Fluidic Force Microscopy and Atomic Force Microscopy Unveil New Insights into the Interactions of Preosteoblasts with 3D-Printed Submicron Patterns.

Physical patterns represent potential surface cues for promoting osteogenic differentiation of stem cells and improving osseointegration of orthopedic implants. Understanding the early cell-surface interactions and their effects on late cellular functions is essential for a rational design of such topographies, yet still elusive. In this work, fluidic force microscopy (FluidFM) and atomic force microscopy (AFM) combined with optical and electron microscopy are used to quantitatively investigate the interaction of preosteoblasts with 3D-printed patterns after 4 and 24 h of culture. The patterns consist of pillars with the same diameter (200 nm) and interspace (700 nm) but distinct heights (500 and 1000 nm) and osteogenic properties. FluidFM reveals a higher cell adhesion strength after 24 h of culture on the taller pillars (32 ± 7 kPa versus 21.5 ± 12.5 kPa). This is associated with attachment of cells partly on the sidewalls of these pillars, thus requiring larger normal forces for detachment. Furthermore, the higher resistance to shear forces observed for these cells indicates an enhanced anchorage and can be related to the persistence and stability of lamellipodia. The study explains the differential cell adhesion behavior induced by different pillar heights, enabling advancements in the rational design of osteogenic patterns.

Microtrap array on a chip for localized electroporation and electro-gene transfection.

We developed a localized single-cell electroporation chip to deliver exogenous biomolecules with high efficiency while maintaining high cell viability. In our microfluidic device, the cells are trapped in a microtrap array by flow, after which target molecules are supplied to the device and electrotransferred to the cells under electric pulses. The system provides the ability to monitor the electrotransfer of exogenous biomolecules in real time. We reveal through numerical simulations that localized electroporation is the mechanism of permeabilization in the microtrap array electroporation device. We demonstrate the simplicity and accuracy of this microtrap technology for electroporation by delivery of both small molecules using propidium iodide and large molecules using plasmid DNA for gene expression, illustrating the potential of this minimally invasive method to be widely used for precise intracellular delivery purposes (from bioprocess engineering to therapeutic applications).

Curvature Induced by Deflection in Thick Meta-Plates.

The design of advanced functional devices often requires the use of intrinsically curved geometries that belong to the realm of non-Euclidean geometry and remain a challenge for traditional engineering approaches. Here, it is shown how the simple deflection of thick meta-plates based on hexagonal cellular mesostructures can be used to achieve a wide range of intrinsic (i.e., Gaussian) curvatures, including dome-like and saddle-like shapes. Depending on the unit cell structure, non-auxetic (i.e., positive Poisson ratio) or auxetic (i.e., negative Poisson ratio) plates can be obtained, leading to a negative or positive value of the Gaussian curvature upon bending, respectively. It is found that bending such meta-plates along their longitudinal direction induces a curvature along their transverse direction. Experimentally and numerically, it is shown how the amplitude of this induced curvature is related to the longitudinal bending and the geometry of the meta-plate. The approach proposed here constitutes a general route for the rational design of advanced functional devices with intrinsically curved geometries. To demonstrate the merits of this approach, a scaling relationship is presented, and its validity is demonstrated by applying it to 3D-printed microscale meta-plates. Several applications for adaptive optical devices with adjustable focal length and soft wearable robotics are presented.

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.

Submicron Patterns-on-a-Chip: Fabrication of a Microfluidic Device Incorporating 3D Printed Surface Ornaments.

Manufacturing high throughput in vitro models resembling the tissue microenvironment is highly demanded for studying bone regeneration. Tissues such as bone have complex multiscale architectures inside which cells reside. To this end, engineering a microfluidic platform incorporated with three-dimensional (3D) microscaffolds and submicron/nanoscale topographies can provide a promising model for 3D cell cultures. There are, however, certain challenges associated with this goal, such as the need to decorate large surfaces area with high-fidelity 3D submicron structures. Here, we succeeded in fabricating a microfluidic platform embedded with a large area (mm range) of reproducible submicron pillar-based topographies. Using the two-photon polymerization (2PP) as a 3D printing technique based on direct laser writing, uniform submicron patterns were created through optimization of the process parameters and writing strategy. To demonstrate the multiscale fabrication capabilities of this approach, submicron pillars of various heights were integrated onto the surfaces of a 3D microscaffold in a single-step 2PP process. The created submicron topography was also found to improve the hydrophilicity of the surface while being able to withstand flow rates of up to 8 mL/min. The material (IP-Dip resin) used for patterning did not have cytotoxic effects against human mesenchymal stromal cells after 3 days of dynamic culture in the microfluidic device. This proof-of-principle study, therefore, marks a significant step forward in manufacturing submicron structure-on-a-chip models for bone regeneration studies.