title: Inhibition of Rac and ROCK signalling influence osteoblast adhesion, differentiation and mineralization on titanium topographies.
abstract: Reducing the time required for initial integration of bone-contacting implants with host tissues would be of great clinical significance. Changes in osteoblast adhesion formation and reorganization of the F-actin cytoskeleton in response to altered topography are known to be upstream of osteoblast differentiation, and these processes are regulated by the Rho GTPases. Rac and RhoA (through Rho Kinase (ROCK)). Using pharmacological inhibitors, we tested how inhibition of Rac and ROCK influenced osteoblast adhesion, differentiation and mineralization on PT (Pre-treated) and SLA (sandblasted large grit, acid etched) topographies. Inhibition of ROCK, but not Rac, significantly reduced adhesion number and size on PT, with adhesion size consistent with focal complexes. After 1 day, ROCK, but not Rac inhibition increased osteocalcin mRNA levels on SLA and PT, with levels further increasing at 7 days post seeding. ROCK inhibition also significantly increased bone sialoprotein expression at 7 days, but not BMP-2 levels. Rac inhibition significantly reduced BMP-2 mRNA levels. ROCK inhibition increased nuclear translocation of Runx2 independent of surface roughness. Mineralization of osteoblast cultures was greater on SLA than on PT, but was increased by ROCK inhibition and attenuated by Rac inhibition on both topographies. In conclusion, inhibition of ROCK signalling significantly increases osteoblast differentiation and biomineralization in a topographic dependent manner, and its pharmacological inhibition could represent a new therapeutic to speed bone formation around implanted metals and in regenerative medicine applications.
authors.affiliation: Department of Anatomy, Graduate Program of Biomedical Engineering, The University of Western Ontario, London, Ontario, Canada.
authors.forename: Paul D H
authors.initials: PD
authors.lastname: Prowse
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Elliott
authors.forename: Jeff
authors.initials: J
authors.lastname: Hutter
authors.forename: Douglas W
authors.initials: DW
authors.lastname: Hamilton
mesh_terms: D000818:Animals; D002113:Calcification, Physiologic / Q000187:drug effects*; D002448:Cell Adhesion / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects*; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D022001:Focal Adhesions / Q000187:drug effects; D006801:Humans; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D010012:Osteogenesis / Q000235:genetics; D011480:Protease Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects*; D013499:Surface Properties; D014025:Titanium / Q000737:chemistry; D020744:rac GTP-Binding Proteins / Q000037:antagonists & inhibitors*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors*
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D011480:Protease Inhibitors; D014025:Titanium; D054460:rho-Associated Kinases; D020744:rac GTP-Binding Proteins