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. 2019 Oct 1:97:578-586.
doi: 10.1016/j.actbio.2019.07.036. Epub 2019 Jul 23.

Osteoblasts grown on microroughened titanium surfaces regulate angiogenic growth factor production through specific integrin receptors

Andrew L Raines  1 , Michael B Berger  2 , Zvi Schwartz  3 , Barbara D Boyan  4
Affiliations

Osteoblasts grown on microroughened titanium surfaces regulate angiogenic growth factor production through specific integrin receptors

Andrew L Raines et al. Acta Biomater. .

Abstract

Cellular attachment and response to biomaterials are mediated by integrin receptor binding to extracellular matrix proteins adsorbed onto the material surface. Osteoblasts interact with their substrates via several integrin complexes including fibronectin-binding α5β1 and collagen-binding α1β1 and α2β1. Knockdown of α2 or β1 integrin subunits inhibits the production of factors that promote an osteogenic microenvironment, including osteocalcin, osteoprotegerin, and TGFβ1. Osteoblasts also secrete several angiogenic growth factors such as VEGF-A (VEGF165), FGF-2, and angiopoietin 1, which are regulated by titanium surface topography and surface energy. Here, we examined whether signaling through integrin receptor complexes regulates production and secretion of angiogenic factors during osteoblast differentiation on microtextured Ti surfaces. To do this, integrin subunits α1, α2, α5, and β1 were stably silenced in MG63 osteoblast-like cells cultured on grit-blasted/acid-etched hydrophobic Ti (SLA) or on hydrophilic SLA (modSLA). VEGF-A production increased in response to Ti surface topography and energy in integrin α2, α5, and β1 silenced cells but decreased in α1-silenced cells. FGF-2 decreased on modSLA substrates in both α1 and α2-silenced cells but was unchanged in response to silencing of either α5 or β1. In integrin α1, α2, and β1-silenced cells, Ang-1 increased on modSLA but α5-silencing did not affect Ang-1 production during surface mediated differentiation. These results suggest that signaling through specific integrin receptor complexes during osteoblast differentiation on microstructured Ti substrates, regulates the production of angiogenic factors by those cells, and this is differentially regulated by surface hydrophilicity. STATEMENT OF SIGNIFICANCE: Successful implantation of synthetic biomaterials into bone depends on the biological process known as osseointegration. Osseointegration is a highly regulated communication of cells that orchestrates the migration of progenitor cells towards the implant site and promotes the deposition and mineralization of extracellular matrix proteins within the implant microenvironment, to tightly join the implant to native bone. In this process, angiogenesis functions as the initiation site of progenitor cell migration and is necessary for matrix deposition by providing the necessary nutrients for bone formation. In the present study, we show a novel regulation of specific angiogenic growth factors by integrin receptor complexes. This research is important to develop biomaterials that promote and maintain osseointegration through proper vascularization and prevent implant failure in patients lacking sufficient angiogenesis.

Keywords: Angiogenesis; Differentiation; Integrins; Osteoblast; Osteocalcin (OCN); Surface; Titanium.

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Figures

Fig. 1.
Fig. 1.
Integrin gene expression in MG63 cells. MG63 cells cultured on control TCPS, PT, SLA and modSLA Ti substrates were examined for expression of specific integrin receptor subunits. (A) Integrin α1, (B) integrin α2, (C) integrin α5, and (D) integrin β1 gene expression levels. *p < 0.05 vs. TCPS; #p < 0.05 vs. PT.
Fig. 2.
Fig. 2.
Total cell number in MG63 cells silenced for integrins α1, α2, α5, and β1. (A) Total cell number for integrin α1, (B) integrin α2, (C) integrin α5, and (D) integrin β1, as well as MG63 cells were determined. Values presented 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. MG63 cultures on matching substrates.
Fig. 3.
Fig. 3.
Osteocalcin levels in the conditioned media in MG63 cells silenced for integrins αl, α2, α5, and 31. (A) Osteocalcin levels in the conditioned media for integrin αl, (B) integrin α2, (C) integrin α5, and (D) integrin (β1, as well as MG63 cells were determined. Values presented 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. MG63 cultures on matching substrates.
Fig. 4.
Fig. 4.
VEGF-A levels in the conditioned media in MG63 cells silenced for integrins αl, α2, α5, and 31. (A) VEGF-A levels in the conditioned media for integrin αl, (B) integrin α2, (C) integrin α5, and (D) integrin 31, as well as MG63 cells were determined. Values presented 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. MG63 cultures on matching substrates.
Fig. 5.
Fig. 5.
FGF-2 levels in the conditioned media in MG63 cells silenced for integrins α1, α2, α5, and β1. (A) FGF-2 levels in the conditioned media for integrin α1, (B) integrin α2, (C) integrin α5, and (D) integrin as well as MG63 cells were determined. Values presented 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. MG63 cultures on matching substrates.
Fig. 6.
Fig. 6.
Ang-1 levels in the conditioned media in MG63 cells silenced for integrins α1, α2, α5, and β1. (A) Ang-1 levels in the conditioned media for integrin αl, (B) integrin α2, (C) integrin α5, and (D) integrin as well as MG63 cells were determined. Values presented 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. MG63 cultures on matching substrates.
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