Participation of integrin β3 in osteoblast differentiation induced by titanium with nano or microtopography

Abstract

The major role of integrins is to mediate cell adhesion but some of them are involved in the osteoblasts-titanium (Ti) interactions. In this study, we investigated the participation of integrins in osteoblast differentiation induced by Ti with nanotopography (Ti-Nano) and with microtopography (Ti-Micro). By using a PCR array, we observed that, compared with Ti-Micro, Ti-Nano upregulated the expression of five integrins in mesenchymal stem cells, including integrin β3, which increases osteoblast differentiation. Silencing integrin β3, using CRISPR-Cas9, in MC3T3-E1 cells significantly reduced the osteoblast differentiation induced by Ti-Nano in contrast to the effect on T-Micro. Concomitantly, integrin β3 silencing downregulated the expression of integrin αv, the parent chain that combines with other integrins and several components of the Wnt/β-catenin and BMP/Smad signaling pathways, all involved in osteoblast differentiation, only in cells cultured on Ti-Nano. Taken together, our results showed the key role of integrin β3 in the osteogenic potential of Ti-Nano but not of Ti-Micro. Additionally, we propose a novel mechanism to explain the higher osteoblast differentiation induced by Ti-Nano that involves an intricate regulatory network triggered by integrin β3 upregulation, which activates the Wnt and BMP signal transductions. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1303-1313, 2019.

Keywords: CRISPR; integrin; nanotopography; osteoblast; titanium.

Figure 1.

Surface characterization of Ti with nanotopography (Ti-Nano) and Ti with microtopography (Ti-Micro). High resolution scanning electron micrographs of Ti-Nano (A and B) and Ti-Micro (C and D) and roughness parameters, average roughness (Ra, E) and average peak-to-valley roughness (Rz, F). The data are presented as mean ± standard deviation (n=15) and the asterisks (*) indicate statistically significant difference (p≤0.05).

Figure 2.

Efficiency of CRISPR-Cas9 to silence integrin β3. Gene (A) and protein expression (B) of integrin β3 of sgITGB3-MC3T3-E1 (sgITGB3) and KRAB-MC3T3-E1 (KRAB) cells on day 3 after cell sorting. The data are presented as mean ± standard deviation (n=3) and the asterisks (*) indicate statistically significant difference (p≤0.05).

Figure 3.

Effect of integrin β3 silencing on gene expression of some osteoblast markers of cells cultured on Ti with nanotopography (Ti-Nano). Gene expression of runt-related transcription factor 2 (Runx2, A), Osterix (Sp7, B), alkaline phosphatase (Alp, C), osteocalcin (Oc, D), bone sialoprotein (Bsp, E) and osteopontin (Opn, F) of sgITGB3-MC3T3-E1 (sgITGB3) and KRAB-MC3T3-E1 (KRAB) cells on day 7. The data are presented as mean ± standard deviation (n=3) and the asterisks (*) indicate statistically significant difference (p≤0.05).

Figure 4.

Effect of integrin β3 silencing on some markers of osteoblast phenotype of cells cultured on Ti with nanotopography (Ti-Nano). Protein expression of runt-related transcription factor 2 (RUNX2), on day 7, (A) and in situ alkaline phosphatase (ALP) activity, on day 10, (B) of sgITGB3-MC3T3-E1 (sgITGB3) and KRAB-MC3T3-E1 (KRAB) cells. The data RUNX2 protein expression (n=3) and of in situ ALP Activity (n=18) are presented as mean ± standard deviation and the asterisks (*) indicate statistically significant difference (p≤0.05).

Figure 5.

Effect of integrin β3 silencing on gene expression of some osteoblast markers of cells cultured on Ti with microtopography (Ti-Micro). Gene expression of runt-related transcription factor 2 (Runx2, A), Osterix (Sp7, B), alkaline phosphatase (Alp, C), osteocalcin (Oc, D), bone sialoprotein (Bsp, E) and osteopontin (Opn, F) of sgITGB3-MC3T3-E1 (sgITGB3) and KRAB-MC3T3-E1 (KRAB) cells on day 7. The data are presented as mean ± standard deviation (n=3) and the asterisks (*) indicate statistically significant difference (p≤0.05).

Figure 6.

Effect of integrin β3 silencing on some markers of osteoblast phenotype of cells cultured on Ti with microtopography (Ti-Micro). Protein expression of runt-related transcription factor 2 (RUNX2), on day 7, (A) and in situ alkaline phosphatase (ALP) activity, on day 10, (B) of sgITGB3-MC3T3-E1 (sgITGB3) and KRAB-MC3T3-E1 (KRAB) cells. The data RUNX2 protein expression (n=3) and of in situ ALP Activity (n=18) are presented as mean ± standard deviation and the asterisk (*) indicates statistically significant difference (p≤0.05).

Figure 7.

Effect of integrin β3 silencing on the integrin αv (Itgav) gene expression of osteoblasts grown on Ti with nanotopography (Ti-Nano) and on Ti with microtopography (Ti-Micro). Gene expression of Itgb3 (A,C) and Itgav (B,D) of sgITGB3-MC3T3-E1 (sgITGB3) and KRAB-MC3T3-E1 (KRAB) cells, on day 7. The data are presented as mean ± standard deviation (n=3) and the asterisks (*) indicate statistically significant difference (p≤0.05).

Figure 8.

Effect of integrin β3 silencing on the gene expression of Wnt and BMP signaling pathway components of osteoblasts cultured on Ti with nanotopography (Ti-Nano) and Ti with microtopography (Ti-Micro). Gene expression of adenomatous polyposis coli (Apc), Axin1, β-Catenin, Cke1, Frizzled 5 (Fzd5), glycogen synthase kinase 3β (Gsk3b) and protein phosphatase 2A (Pp2a) (A,C) and of activin receptor-like kinase 2 (Alk2), bone morphogenetic protein receptor type 1A (Bmpr1a), bone morphogenetic protein receptor type 2A (Bmpr2a), Smad1 and Smad4 (B,D) of sgITGB3-MC3T3-E1 (sgITGB3) and KRAB-MC3T3-E1 (KRAB) cells, on day 7. The data are presented as mean ± standard deviation (n=3) and the asterisks (*) indicate statistically significant difference (p≤0.05).

Figure 9.

Schematic representation of the participation of integrin β3 on osteoblast differentiation induced by Ti with nanotopography (Ti-Nano) and Ti with microtopography (Ti-Micro).