Trick and treat: Cell-instructive and bactericidal nanopatterns for bone implants

Biomaterials & Tissue Biomechanic

Nanoimprinting for high-throughput replication of geometrically precise pillars in fused silica to regulate cell behavior

Developing high-throughput nanopatterning techniques that also allow for precise control over the dimensions of the fabricated features is essential for the study of cell-nanopattern interactions. Here, we developed a process that fulfills both of these criteria. Firstly, we used electron-beam lithography (EBL) to fabricate precisely controlled arrays of submicron pillars with varying values of interspacing on a large area of fused silica. Two types of etching procedures with two different systems were developed to etch the fused silica and create the final desired height. We then studied the interactions of preosteoblasts (MC3T3-E1) with these pillars. Varying interspacing was observed to significantly affect the morphological characteristics of the cell, the organization of actin fibers, and the formation of focal adhesions. The expression of osteopontin (OPN) significantly increased on the patterns, indicating the potential of the pillars for inducing osteogenic differentiation. The EBL pillars were thereafter used as master molds in two subsequent processing steps, namely soft lithography and thermal nanoimprint lithography for high-fidelity replication of the pillars on the substrates of interest. The molding parameters were optimized to maximize the fidelity of the generated patterns and minimize the wear and tear of the master mold. Comparing the replicated feature with those present on the original mold confirmed that the geometry and dimensions of the replicated pillars closely resemble those of the original ones. The method proposed in this study, therefore, enables the precise fabrication of submicron- and nanopatterns on a wide variety of materials that are relevant for systematic cell studies. Statement of significance: Submicron pillars with specific dimensions on the bone implants have been proven to be effective in controlling cell behaviors. Nowadays, numerous methods have been proposed to produce bio-instructive submicron-topographies. However, most of these techniques are suffering from being low-throughput, low-precision, and expensive. Here, we developed a high-throughput nanopatterning technique that allows for control over the dimensions of the features for the study of cell-nanotopography interactions. Assessing the adaptation of preosteoblast cells showed the potential of the pillars for inducing osteogenic differentiation. Afterward, the pillars were used for high-fidelity replication of the bio-instructive features on the substrates of interest. The results show the advantages of nanoimprint lithography as a unique technique for the patterning of large areas of bio-instructive surfaces.Biomaterials & Tissue Biomechanic

Automated Folding of Origami Lattices: From Nanopatterned Sheets to Stiff Meta-Biomaterials

Folding nanopatterned flat sheets into complex 3D structures enables the fabrication of meta-biomaterials that combine a rationally designed 3D architecture with nanoscale surface features. Self-folding is an attractive approach for realizing such materials. However, self-folded lattices are generally too compliant as there is an inherent competition between ease-of-folding requirements and final load-bearing characteristics. Inspired by sheet metal forming, an alternative route is proposed for the fabrication of origamilattices. This ‘automated-folding’ approach allows for the introduction of sharp folds into thick metal sheets, thereby enhancing their stiffness. The first time realization of automatically folded origami lattices with bone-mimicking mechanical properties is demonstrated. The proposed approach is highly scalable given that the unit cells making up the meta-biomaterial can be arbitrarily large in number and small in dimensions. To demonstrate the scalability and versatility of the proposed approach, it is fabricated origamilattices with > 100 unit cells, lattices with unit cells as small as 1.25 mm, and auxetic lattices. The nanopatterned the surface of the sheets prior to folding. Protected by a thin coating layer, these nanoscale features remained intact during the folding process. It is found that the nanopatterned folded specimens exhibits significantly increased mineralization as compared to their non-patterned counterparts.Support Biomechanical EngineeringBiomaterials & Tissue Biomechanic

Reactive ion etching for fabrication of biofunctional titanium nanostructures

One of the major problems with the bone implant surfaces after surgery is the competition of host and bacterial cells to adhere to the implant surfaces. To keep the implants safe against implant-associated infections, the implant surface may be decorated with bactericidal nanostructures. Therefore, fabrication of nanostructures on biomaterials is of growing interest. Here, we systematically studied the effects of different processing parameters of inductively coupled plasma reactive ion etching (ICP RIE) on the Ti nanostructures. The resultant Ti surfaces were characterized by using scanning electron microscopy and contact angle measurements. The specimens etched using different chamber pressures were chosen for measurement of the mechanical properties using nanoindentation. The etched surfaces revealed various morphologies, from flat porous structures to relatively rough surfaces consisting of nanopillars with diameters between 26.4 ± 7.0 nm and 76.0 ± 24.4 nm and lengths between 0.5 ± 0.1 μm and 5.2 ± 0.3 μm. The wettability of the surfaces widely varied in the entire range of hydrophilicity. The structures obtained at higher chamber pressure showed enhanced mechanical properties. The bactericidal behavior of selected surfaces was assessed against Staphylococcus aureus and Escherichia coli bacteria while their cytocompatibility was evaluated with murine preosteoblasts. The findings indicated the potential of such ICP RIE Ti structures to incorporate both bactericidal and osteogenic activity, and pointed out that optimization of the process conditions is essential to maximize these biofunctionalities.Biomaterials & Tissue BiomechanicsMaterials and EnvironmentBT/Biocatalysi

Bactericidal effects of nanopatterns: a systematic review

We systematically reviewed the currently available evidence on how the design parameters of surface nanopatterns (e.g. height, diameter, and interspacing) relate to their bactericidal behavior. The systematic search of the literature resulted in 46 studies that satisfied the inclusion criteria of examining the bactericidal behavior of nanopatterns with known design parameters in absence of antibacterial agents. Twelve of the included studies also assessed the cytocompatibility of the nanopatterns. Natural and synthetic nanopatterns with a wide range of design parameters were reported in the included studies to exhibit bactericidal behavior. However, most design parameters were in the following ranges: heights of 100–1000 nm, diameters of 10–300 nm, and interspacings of <500 nm. The most commonly used type of nanopatterns were nanopillars, which could kill bacteria in the following range of design parameters: heights of 100–900 nm, diameters of 20–207 nm, and interspacings of 9–380 nm. The vast majority of the cytocompatibility studies (11 out of 12) showed no adverse effects of bactericidal nanopatterns with the only exception being nanopatterns with extremely high aspect ratios. The paper concludes with a discussion on the evidence available in the literature regarding the killing mechanisms of nanopatterns and the effects of other parameters including surface affinity of bacteria, cell size, and extracellular polymeric substance (EPS) on the killing efficiency. Statement of significance: The use of nanopatterns to kill bacteria without the need for antibiotics represents a rapidly growing area of research. However, the optimum design parameters to maximize the bactericidal behavior of such physical features need to be fully identified. The present manuscript provides a systematic review of the bactericidal nanopatterned surfaces. Identifying the effective range of dimensions in terms of height, diameter, and interspacings, as well as covering their impact on mammalian cells, has enabled a comprehensive discussion including the bactericidal mechanisms and the factors controlling the bactericidal efficiency. Overall, this review helps the readers have a better understanding of the state-of-the-art in the design of bactericidal nanopatterns, serving as a design guideline and contributing to the design of future experimental studies.Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.Biomaterials & Tissue BiomechanicsMedical Instruments & Bio-Inspired Technolog

On the Use of Black Ti as a Bone Substituting Biomaterial: Behind the Scenes of Dual-Functionality

Despite the potential of small-scale pillars of black titanium (bTi) for killing the bacteria and directing the fate of stem cells, not much is known about the effects of the pillars’ design parameters on their biological properties. Here, three distinct bTi surfaces are designed and fabricated through dry etching of the titanium, each featuring different pillar designs. The interactions of the surfaces with MC3T3-E1 preosteoblast cells and Staphylococcus aureus bacteria are then investigated. Pillars with different heights and spatial organizations differently influence the morphological characteristics of the cells, including their spreading area, aspect ratio, nucleus area, and cytoskeletal organization. The preferential formation of focal adhesions (FAs) and their size variations also depend on the type of topography. When the pillars are neither fully separated nor extremely tall, the colocalization of actin fibers and FAs as well as an enhanced matrix mineralization are observed. However, the killing efficiency of these pillars against the bacteria is not as high as that of fully separated and tall pillars. This study provides a new perspective on the dual-functionality of bTi surfaces and elucidates how the surface design and fabrication parameters can be used to achieve a surface topography with balanced bactericidal and osteogenic properties.Biomaterials & Tissue BiomechanicsMicro and Nano EngineeringBT/Biocatalysi

Controlled metal crumpling as an alternative to folding for the fabrication of nanopatterned meta-biomaterials

We designed and fabricated a simple setup for the controlled crumpling of nanopatterned, surface-porous flat metallic sheets for the fabrication of volume-porous biomaterials and showed that crumpling can be considered as an efficient alternative to origami-inspired folding. Before crumpling, laser cutting was used to introduce pores to the sheets. We then fabricated titanium (Ti) nanopatterns through reactive ion etching on the polished Ti sheets. Thereafter, nanopatterned porous Ti sheets were crumpled at two deformation velocities (i.e., 2 and 100 mm/min). The compression tests of the scaffolds indicated that the elastic modulus of the specimens vary in the range of 11.8–13.9 MPa. Micro-computed tomography scans and computational simulations of crumpled scaffolds were performed to study the morphological properties of the resulting meta-biomaterials. The porosity and pore size of the scaffolds remained within the range of those reported for trabecular bone. Finally, the in vitro cell preosteoblasts culture demonstrated the cytocompatibility of the nanopatterned scaffolds. Moreover, the aspect ratio of the cells residing on the nanopatterned surfaces was found to be significantly higher than those cultured on the control scaffolds, indicating that the nanopatterned surface may have a higher potential for inducing the osteogenic differentiation of the preosteoblasts.Biomaterials & Tissue BiomechanicsSupport Biomechanical Engineerin

Quantitative mechanics of 3D printed nanopillars interacting with bacterial cells

One of the methods to create sub-10 nm resolution metal-composed 3D nanopillars is electron beam-induced deposition (EBID). Surface nanotopographies (e.g., nanopillars) could play an important role in the design and fabrication of implantable medical devices by preventing the infections that are caused by the bacterial colonization of the implant surface. The mechanical properties of such nanoscale structures can influence their bactericidal efficiency. In addition, these properties are key factors in determining the fate of stem cells. In this study, we quantified the relevant mechanical properties of EBID nanopillars interacting with Staphylococcus aureus (S. aureus) using atomic force microscopy (AFM). We first determined the elastic modulus (17.7 GPa) and the fracture stress (3.0 ± 0.3 GPa) of the nanopillars using the quantitative imaging (QI) mode and contact mode (CM) of AFM. The displacement of the nanopillars interacting with the bacteria cells was measured by scanning electron microscopy (50.3 ± 9.0 nm). Finite element method based simulations were then applied to obtain the force-displacement curve of the nanopillars (considering the specified dimensions and the measured value of the elastic modulus) based on which an interaction force of 88.7 ± 36.1 nN was determined. The maximum von Mises stress of the nanopillars subjected to these forces was also determined (3.2 ± 0.3 GPa). These values were close to the maximum (i.e., fracture) stress of the pillars as measured by AFM, indicating that the nanopillars were close to their breaking point while interacting with S. aureus. These findings reveal unique quantitative data regarding the mechanical properties of nanopillars interacting with bacterial cells and highlight the possibilities of enhancing the bactericidal activity of the investigated EBID nanopillars by adjusting both their geometry and mechanical properties.Biomaterials & Tissue BiomechanicsMicro and Nano EngineeringImPhys/Microscopy Instrumentation & TechniquesBT/Biocatalysi

Mechanotransduction in high aspect ratio nanostructured meta-biomaterials: The role of cell adhesion, contractility, and transcriptional factors

Black Ti (bTi) surfaces comprising high aspect ratio nanopillars exhibit a rare combination of bactericidal and osteogenic properties, framing them as cell-instructive meta-biomaterials. Despite the existing data indicating that bTi surfaces induce osteogenic differentiation in cells, the mechanisms by which this response is regulated are not fully understood. Here, we hypothesized that high aspect ratio bTi nanopillars regulate cell adhesion, contractility, and nuclear translocation of transcriptional factors, thereby inducing an osteogenic response in the cells. Upon the observation of significant changes in the morphological characteristics, nuclear localization of Yes-associated protein (YAP), and Runt-related transcription factor 2 (Runx2) expression in the human bone marrow-derived mesenchymal stem cells (hMSCs), we inhibited focal adhesion kinase (FAK), Rho-associated protein kinase (ROCK), and YAP in separate experiments to elucidate their effects on the subsequent expression of Runx2. Our findings indicated that the increased expression of Runx2 in the cells residing on the bTi nanopillars compared to the flat Ti is highly dependent on the activity of FAK and ROCK. A mechanotransduction pathway is then postulated in which the FAK-dependent adhesion of cells to the extreme topography of the surface is in close relation with ROCK to increase the endogenous forces within the cells, eventually determining the cell shape and area. The nuclear translocation of YAP may also enhance in response to the changes in cell shape and area, resulting in the translation of mechanical stimuli to biochemical factors such as Runx2.Biomaterials & Tissue Biomechanic

3D-Printed Submicron Patterns Reveal the Interrelation between Cell Adhesion, Cell Mechanics, and Osteogenesis

The surface topography of implantable devices is of crucial importance for guiding the cascade of events that starts from the initial contact of the cells with the surface and continues until the complete integration of the device in its immediate environment. There is, however, limited quantitative information available regarding the relationships between the different stages of such cascade(s) and how the design of surface topography influences them. We, therefore, used direct laser writing to 3D-print submicron pillars with precisely controlled dimensions and spatial arrangements to perform a systematic study of such relationships. Using single-cell force spectroscopy, we measured the adhesion force and the work of adhesion of the preosteoblast cells residing on the different types of surfaces. Not only the adhesion parameters (after 2-60 s) but also the formation of focal adhesions was strongly dependent on the geometry and arrangement of the pillars: sufficiently tall and dense pillars enhanced both adhesion parameters and the formation of focal adhesions. Our morphological study of the cells (after 24 h) showed that those enhancements were associated with a specific way of cell settlement onto the surface (i.e., "top state"). The cells interacting with tall and dense pillars were also characterized by numerous thick actin stress fibers in the perinuclear region and possibly high internal stresses. Furthermore, living cells with highly organized cytoskeletal networks exhibited greater values of the elastic modulus. The early responses of the cells predicted their late response including matrix mineralization: tall and dense submicron pillars significantly upregulated the expression of osteopontin after 21 days of culture under both osteogenic and nonosteogenic conditions. Our findings paint a detailed picture of at least one possible cascade of events that starts from initial cell adhesion and continues to subsequent cellular functions and eventual matrix mineralization. These observations could inform the future developments of instructive surfaces for medical devices based on physical surface cues and early markers. Biomaterials & Tissue BiomechanicsMicro and Nano EngineeringChemE/Product and Process Engineerin