title: Integrin-mediated signaling in osteoblasts on titanium implant materials.
abstract: The intracellular signaling pathway for osteoblast adhesion to the orthopedic implant material Ti6Al4V (TIV) was investigated and compared to integrin-mediated adhesion to extracellular matrix proteins. Primary osteoblasts from fetal rat calvaria were plated onto TIV, fibronectin (FN), and poly-L-lysine (PLL) and the levels of focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK), and AP-1 transcription factors, c-fos and c-jun, were compared by Western and Northern blots. Cells on all substrates showed maximum FAK phosphorylation within 60 min and then a decrease at 2 and 24 h. However, the subsequent signal transduction pathway differed on PLL compared to TIV and FN. MAPK was phosphorylated similarly in osteoblasts attached to FN and TIV, whereas cells on PLL demonstrated no MAPK phosphorylation. On TIV and FN, c-fos and c-jun mRNA levels were maximal within 1 h and then plateaued or declined by 2 h. On PLL, they increased at 2 h. Within 1 h, c-fos protein was stimulated in cells attached to TIV and FN and decreased in cells on PLL. c-jun protein increased on all substrates compared to unplated cells. Cytoskeletal changes visualized by phalloidin fluorescence microscopy at 4 h of culture were delayed on TIV compared to FN. In addition, approximately 50% fewer cells adhered to TIV compared to FN or PLL. By 24 h, a well-spread cytoskeleton with focal adhesion sites was apparent on TIV and FN, but cells on PLL were rounded with minimal cell spreading. During 6 days of culture, cells on FN and TIV proliferated, whereas the number of cells on PLL remained the same or decreased, depending on the initial plating density. We conclude that osteoblast adhesion to TIV implants is similar to osteoblast adhesion to FN and leads to osteoblast proliferation. These data provide evidence for the biocompatibility of TIV at a molecular level.
authors.affiliation: Department of Orthopaedic Surgery, MC 1110, University of Connecticut Health Center, Farmington, Connecticut 06032, USA.
authors.forename: A
authors.initials: A
authors.lastname: Krause
authors.forename: E A
authors.initials: EA
authors.lastname: Cowles
authors.forename: G
authors.initials: G
authors.lastname: Gronowicz
mesh_terms: D000497:Alloys; D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry*; D002448:Cell Adhesion; D002455:Cell Division; D002478:Cells, Cultured; D020099:Coated Materials, Biocompatible; D003599:Cytoskeleton / Q000648:ultrastructure; D005353:Fibronectins; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D016762:Genes, fos; D016758:Genes, jun; D016023:Integrins / Q000502:physiology*; D007365:Intercellular Junctions / Q000648:ultrastructure; D020935:MAP Kinase Signaling System; D008422:Materials Testing; D008856:Microscopy, Fluorescence; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D010766:Phosphorylation; D011107:Polylysine; D019736:Prostheses and Implants; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D015398:Signal Transduction; D013997:Time Factors; D014025:Titanium / Q000737:chemistry*; D018808:Transcription Factor AP-1 / Q000096:biosynthesis
chemical_list: D000497:Alloys; D001672:Biocompatible Materials; D020099:Coated Materials, Biocompatible; D005353:Fibronectins; D016023:Integrins; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; C031462:titanium alloy (TiAl6V4); D011107:Polylysine; D014025:Titanium; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496052:Ptk2 protein, rat