Graded functionalization of biomaterial surfaces using mussel-inspired adhesive coating of polydopamine

Highlights

• A method for generating polydopamine (PD) gradient on biomaterial has been reported.

• PD gradient substrates showed gradient in surface chemistry and hydrophilicity.

• PD gradient substrates were able to modulate the spatial distribution of biomolecules.

Abstract

Biomaterials with graded functionality have various applications in cell and tissue engineering. In this study, by controlling oxidative polymerization of dopamine, we demonstrated universal techniques for generating chemical gradients on various materials with adaptability for secondary molecule immobilization. Diffusion-controlled oxygen supply was successfully exploited for coating of polydopamine (PD) in a gradient manner on different materials, regardless of their surface chemistry, which resulted in gradient in hydrophilicity and surface roughness. The PD gradient controlled graded adhesion and spreading of human mesenchymal stem cells (hMSCs) and endothelial cells. Furthermore, the PD gradient on these surfaces served as a template to allow for graded immobilization of different secondary biomolecules such as cell adhesive arginine-glycine-aspartate (RGD) peptides and siRNA lipidoid nanoparticles (sLNP) complex, for site-specific adhesion of human mesenchymal stem cells, and silencing of green fluorescent protein (GFP) expression on GFP-HeLa cells, respectively. In addition, the same approach was adapted for generation of nanofibers with surface in graded biomineralization under simulated body fluid (SBF). Collectively, oxygen-dependent generation of PD gradient on biomaterial substrates can serve as a simple and versatile platform that can be used for various applications realizing in vivo tissue regeneration and in vitro high-throughput screening of biomaterials.

Introduction

There is currently a great interest in graded presentation of biomolecules on a material surface for regulating cell functions both in vitro and in vivo or to screen and optimize cell-biomolecule interactions [1], [2], [3]. Several recent reports have implemented biomolecule gradient platform to study biological process such as cell migration, stem cell differentiation, angiogenesis, or spatially defined cell adhesion and spreading [4], [5], [6], [7]. For instance, a linear cyclic arginine-glycine-aspartate (RGDfK) peptide gradient was formed to analyze the role of ECM concentration in cell polarization and migration [8]. Different aspects of stem cell responses were screened on an amine functional gradient platform and optimal surface chemistry for retention of pluripotency were identified [9]. A nanofiber scaffold with hydroxyapatite gradient or three-dimensional scaffolds with a gradient distribution of osteogenic gene were also reported for engineering bone-soft tissue interfacial site [10], [11].

Presently, most technologies for the graded biomolecule immobilization are based on the surface modification of substrates with certain functional groups that have specific interactions with the biomolecules [12], [13], [14]. Some representative systems involve the use of self-assembled monolayers (SAMs) or plasma-based approaches and, thereby, demonstrating the fabrication of tunable surfaces chemistries and the secondary attachment of various biomolecules. For example, gradients in the densities of amine or carboxyl groups have been prepared via controlled plasma treatment using a covered mask, or by moving the sample stage to control the differentiation of stem cells or the growth of rat neuronal progenitor cells (PC-12) [15], [16], [17]. Similarly, gradient surfaces varying from high hydroxyl group density to high aldehyde group density were prepared through diffusion-controlled plasma polymerization for the gradient immobilization of nerve growth factor (NGF) [18]. Different fabrication techniques such as nanolithography, programmed inkjet printing, or controlled UV exposure, have also been combined with SAMs to generate substrates with gradients of laminin or bone morphogenetic protein-2 (BMP-2) [19], [20], [21], [22]. Although the aforementioned technologies have been successful in generating specific gradients for each objective, these processes are mostly time-consuming and rely on relatively complex setups. Most importantly, the gradient formation is often limited to certain substrate chemistries and types of target molecules, which require individualized designs depending on their applications. Until now, there has been no report on a facile strategy through which a gradient of desirable signals for the modulation of cell or tissue function could be adaptable for different materials.

Previously, inspired by the strong adhesiveness of marine mussels, it was demonstrated that dopamine could undergo oxidative self-polymerization to synthesize polydopamine (PD) under alkaline conditions; this PD could then be coated onto various materials independent of their surface properties, and subsequently, could facilitate the secondary immobilization of biomolecules, genes, and minerals [23], [24], [25], [26]. This polymerization is controlled by various parameters such as pH, dopamine concentration, temperature, and oxygen concentration [25], [27], [28], [29]. In this study, we hypothesized that (1) a controlled supply of oxygen could control dopamine polymerization in a spatially controlled manner and (2) the resulting graded PD coating on substrates could then be amenable for generation of a gradient of various biomolecules. Given that, we developed a system in which a gradient in oxygen concentration was created across the depth of the reaction solution and investigated its effect on PD gradient formation on different substrates including parafilm, polydimethylsiloxane (PDMS), and poly (L-lactic acid) (PLLA) nanofibers. We then investigated the effect of PD gradient platform on the adhesion of stem cells and endothelial cells. In addition, the effect of gradient in PD coating on graded functionalization of various biomaterials surfaces with different biomolecules was also investigated. Specifically, a cell-instructive peptide, and siRNAs were immobilized in a gradient manner, which were confirmed to control cell functions in a gradient manner. Finally, we also investigated the potential for fabricating a triple-sandwich gradient on a nanofiber surface to regulate spatially controlled biomineralization.