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. 2005 Apr 26;102(17):5953-7.
doi: 10.1073/pnas.0407356102. Epub 2005 Apr 12.

Integrin binding specificity regulates biomaterial surface chemistry effects on cell differentiation

Benjamin G Keselowsky  1 , David M Collard, Andrés J García
Affiliations

Integrin binding specificity regulates biomaterial surface chemistry effects on cell differentiation

Benjamin G Keselowsky et al. Proc Natl Acad Sci U S A. .

Abstract

Biomaterial surface chemistry has profound consequences on cellular and host responses, but the underlying molecular mechanisms remain poorly understood. Using self-assembled monolayers as model biomaterial surfaces presenting well defined chemistries, we demonstrate that surface chemistry modulates osteoblastic differentiation and matrix mineralization independently from alterations in cell proliferation. Surfaces were precoated with equal densities of fibronectin (FN), and surface chemistry modulated FN structure to alter integrin adhesion receptor binding. OH- and NH(2)-terminated surfaces up-regulated osteoblast-specific gene expression, alkaline phosphatase enzymatic activity, and matrix mineralization compared with surfaces presenting COOH and CH(3) groups. These surface chemistry-dependent differences in cell differentiation were controlled by binding of specific integrins to adsorbed FN. Function-perturbing antibodies against the central cell binding domain of FN completely inhibited matrix mineralization. Furthermore, blocking antibodies against beta(1) integrin inhibited matrix mineralization on the OH and NH(2) surfaces, whereas function-perturbing antibodies specific for beta(3) integrin increased mineralization on the COOH substrate. These results establish surface-dependent differences in integrin binding as a mechanism regulating differential cellular responses to biomaterial surfaces. This mechanism could be exploited to engineer materials that control integrin binding specificity to elicit desired cellular activities to enhance the integration of biomaterials and improve the performance of biotechnological culture supports.

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Figures

Fig. 1.
Fig. 1.
Surface chemistry does not alter osteoblast proliferation. MC3T3-E1 cells were cultured on SAMs coated with equivalent FN surface densities (40 ng/cm2) for 16 h and pulsed with BrdUrd for 4 h before analysis.
Fig. 2.
Fig. 2.
Surface chemistry modulates osteoblastic gene expression. MC3T3-E1 cells were cultured on SAMs coated with equivalent FN surface densities (40 ng/cm2). Osteoblast-specific gene expression at 7 days was quantified by real-time RT-PCR (* vs. CH3 and COOH, P < 8 × 10-6). Data are plotted as mean ± SE.
Fig. 3.
Fig. 3.
Surface chemistry modulates ALP enzymatic activity and matrix mineralization. MC3T3-E1 cells were cultured on SAMs coated with equivalent FN surface densities (40 ng/cm2) and analyzed at 14 days. (A) ALP enzymatic activity exhibits surface chemistry-dependent differences. (B) von Kossa staining (Upper) for mineralized deposits (black) and quantification (Lower; mean ± SE) showing differences among surface chemistries (* vs. CH3 and COOH, P < 0.01). (C) FTIR spectra showing biological hydroxyapatite on OH and NH2 SAMs. The doublet at 560 cm-1/605 cm-1 corresponds to crystalline phosphate phase representative of hydroxyapatite. The peak at 870 cm-1 is assigned to carbonate substitution in hydroxyapatite phase. Peaks at 1,100 cm-1 and 1,650 cm-1 are assigned to phosphate and amide I (corresponding to protein) vibrations, respectively.
Fig. 4.
Fig. 4.
Integrin binding specificity for adsorbed FN regulates the effects of surface chemistry on matrix mineralization. (A) Human FN-specific HFN7.1 antibody completely blocks mineralization on OH and NH2 SAMs, demonstrating that binding to preadsorbed FN is critical for mineralization on these surfaces (mean ± SE; * vs. control, P < 0.01). (B) Integrin-specific antibodies alter mineralization in a surface chemistry-dependent fashion. Antibodies against β1 integrin block mineralization on OH and NH2 SAMs, whereas anti-β3 integrin antibodies up-regulate mineralization on COOH and, to a lesser extent, NH2 (mean ± SE; * vs. control, P < 0.05).
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