title: Osteoblast differentiation in vitro and in vivo promoted by Osterix.
abstract: C3H10T1/2/Osx, a stably transfected cell line with Osterix (Osx), was produced and chondrocytic and osteoblastic differentiation were studied in vitro. Osx promoted osteoblastic lineage that was dexamethasone dependent. Furthermore, in vivo, Osx induced ectopic mineralization in a heterotopic mouse muscle model. Skeletogenesis involves a cascade of molecular activities sequentially performed by osteoblasts and chondroblasts. A transcriptional factor gene Osx appears to influence cell disposition toward the chondrocytic or osteoblastic phenotype and therefore may be an important signaling cue for bone formation. Understanding the molecular conditions involved with Osx promoted osteoblast differentiation will facilitate therapeutic applications of Osx. Consequently, the objective of this study was to investigate chondrocytic and osteoblastic phenotype differentiation using an Osx plasmid DNA exploiting both in vitro and in vivo methodologies. In vitro, a stably transfected C3H10T1/2/Osx cell line was established and promotion of either an osteoblast or chondroblast phenotype was performed by selectively introducing dexamethasone (dex) and assaying mRNA content and phenotype markers. In vivo, a mouse muscle model was used to determine heterotopic ossification using designated Osx plasmid DNA doses incorporated in a (50:50 Poly (D,L-lactide-co-glycolide) (i.e., PLGA) 3D scaffold. Histological assessment was used to determine implant responses. Quantitative real-time polymerase chain reaction (q-RT-PCR) showed a significant increase in mRNA expression of osteocalcin (Ocn), Runx2, osteonectin (On) and osteopontin (Op) (p < 0.05) in the C3H10T1/2/Osx cells compared to the empty vector transfected cell control. At day 21, mineralization was demonstrated in the cultures of C3H10T1/2/Osx exposed to dex, but neither in cultures lacking dex nor controls. In the absence of dex, C3H10T1/2/Osx cells revealed a significantly higher expression of Sox9 and Aggrecan (Agc). In vivo, 80 microg of Osx plasmid DNA induced heterotopic mineralization 4 weeks following implantation in a mouse muscle model and the effect was dependent on the Osx plasmid DNA dose delivered in the PLGA scaffold. Using a non-committed cell line (C3H10T1/2), cell differentiation to an osteoblast phenotype appears to be dependent upon an interaction between intracellular events initiated by the transcriptional factor Osx and the presence of dex. The in vivo findings suggest Osx may promote osteoblast differentiationand mineralization at a heterotopic site.
authors.affiliation: Bone Tissue Engineering Center, Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Huihua
authors.initials: H
authors.lastname: Fu
authors.forename: Bruce
authors.initials: B
authors.lastname: Doll
authors.forename: Tim
authors.initials: T
authors.lastname: McNelis
authors.forename: Jeffrey O
authors.initials: JO
authors.lastname: Hollinger
mesh_terms: D000818:Animals; D000943:Antigens, Differentiation / Q000096:biosynthesis; D002113:Calcification, Physiologic / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism; D005260:Female; D051379:Mice; D008813:Mice, Inbred ICR; D041681:NIH 3T3 Cells; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids / Q000737:chemistry; D011098:Polyglactin 910 / Q000737:chemistry; D000076182:Sp7 Transcription Factor; D054457:Tissue Scaffolds / Q000737:chemistry; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D000943:Antigens, Differentiation; D000076182:Sp7 Transcription Factor; C479466:Sp7 protein, mouse; D014157:Transcription Factors; D011098:Polyglactin 910