Rapid cell culture and pre-clinical screening of a transforming growth factor-beta (TGF-beta) inhibitor for orthopaedics.
BACKGROUND
Transforming growth factor-beta (TGF-beta) and bone morphogenetic proteins (BMPs) utilize parallel and related signaling pathways, however the interaction between these pathways in bone remains unclear. TGF-beta inhibition has been previously reported to promote osteogenic differentiation in vitro, suggesting it may have a capacity to augment orthopaedic repair. We have explored this concept using an approach that represents a template for the testing of agents with prospective orthopaedic applications.


METHODS
The effects of BMP-2, TGF-beta1, and the TGF-beta receptor (ALK-4/5/7) inhibitor SB431542 on osteogenic differentiation were tested in the MC3T3-E1 murine pre-osteoblast cell line. Outcome measures included alkaline phosphatase staining, matrix mineralization, osteogenic gene expression (Runx2, Alp, Ocn) and phosphorylation of SMAD transcription factors. Next we examined the effects of SB431542 in two orthopaedic animal models. The first was a marrow ablation model where reaming of the femur leads to new intramedullary bone formation. In a second model, 20 microg rhBMP-2 in a polymer carrier was surgically introduced to the hind limb musculature to produce ectopic bone nodules.


RESULTS
BMP-2 and SB431542 increased the expression of osteogenic markers in vitro, while TGF-beta1 decreased their expression. Both BMP-2 and SB431542 were found to stimulate pSMAD1 and we also observed a non-canonical repression of pSMAD2. In contrast, neither in vivo system was able to provide evidence of improved bone formation or repair with SB431542 treatment. In the marrow ablation model, systemic dosing with up to 10 mg/kg/day SB431542 did not significantly increase reaming-induced bone formation compared to vehicle only controls. In the ectopic bone model, local co-administration of 38 microg or 192 microg SB431542 did not increase bone formation.


CONCLUSIONS
ALK-4/5/7 inhibitors can promote osteogenic differentiation in vitro, but this may not readily translate to in vivo orthopaedic applications.
Schindeler
Aaron
A
Department of Orthopaedic Research & Biotechnology, The Children's Hospital at Westmead, Locked Bag 4001, Westmead, NSW 2145, Australia. AaronS@chw.edu.au
Morse
Alyson
A
Peacock
Lauren
L
Mikulec
Kathy
K
Yu
Nicole Y C
NY
Liu
Renjing
R
Kijumnuayporn
Sandy
S
McDonald
Michelle M
MM
Baldock
Paul A
PA
Ruys
Andrew J
AJ
Little
David G
DG
D000818:Animals; D001549:Benzamides / Q000627:therapeutic use*; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D002460:Cell Line; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000096:biosynthesis / Q000235:genetics; D004149:Dioxoles / Q000627:therapeutic use*; D004195:Disease Models, Animal; D051379:Mice; D008810:Mice, Inbred C57BL; D019637:Orthopedic Procedures / Q000379:methods; D010012:Osteogenesis / Q000235:genetics / Q000502:physiology*; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051785:Smad Proteins / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D001549:Benzamides; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D050659:Core Binding Factor Alpha 1 Subunit; D004149:Dioxoles; D018125:Receptors, Transforming Growth Factor beta; C493716:Runx2 protein, mouse; D051785:Smad Proteins; D053773:Transforming Growth Factor beta1