Expanding the Applicability of Electroactive Polymers for Tissue Engineering Through Surface Biofunctionalization

Abstract

Polyvinylidene fluoride (PVDF) is a synthetic semicrystalline fluoropolymer with great potential for tissue engineering applications. In addition to its excellent mechanical strength, thermal stability, biocompatibility and simple processability into different morphologies, the relevance of PVDF-based materials for tissue engineering applications comes for its electroactive properties, which include piezo-, pyro- and ferroelectricity. Nevertheless, its synthetic nature and inherent hydrophobicity strongly limit the applicability of this polymer for certain purposes, particularly those involving cell attachment. In addition, the variable adhesion of cells and proteins to PVDF surfaces with different net surface charge makes it difficult to accurately compare the biological response in each case. In this work, we describe a method for the surface functionalization of PVDF films with biological molecules. After an initial chemical modification, and, independently of its polarization state, the PVDF films covalently bind equivalent amounts of cell-binding proteins. In addition, the materials retain their properties, including piezoelectric activity, representing a very promising method for the functionalization of PVDF-based tissue engineering approaches.

Keywords: PVDF; biofunctionalization; collagen; electroactivity; piezoelectricity.

Figure 1

Schematic representation of the functionalization strategy: (A) Incubation of the activated PVDF surface with MAA solution. (B) PMAA polymerization by ester bond formation. (C) Acid activation with EDC/NHS. (D) Amide bond formation with the protein of choice. (E) Incubation of the activated PVDF surface with AAC solution. (F) PAAC polymerization by ester bond formation. (G) Membrane activation by EDC/NHS method. (H) Amide bond formation with the protein of choice.

Figure 2

Physicochemical characterization of the PVDF-PMAA and PVDF-PAAC surfaces: (A) Surface functionalization with both acids is estimated from the absorption at 207 nm and 193 nm for PMAA and PAAC, respectively. “NP” stands for PVDF (non-poled), “+/–” for poled PVDF, “+” for positively poled PVDF, and “–” for negatively poled PVDF. (B) Images acquired for contact angle measurements and the corresponding quantification (C). In (A,C), grey corresponds to PVDF (non-poled), white to PVDF (poled+), and black to PVDF (poled−). Statistical significances are denoted with * for p ≤ 0.05, ** for p ≤ 0.01 and *** for p ≤ 0.001.

Figure 3

Quantification of the collagen immobilized on the PVDF surfaces and estimation of the piezoelectric properties: (A) Quantification of the amount of immobilized collagen type I on the functionalized PVDF substrates is measured by the BCA assay and reveals higher protein densities on the PVDF-PMAA than on the PVDF-PAAC surfaces. Nevertheless, similar protein densities are detected across the surfaces with different electric potentials. (B) Although there is a slight decay in the modulus of the piezoelectric coefficient (|d₃₃|) of the functionalized PVDF films after the biofunctionalization process, particularly in case of PVDF (poled−), the resulting materials retain their piezoelectric properties to a great extent. Statistical significances are denoted with * for p ≤ 0.05, ** for p ≤ 0.01 and *** for p ≤ 0.001.

Figure 4

Scanning electron microscopy images of the samples with immobilized collagen type I. Collagen fibres appeared immobilized on the PVDF membranes functionalized with PMAA (left column) and PAAC (right column), forming a homogenous network across the surface, independently of its polarity. Scale bar represents 50 µm. Insets represent a 10-fold magnification.

Figure 5

Fluorescently stained primary hDPSCs adhering to bare (left column) and functionalized PVDF surfaces (middle and right columns show PMAA-functionalized and AAC functionalized surfaces, respectively). hDPSCs are stained with Hoechst33342 (nuclei in blue) and phalloidin (filamentous actin in red). The morphological features of the cells reveal the good biocompatibility of the collagen-functionalized PVDF surfaces. Scale bar represents 150 µm.