title: Covalent attachment of P15 peptide to titanium surfaces enhances cell attachment, spreading, and osteogenic gene expression.
abstract: P15, a synthetic 15 amino acid peptide, mimics the cell-binding domain within the alpha-1 chain of human collagen is being tested in clinical trials to determine if it enhances bone formation in spinal fusions. We hypothesize that covalent attachment of P15 to titanium implants may also serve to promote osseointegration. To test this hypothesis, we measured osteoblast and mesenchymal cell adhesion, proliferation, and maturation on P15 tethered to a titanium (Ti-P15) surface. P15 peptide was covalently bonded to titanium alloy surfaces and incubated with osteoblast like cells. Cell toxicity, adhesion, spreading, and differentiation was then evaluated. Real-time quantitative PCR, Western blot analysis, and fluorescent immunohistochemistry was performed to measure osteoblast gene expression and differentiation. There was no evidence of toxicity. Significant increases in early cell attachment, spreading, and proliferation were observed on the Ti-P15 surface. Increased filapodial attachments, α(2) integrin expression, and phosphorylated focal adhesion kinase immunostaining indicated activation of integrin signaling pathways. qRT-PCR analysis indicated there was significant increase in osteogenic differentiation markers in cells grown on Ti-P15 compared to control-Ti. Western blotting confirmed these findings. Surface modification of titanium with P15 significantly increased cell attachment, spreading, osteogenic gene expression, and differentiation. Results of this study suggest that Ti-P15 has the potential to safely enhance bone formation and promote osseointegration of titanium implants.
authors.affiliation: Department of Orthopedics, The Second Hospital of Jilin University, Chang Chun, Jilin, China.
authors.forename: Qinyi
authors.initials: Q
authors.lastname: Liu
authors.forename: Worawat
authors.initials: W
authors.lastname: Limthongkul
authors.forename: Gursukhmandeep
authors.initials: G
authors.lastname: Sidhu
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.forename: Alexander
authors.initials: A
authors.lastname: Vaccaro
authors.forename: Ryan
authors.initials: R
authors.lastname: Shenck
authors.forename: Noreen
authors.initials: N
authors.lastname: Hickok
authors.forename: Irving
authors.initials: I
authors.lastname: Shapiro
authors.forename: Theresa
authors.initials: T
authors.lastname: Freeman
mesh_terms: D000818:Animals; D002448:Cell Adhesion; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival; D003094:Collagen / Q000494:pharmacology*; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D039421:Integrin alpha2 / Q000378:metabolism; D051379:Mice; D016348:Osseointegration / Q000187:drug effects*; D010006:Osteoblasts / Q000187:drug effects; D010012:Osteogenesis; D010446:Peptide Fragments / Q000494:pharmacology*; D019736:Prostheses and Implants; D015398:Signal Transduction; D014025:Titanium
chemical_list: D039421:Integrin alpha2; D010446:Peptide Fragments; C407062:cell-binding peptide P-15; D003094:Collagen; D014025:Titanium; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human