Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Jun;31(18):4909-17.
doi: 10.1016/j.biomaterials.2010.02.071. Epub 2010 Mar 30.

Regulation of angiogenesis during osseointegration by titanium surface microstructure and energy

Andrew L Raines  1 , Rene Olivares-Navarrete, Marco Wieland, David L Cochran, Zvi Schwartz, Barbara D Boyan
Affiliations

Regulation of angiogenesis during osseointegration by titanium surface microstructure and energy

Andrew L Raines et al. Biomaterials. 2010 Jun.

Abstract

Rough titanium (Ti) surface microarchitecture and high surface energy have been shown to increase osteoblast differentiation, and this response occurs through signaling via the alpha(2)beta(1) integrin. However, clinical success of implanted materials is dependent not only upon osseointegration but also on neovascularization in the peri-implant bone. Here we tested the hypothesis that Ti surface microtopography and energy interact via alpha(2)beta(1) signaling to regulate the expression of angiogenic growth factors. Primary human osteoblasts (HOB), MG63 cells and MG63 cells silenced for alpha(2) integrin were cultured on Ti disks with different surface microtopographies and energies. Secreted levels of vascular endothelial growth factor-A (VEGF-A), basic fibroblast growth factor (FGF-2), epidermal growth factor (EGF), and angiopoietin-1 (Ang-1) were measured. VEGF-A increased 170% and 250% in MG63 cultures, and 178% and 435% in HOB cultures on SLA and modSLA substrates, respectively. In MG63 cultures, FGF-2 levels increased 20 and 40-fold while EGF increased 4 and 6-fold on SLA and modSLA surfaces. These factors were undetectable in HOB cultures. Ang-1 levels were unchanged on all surfaces.Media from modSLA MG63 cultures induced more rapid differentiation of endothelial cells and this effect was inhibited by anti-VEGF-A antibodies. Treatment of MG63 cells with 1 alpha,25(OH)(2)D3 enhanced levels of VEGF-A on SLA and modSLA.Silencing the alpha(2) integrin subunit increased VEGF-A levels and decreased FGF-2 levels. These results show that Ti surface microtopography and energy modulate secretion of angiogenic growth factors by osteoblasts and that this regulation is mediated at least partially via alpha(2)beta(1) integrin signaling.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Response of MG63 cells cultured on tissue culture polystyrene and Ti surfaces: (A) cell number, (B) alkaline phosphatase specific activity in the cell lysate, and (C) osteocalcin levels in the conditioned medium. Cells were cultured on control (TCPS), PT, SLA, and modSLA Ti surfaces. Values presented are mean ± SEM of six independent cultures. The data presented are from one of two separate experiments with comparable results. Data were analyzed using ANOVA and statistical significance between groups was determined using Bonferroni's modification of Student's t-test. *p< 0.05 vs. TCPS; ·p<0.05 vs. PT; Φp<0.05 vs. SLA.
Figure 2
Figure 2
Production of angiogenic growth factors by MG63 cells on tissue culture polystyrene and Ti surfaces: (A) VEGF-A, (B) FGF-2, (C) EGF, and (D) Ang-1 levels were determined in the conditioned medium by ELISA. Cells were cultured on control (TCPS), PT, SLA, and modSLA Ti surfaces. Values presented are mean ± SEM of six independent cultures. The data presented are from one of two separate experiments with comparable results. Data were analyzed using ANOVA and statistical significance between groups was determined using Bonferroni's modification of Student's t-test. *p< 0.05, vs. TCPS; ·p<0.05, vs. PT; Φp<0.05, vs. SLA.
Figure 3
Figure 3
Characterization of normal human osteoblasts cultured on TCPS, PT, SLA, and modSLA Ti surfaces: (A) cell number, (B) osteocalcin, and (C) VEGF-A levels in the conditioned medium. Values presented are mean ± SEM of six independent cultures. The data presented are from one of two separate experiments with comparable results. Data were analyzed using ANOVA and statistical significance between groups was determined using Bonferroni's modification of Student's t-test. *p< 0.05 vs. TCPS; ·p<0.05 vs. PT; Φp<0.05 vs. SLA.
Figure 4
Figure 4
Effect of 1α,25 (OH)2D3 treatment on MG63 cell differentiation. MG63 cells were cultured on TCPS, PT, SLA, and modSLA Ti surfaces. At confluence, cultures were treated with 10-9M or 10-8M 1α,25 (OH)2D3 for 24 hours. (A) Osteocalcin and (B) VEGF-A levels in the conditioned medium were determined. Values are mean ± SEM of six independent cultures. The data presented are from one of two experiments with comparable results. Data were analyzed using ANOVA and statistical significance between groups was determined using Bonferroni's modification of Student's t-test. *p< 0.05 vs. TCPS; ·p<0.05 vs. PT; Φp<0.05 vs. SLA.
Figure 5
Figure 5
Endothelial cell differentiation. Representative images of endothelial cells cultured on a fibrin gel matrix with conditioned media from (A) TCPS, (B) PT, (C) SLA, and (D) modSLA cultures of MG63 cells.
Figure 6
Figure 6
Endothelial cell differentiation. Endothelial tube formation was assessed using a Matrigel® tube formation assay with conditioned medium from MG63 cells cultured on TCPS, PT, SLA, and modSLA surfaces. (A) Total endothelial tube length and total number of branch points at 4h. Values are mean ± SEM of six independent cultures. Data were analyzed using ANOVA and statistical significance between groups was determined using Bonferroni's modification of Student's t-test. *p< 0.05 vs. TCPS; ·p<0.05 vs. PT; #p<0.05 vs. SLA. (B) Addition of VEGF-A neutralization antibody inhibits endothelial cell differentiation in response to MG63 conditioned media. Endothelial tube formation was assessed using conditioned medium from MG63 cells cultured on TCPS, PT, SLA, and modSLA surfaces in the presence of a VEGF-A neutralization antibody. Total endothelial tube length is presented for cultures with and without the addition of neutralization antibody. *p<0.05 vs. no antibody. (C) Endothelial cell differentiation was further assessed using a fibrin gel assay to determine total endothelial tube length within a fibrin gel matrix. *p< 0.05 vs. TCPS.
Figure 7
Figure 7
Integrin α2 signaling regulates pro-angiogenic growth factor production. Stably silenced α2 cells and MG63 cells were cultured on TCPS, PT, SLA, and modSLA substrates. Total cell number (A), osteocalcin (B), VEGF (C), and FGF-2 (D) levels were determined in the conditioned media. Values are mean ± SEM of six independent cultures. Data were analyzed using ANOVA and Student's t-test. *p<0.05 vs. MG63 wild-type on TCPS; #p<0.05 vs. MG63 wild-type.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Hench LL, Polak JM. Third-generation biomedical materials. Science. 2002;295:1014–7. - PubMed
    1. Jell G, Stevens MM. Gene activation by bioactive glasses. J Mater Sci Mater Med. 2006;17:997–1002. - PubMed
    1. Kriparamanan R, Aswath P, Zhou A, Tang L, Nguyen KT. Nanotopography: cellular responses to nanostructured materials. J Nanosci Nanotechnol. 2006;6:1905–19. - PubMed
    1. Goto T, Yoshinari M, Kobayashi S, Tanaka T. The initial attachment and subsequent behavior of osteoblastic cells and oral epithelial cells on titanium. Biomed Mater Eng. 2004;14:537–44. - PubMed
    1. Boyan BD, Schwartz Z, Lohmann CH, Sylvia VL, Cochran DL, Dean DD, Puzas JE. Pretreatment of bone with osteoclasts affects phenotypic expression of osteoblast-like cells. J Orthop Res. 2003;21:638–47. - PubMed

Publication types

MeSH terms