Mechanistic Insight in Surface Nanotopography Driven Cellular Migration

Fibroblast recruitment and migration is an integral part of homeostasis, embryogenesis, immune surveillance, inflammation, and wound healing. The recruitment of fibroblasts to a wound site is governed by the concentration gradient of growth factors, chemokines, and other signaling molecules secreted by various types of cells including macrophages, neutrophils, endothelial cells, and platelets. However, the migration of fibroblasts also depends on the topography of the extracellular matrix (ECM) which is made from a network of components such as collagen, fibrin, hyaluronic acid, laminin, fibronectin, vitronectin, and proteoglycans. ECM generated topography such as pores, grooves, pits, and protrusions directly affects the way the adhesion and migration of cells takes place. To form focal adhesions with the ECM, cells probe the proteins in the ECM using specialized receptors called “Integrins”, which are made from noncovalently bonded heterodimers of α and β subunits. On the basis of the topography of the ECM, integrins transmit chemical and mechanical signals between the extracellular and intracellular microenvironment, which then control cellular behaviors such as adhesion, migration, differentiation, survival, and conformation. The phenomena underlying these chemical signals include the transfer of extracellular information from integrins to actin filaments via adapter proteins such as talin and vinculin, leading to the mechanical recruitment of several structural molecules to the cell membrane to form focal adhesions. Furthermore, integrins and vinculin bind with focal adhesion proteins (FA) including F-actin, talin, phaxillin, and α-actinin to regulate cellular interaction.

Recent studies by our group, and others, have shown that cellular behavior such as adhesion, proliferation, migration, and differentiation can be modulated by tailoring surface topography (hills, grooves, pillars, wells, pores, spheres, waves, and ridges) and chemistry (amines, carboxyl, hydroxyl, and hydrocarbon). However, only a few of these studies demonstrated and discriminated the effect of nanotopography on cell migration. Cui et al. demonstrated that nano grooves simulating the topography of ECM enhanced migration and elongation of fibroblasts along the grooves. Dalby et al. investigated the morphology of fibroblasts on cylindrical nanocolumn topography where enhanced filopodia formation was reported on the topographic surface. Kim et al. reported guided migration of cells on vertical nanopatterns compared to parallel nanopatterns. Cui et al. studied real-time cell migration using impedance values generated from an electric cell–substrate impedance sensing device. They observed that surface topography has an effect on fibroblast orientation, proliferation, and migration.

Down Signaling Pathway Involved in Cell-Surface Interaction

These studies clearly demonstrate that biomaterial surface nanotopography has a direct influence on cellular behavior.However, none of these studies shows the relationship and interplay between surface nanotopography and chemistry. The issue is rather related to developing a technology that allows for precise tailoring of the outermost surface chemistry while at the same time preserving the desired nanotopography range. Our previous work addressed this issue by utilizing the combination of plasma polymer deposition and surface nanoengineering. We showed that our technology can be successfully utilized in modulating cell adhesion, proliferation, differentiation, and inflammatory responses.

Fabrication of Well-Defined Surface Nanotopography and Chemistry

Furthermore, we explored the mechanisms responsible for the modulation of cell behavior and reported that nanotopography induces conformational changes to fibrinogen leads to unfolding and exposure of normally hidden receptors. On the basis of these studies, they hypothesize that the fabrication of biomaterial surfaces with specific nanotopography and chemistry can be utilized to understand the mechanisms underlying cellular migration which has direct implications on designing future biomaterial constructs that provide improved wound healing.

In this work, they evaluate the effect of well-defined surface nanotopography and tailored outermost surface chemistry on fibroblast adhesion, proliferation, migration, and gene expression of FA proteins.

Surface characterization of nanotopography and chemistry modified surfaces. AFM/SEM images showing nanoparticle distribution on surfaces, scale bar in SEM images = 1 μm (1a), number of nanoparticles per μm2 calculated from AFM images (1b), surface roughness (nm) (1c), percentage increase in surface area (1d), percentage of atomic concentration obtained from XPS (1e), and advancing water contact angle of the modified surfaces (1f).

As model substrata,they have employed “nanohills” fabricated using gold nanoparticles (AuNPs) of predetermined size to obtain the desired number of surface nanoprotrusions. To differentiate between the interplay of surface nanotopography and chemistry, the outermost surface chemistry of the nanohills was tailored using plasma polymerized 2-methyl-2-oxazoline (pOX). pOX was chosen for its low bacteria fouling properties and high biocompatibility. Fibroblasts were cultured for 24 h on these model substrates and assays for wound closure, cell proliferation, cell migration, and gene expression of FA sites were performed. Tahey found using the scratch wound closure assay that wound closure is significantly faster on the nanotopography modified surfaces. Moreover, wound closure on these modified surfaces was independent from the rate of cell proliferation and can be mainly attributed to enhanced cell migration. Finally, they have demonstrated that surface nanotopography can modulate the FA gene expression, leading to faster wound closure.

Representative images of gap closure on different topography height at different time points, scale bar = 300 μm (a). Scratch confluency measured over time on different topography modified surfaces (b).

Representative fluorescence microscopy images for vinculin (red), F-actin (green) and nuclei (blue) on modified surfaces, scale bar = 50 μm (a), F-actin mean intensity per cell (b), vinculin mean intensity per cell (c), and vinculin active sites per cell (d), * = p < 0.05, **** = p < 0.0001.

Vinculin Structure and Inactive and Active States of Vinculin

  1. Mechanistic Insight in Surface Nanotopography Driven Cellular Migration Panthihage Ruvini L. Dabare, Akash Bachhuka, Rahul M. Visalakshan, Hanieh S. Shirazi, Kostya Ostriko, Louise E. Smith, and Krasimir Vasilev ACS Biomaterials Science & Engineering 2021 7 (10), 4921-4932 DOI: 10.1021/acsbiomaterials.1c00853