Inhibition of Rac and ROCK Signalling Influence Osteoblast Adhesion, Differentiation and Mineralization on Titanium Topographies

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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.

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Most cited references 53

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Surface treatments of titanium dental implants for rapid osseointegration.

L Le Guéhennec, A Soueidan, P Layrolle … (2007)

The osseointegration rate of titanium dental implants is related to their composition and surface roughness. Rough-surfaced implants favor both bone anchoring and biomechanical stability. Osteoconductive calcium phosphate coatings promote bone healing and apposition, leading to the rapid biological fixation of implants. The different methods used for increasing surface roughness or applying osteoconductive coatings to titanium dental implants are reviewed. Surface treatments, such as titanium plasma-spraying, grit-blasting, acid-etching, anodization or calcium phosphate coatings, and their corresponding surface morphologies and properties are described. Most of these surfaces are commercially available and have proven clinical efficacy (>95% over 5 years). The precise role of surface chemistry and topography on the early events in dental implant osseointegration remain poorly understood. In addition, comparative clinical studies with different implant surfaces are rarely performed. The future of dental implantology should aim to develop surfaces with controlled and standardized topography or chemistry. This approach will be the only way to understand the interactions between proteins, cells and tissues, and implant surfaces. The local release of bone stimulating or resorptive drugs in the peri-implant region may also respond to difficult clinical situations with poor bone quality and quantity. These therapeutic strategies should ultimately enhance the osseointegration process of dental implants for their immediate loading and long-term success.

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The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors.

A J Ridley, A. Hall (1992)

Actin stress fibers are one of the major cytoskeletal structures in fibroblasts and are linked to the plasma membrane at focal adhesions. rho, a ras-related GTP-binding protein, rapidly stimulated stress fiber and focal adhesion formation when microinjected into serum-starved Swiss 3T3 cells. Readdition of serum produced a similar response, detectable within 2 min. This activity was due to a lysophospholipid, most likely lysophosphatidic acid, bound to serum albumin. Other growth factors including PDGF induced actin reorganization initially to form membrane ruffles, and later, after 5 to 10 min, stress fibers. For all growth factors tested the stimulation of focal adhesion and stress fiber assembly was inhibited when endogenous rho function was blocked, whereas membrane ruffling was unaffected. These data imply that rho is essential specifically for the coordinated assembly of focal adhesions and stress fibers induced by growth factors.

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Rational design and characterization of a Rac GTPase-specific small molecule inhibitor.

Yuan Gao, J Bradley Dickerson, Fukun Guo … (2004)

The signaling pathways mediated by Rho family GTPases have been implicated in many aspects of cell biology. The specificity of the pathways is achieved in part by the selective interaction between Dbl family guanine nucleotide exchange factors (GEFs) and their Rho GTPase substrates. Here, we report a first-generation small-molecule inhibitor of Rac GTPase targeting Rac activation by GEF. The chemical compound NSC23766 was identified by a structure-based virtual screening of compounds that fit into a surface groove of Rac1 known to be critical for GEF specification. In vitro it could effectively inhibit Rac1 binding and activation by the Rac-specific GEF Trio or Tiam1 in a dose-dependent manner without interfering with the closely related Cdc42 or RhoA binding or activation by their respective GEFs or with Rac1 interaction with BcrGAP or effector PAK1. In cells, it potently blocked serum or platelet-derived growth factor-induced Rac1 activation and lamellipodia formation without affecting the activity of endogenous Cdc42 or RhoA. Moreover, this compound reduced Trio or Tiam1 but not Vav, Lbc, Intersectin, or a constitutively active Rac1 mutant-stimulated cell growth and suppressed Trio, Tiam1, or Ras-induced cell transformation. When applied to human prostate cancer PC-3 cells, it was able to inhibit the proliferation, anchorage-independent growth and invasion phenotypes that require the endogenous Rac1 activity. Thus, NSC23766 constitutes a Rac-specific small-molecule inhibitor that could be useful to study the role of Rac in various cellular functions and to reverse tumor cell phenotypes associated with Rac deregulation.

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Author and article information

Contributors

Luc Malaval: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, USA )

ISSN (Electronic): 1932-6203

Publication date Collection: 2013

Publication date (Electronic): 7 March 2013

Volume: 8

Issue: 3

Electronic Location Identifier: e58898

Affiliations

[1 ]Department of Anatomy, Graduate Program of Biomedical Engineering, The University of Western Ontario, London, Ontario, Canada

[2 ]Department of Cell Biology, Graduate Program of Biomedical Engineering, The University of Western Ontario, London, Ontario, Canada

[3 ]Department of Physics and Astronomy, The University of Western Ontario, London, Ontario, Canada

[4 ]Division of Oral Biology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada

INSERM U1059/LBTO, Université Jean Monnet, France

Author notes

* E-mail: dhamil2@ 123456uwo.ca

Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: DWH PDHP. Performed the experiments: PDHP CGE JH DWH. Analyzed the data: PDHP CGE JH DWH. Wrote the paper: PDHP DWH.

Article

Publisher ID: PONE-D-12-23750

DOI: 10.1371/journal.pone.0058898

PMC ID: 3591363

PubMed ID: 23505566

SO-VID: 517838a6-1da1-4b16-ab69-1adcc00182dc

License:

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 8 August 2012

Date accepted : 8 February 2013

Page count

Pages: 13

Funding

This work was funded by the Canadian Foundation for Innovation Leaders Opportunity Fund(Grant no: 18742) and the Natural Sciences and Engineering Research Council of Canada(Grant Number: 355615-2009) to DWH. DWH is a recipient of the Ontario Ministry of Research and Innovation Early Researcher Award. This work was also supported by the Joint Motion Program(JuMP) – A CIHR Training Program in Musculoskeletal Health Research and Leadership. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Inhibition of Rac and ROCK Signalling Influence Osteoblast Adhesion, Differentiation and Mineralization on Titanium Topographies

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PLOS: COVID-19 pandemic

Most cited references 53

Surface treatments of titanium dental implants for rapid osseointegration.

The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors.

Rational design and characterization of a Rac GTPase-specific small molecule inhibitor.

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