering; C493716:Runx2 protein, mouse; D000076182:Sp7 Transcription Factor; C479466:Sp7 protein, mouse; D014157:Transcription Factors; D017886:Durapatite; D000469:Alkaline Phosphatase