The behavior of osteoblast-like cells on various substrates with functional blocking of integrin-beta1 and integrin-beta3

Abstract

This study was designed to examine the influence of integrin subunit-beta1 and subunit-beta3 on the behavior of primary osteoblast-like cells, cultured on calcium phosphate (CaP)-coated and non coated titanium (Ti). Osteoblast-like cells were incubated with specific monoclonal antibodies against integrin-beta1 and integrin-beta3 to block the integrin function. Subsequently, cells were seeded on Ti discs, either non coated or provided with a 2 microm carbonated hydroxyapatite coating using Electrostatic Spray Deposition. Results showed that on CaP coatings, cellular attachment was decreased after a pre-treatment with either anti-integrin-beta1 or anti-integrin-beta3 antibodies. On Ti, cell adhesion was only slightly affected after a pre-treatment with anti-integrin-beta3 antibodies. Scanning electron microscopy showed that on both types of substrate, cellular morphology was not changed after a pre-treatment with either antibody. With quantitative PCR, it was shown for both substrates that mRNA expression of integrin-beta1 was increased after a pre-treatment with either anti-integrin-beta1 or anti-integrin-beta3 antibodies. Furthermore, after a pre-treatment with either antibody, mRNA expression of integrin-beta3 and ALP was decreased, on both types of substrate. In conclusion, osteoblast-like cells have the ability to compensate to great extent for the blocking strategy as applied here. Still, integrin-beta1 and beta3 seem to play different roles in attachment, proliferation, and differentiation of osteoblast-like cells, and responses on CaP-coated substrates differ to non coated Ti. Furthermore, the influence on ALP expression suggests involvement of both integrin subunits in signal transduction for cellular differentiation.

Fig. 1

Cellular attachment in the first run. (A) and (B) CaP-coated substrates derived with Electrostatic Spray Deposition (ESD); (C) and (D) non-coated Ti. * indicates a significant difference between the control group and the experimental group with the pre-treatment with anti-integrin-β1 antibodies. ^# indicates a significant difference between the control group and the experimental group that was pre-treated with anti-integrin-β3 antibodies

Fig. 2

Cellular attachment in the second run. (A) and (B) CaP-coated substrates derived with Electrostatic Spray Deposition (ESD); (C) and (D) non-coated Ti. * indicates a significant difference between the control group and the experimental group with the pre-treatment with anti-integrin-β1 antibodies. ^# indicates a significant difference between the control group and the experimental group that was pre-treated with anti-integrin-β3 antibodies

Fig. 3

Scanning electron micrographs of osteoblast-like cells on CaP-coated (A, B, and C) substrates, and non-coated (D, E, and F) Ti substrates after 1 day of culture. There were no morphological differences between the control group (A and D), or the experimental groups that were either pre-treated with anti-integrin-β1 antibodies (B and E), or anti-integrin-β3 antibodies (C and F)