In vitro and in vivo osteoblastic differentiation of BMP-2- and Runx2-engineered skeletal myoblasts.
Genetic engineering with osteogenic factors is a promising approach for cell-based therapeutics and orthopedic regeneration. However, the relative efficacy of different strategies for inducing osteoblastic differentiation remains unclear and is further complicated by varied delivery vehicles, cell types, and evaluation criteria. In order to elucidate the effects of distinct gene-based strategies, we quantitatively evaluated osteoblastic differentiation and mineralization of primary skeletal myoblasts overexpressing either the BMP-2 growth factor or Runx2 transcription factor. Retroviral delivery of BMP-2 or Runx2 stimulated differentiation into an osteoblastic phenotype, as demonstrated by the induction of osteogenic gene expression, alkaline phosphatase activity, and matrix mineralization in monolayer culture and on collagen scaffolds both in vitro and in an intramuscular site in vivo. In general, BMP-2 stimulated osteoblastic markers faster and to a greater extent than Runx2, although we also identified experimental conditions under which these two factors produced similar effects. Additionally, Runx2-engineered cells did not utilize paracrine signaling via secreted osteogenic factors, in contrast to cells overexpressing BMP-2, as demonstrated by conditioned media studies and activation of Smad signaling. These results emphasize the complexity of gene therapy-based orthopedic therapeutics as an integrated relationship of differentiation state, construct maturation, and paracrine signaling of osteogenic cells. This study is significant in evaluating proposed therapeutic systems and defining a successful strategy for integrating gene medicine and orthopedic regeneration.
Gersbach
Charles A
CA
Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30332, USA.
Guldberg
Robert E
RE
García
Andrés J
AJ
D000818:Animals; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016513:Mice, SCID; D024510:Muscle Development; D032448:Myoblasts, Skeletal / Q000166:cytology* / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism; D010012:Osteogenesis / Q000502:physiology; D019899:Paracrine Communication; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D015398:Signal Transduction; D051785:Smad Proteins / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism
C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D050659:Core Binding Factor Alpha 1 Subunit; D012333:RNA, Messenger; C493716:Runx2 protein, mouse; D051785:Smad Proteins; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1