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

Abstract

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.

In situ construction of flower-like nanostructured calcium silicate bioceramics for enhancing bone regeneration mediated via FAK/p38 signaling pathway

“…S7b), indicating the remarkable protein loading capacity of nCS. Preferable protein adsorption capacity was beneficial for the formation of focal adhesions, which was conducive to osteogenesis and vascularization [ 38 ].

Fig.

…”

Osteoimmunomodulatory Potential of 3D Printed Submicron Patterns Assessed in a Direct Co-Culture Model

“…A Nanoscribe Photonic Professional GT laser lithography system (Nanoscribe, Germany) working on the basis of two-photon polymerization was used to fabricate the patterns. The general writing language (GWL) file of the designed pillars (diameter = 300 nm, height = 500 nm, and interspacing = 700 nm) was prepared as previously described [19,23,24]. A curable acrylate-based photosensitive resin, namely IP-L780, was polymerized under the exposure of an infrared laser beam, and submicron pillars were fabricated using a previously described protocol [22].…”

“…We further investigated the influence of specific physical patterns on the response of both types of cells in the direct co-culture model established above. In our previous studies, we have extensively examined the effects of the spatial arrangement of submicron pillars on preosteoblasts [23] and macrophages [22]. Here, we studied whether submicron pillars can modulate the pro-inflammatory response observed in the co-culture towards stimulating the osteogenic response.…”

“…In our previous studies, we investigated the role of 3D printed submicron pillars [19] on the inflammatory response of macrophages [20] and the osteogenic response of preosteoblasts [21] in mono-cultures. We observed that arrays of submicron pillars with specific sizes and spatial arrangement could promote the polarization of M1 macrophages towards prohealing M2 phenotype and enhance the expression of osteopontin (OPN) in both osteogenic and non-osteogenic media [20,21]. Therefore, in this study, we assessed the OIM potential of such patterns.…”

Physico-Chemical Characteristics and Posterolateral Fusion Performance of Biphasic Calcium Phosphate with Submicron Needle-Shaped Surface Topography Combined with a Novel Polymer Binder

“…In high-throughput screening studies of surfaces consisting of needles, it was shown than only a subset of needles' features promoted the favorable polarization towards M2 macrophages from murine and human origins [14,15]. The dimensional and density aspect of the needle features was also shown to influence adhesion, mechanics, and osteogenic differentiation from murine and human cells [16][17][18]. The submicron needle-shaped surface topography feature of BCP is, therefore, pivotal for predictable fusion and should be preserved when designing bone graft formulations suitable for the end-users.…”