J. te Riet

The influence of nanoscale grooved substrates on osteoblast behavior and extracellular matrix deposition

To fight bone diseases characterized by poor bone quality like osteoporosis and osteoarthritis, as well as in reconstructive surgery, there is a need for a new generation of implantable biomaterials. It is envisioned that implant surfaces can be improved by mimicking the natural extracellular matrix of bone tissue, which is highly a organized nano-composite. In this study we aimed to get a better understanding of osteoblast response to nanometric grooved substrates varying in height, width and spacing. A throughput screening biochip was created using electron beam lithography. Subsequently, uniform large-scale nanogrooved substrates were created using laser interference lithography and reactive ion etching. Results showed that osteoblasts were responsive to nanopatterns down to 75 nm in width and 33nm in depth. SEM and TEM studies showed that an osteoblast-driven calcium phosphate (CaP) mineralization was observed to follow the surface pattern dimensions. Strikingly, aligned mineralization was found on even smaller nanopatterns of 50 nm in width and 17 nm in depth. A single cell based approach for real time PCR demonstrated that osteoblast-specific gene expression was increased on nanopatterns relative to a smooth control. The results indicate that nanogrooves can be a very promising tool to direct the bone response at the interface between an implant and the bone tissue.

The interaction between nanoscale surface features and mechanical loading and its effect on osteoblast-like cells behavior

Osteoblasts respond to mechanical stimulation by changing morphology, gene expression and matrix mineralization. Introducing surface topography on biomaterials, independently of mechanical loading, has been reported to give similar effects. In the current study, using a nanotextured surface, and mechanical loading, we aimed to develop a multi-factorial model in which both parameters interact. Mechanical stimulation to osteoblast-like cells was applied by longitudinal stretch in parallel direction to the nanotexture (300 nm wide and 60 nm deep grooves), with frequency of 1 Hz and stretch magnitude varying from 1% to 8%. Scanning electron microscopy showed that osteoblast-like cells subjected to mechanical loading oriented perpendicularly to the stretch direction. When cultured on nanotextured surfaces, cells aligned parallel to the texture. However, the parallel cell direction to the nanotextured surface was lost and turned to perpendicular when parallel stretch to the nanotexture, greater than 3% was applied to the cells. This phenomenon could not be achieved when a texture with micro-sized dimensions was used. Moreover, a significant synergistic effect on upregulation of fibronectin and Cfba was observed when dual stimulation was used. These findings can lead to a development of new biomimetic materials that can guide morphogenesis in tissue repair and bone remodeling.

Distinct kinetic and mechanical properties govern ALCAM-mediated interactions as shown by single-molecule force spectroscopy.

The activated leukocyte cell adhesion molecule (ALCAM) mediates dynamic homotypic and heterotypic cellular interactions. Whereas homotypic ALCAM-ALCAM interactions have been implicated in the development and maintenance of tissue architecture and tumor progression, heterotypic ALCAM-CD6 interactions act to initiate and stabilize T-cell–dendritic-cell interactions affecting T-cell activation. The ability to resist the forces acting on the individual bonds during these highly dynamic cellular contacts is thought to be crucial for the (patho)physiology of ALCAM-mediated cell adhesion. Here, we used atomic force microscopy to characterize the relationship between affinity, avidity and the stability of ALCAM-mediated interactions under external loading, at the single-molecule level. Disruption of the actin cytoskeleton resulted in enhanced ALCAM binding avidity, without affecting the tensile strength of the individual bonds. Force spectroscopy revealed that the ALCAM-CD6 bond displayed a significantly higher tensile strength, a smaller reactive compliance and an up to 100-fold lower dissociation rate in the physiological force window in comparison to the homotypic interaction. These results indicate that homotypic and heterotypic ALCAM-mediated adhesion are governed by significantly distinct kinetic and mechanical properties, providing novel insight into the role of ALCAM during highly dynamic cellular interactions.

Dynamic coupling of ALCAM to the actin cortex strengthens cell adhesion to CD6.

ABSTRACT At the immunological synapse, the activated leukocyte cell adhesion molecule (ALCAM) on a dendritic cell (DC) and CD6 molecules on a T cell contribute to sustained DC–T-cell contacts. However, little is known about how ALCAM–CD6 bonds resist and adapt to mechanical stress. Here, we combine single-cell force spectroscopy (SCFS) with total-internal reflection fluorescence microscopy to examine ALCAM–CD6-mediated cell adhesion. The combination of cells expressing ALCAM constructs with certain cytoplasmic tail mutations and improved SCFS analysis processes reveal that the affinity of ALCAM–CD6 bonds is not influenced by the linking of the intracellular domains of ALCAM to the actin cortex. By contrast, the recruitment of ALCAM to adhesion sites and the propensity of ALCAM to anchor plasma membrane tethers depend on actin cytoskeletal interactions. Furthermore, linking ALCAM to the actin cortex through adaptor proteins stiffens the cortex and strengthens cell adhesion. We propose a framework for how ALCAMs contribute to DC–T-cell adhesion, stabilize DC–T-cell contacts and form a mechanical link between CD6 and the actin cortex to strengthen cell adhesion at the immunological synapse.

Molecular Friction as a Tool to Identify Functionalized Alkanethiols

By using the nanografting method, well-defined nanoscale patches of alkanethiols were constructed in a self-assembled monolayer (SAM) matrix on an atomically flat gold (Au(111)) surface. A series of nanografted patches, composed of alkanethiols with different end groups (-CH(3), -CF(3), -OH, -SH, -COOH, and -NH(2)), were analyzed in detail by a combination of atomic force microscopy (AFM) height and quantitative lateral friction measurements. By constructing a series of nanografted patches of methyl-terminated thiols with various chain lengths, it was shown that the absolute friction of the nanografted patches was always smaller than that of the surrounding SAM matrix, demonstrating that, because of the spatially confined self-assembly during nanografting, SAMs show less defects. In addition, the friction gradually increased for decreasing alkane chain length as expected, although a subtle odd-even effect was observed. The study of thiols with functionalized end groups (-CF(3), -OH, -SH, -COOH, and -NH(2)) gave specific insights in orientation, packing, and structure of the molecules in the SAMs. Depending on the thiol end groups, these nanografted patches exhibited large and specific differences in lateral friction force, which offers the unique possibility to use the friction as a molecular recognition tool for thiol-based self-assembled monolayers.

Influence of nanostructural environment and fluid flow on osteoblast-like cell behavior: A model for cell-mechanics studies

Introducing nanoroughness on various biomaterials has been shown to profoundly effect cell-material interactions. Similarly, physical forces act on a diverse array of cells and tissues. Particularly in bone, the tissue experiences compressive or tensile forces resulting in fluid shear stress. The current study aimed to develop an experimental setup for bone cell behavior, combining a nanometrically grooved substrate (200 nm wide, 50 nm deep) mimicking the collagen fibrils of the extracellular matrix, with mechanical stimulation by pulsatile fluid flow (PFF). MC3T3-E1 osteoblast-like cells were assessed for morphology, expression of genes involved in cell attachment and osteoblastogenesis and nitric oxide (NO) release. The results showed that both nanotexture and PFF did affect cellular morphology. Cells aligned on nanotexture substrate in a direction parallel to the groove orientation. PFF at a magnitude of 0.7 Pa was sufficient to induce alignment of cells on a smooth surface in a direction perpendicular to the applied flow. When environmental cues texture and flow were interacting, PFF of 1.4 Pa applied parallel to the nanogrooves initiated significant cellular realignment. PFF increased NO synthesis 15-fold in cells attached to both smooth and nanotextured substrates. Increased collagen and alkaline phosphatase mRNA expression was observed on the nanotextured substrate, but not on the smooth substrate. Furthermore, vinculin and bone sialoprotein were up-regulated after 1 h of PFF stimulation. In conclusion, the data show that interstitial fluid forces and structural cues mimicking extracellular matrix contribute to the final bone cell morphology and behavior, which might have potential application in tissue engineering.

Nanostructured substrate conformation can decrease osteoblast-like cell dysfunction in simulated microgravity conditions

Cells in situ are surrounded with defined structural elements formed by the nanomolecular extracellular matrix (ECM), and at the same time subjected to different mechanical stimuli arising from variety of physiological processes. In this study, using a nanotextured substrate mimicking the structural elements of the ECM and simulated microgravity, we wanted to develop a multifactorial model and understand better what guides cells in determining the morphological cell response. In our set‐up, bone precursor cells from rat bone marrow were isolated and cultured on nanotextured polystyrene substrate (pitch 200 nm, depth 50 nm). Simulated microgravity was applied to the cells, using a random positioning machine (RPM). The results demonstrated that cells cultured on nanotextured substrate align parallel to the grooves and re‐align significantly, but not completely, when subjected to simulated microgravity. The nanotextured substrate increased cell number and alkaline phosphatase (ALP) activity, whereas simulated microgravity decreased cells number and ALP activity. When the nanotextured substrate and simulated microgravity were combined together, the negative effect of the simulated microgravity ALP and cell number was reversed. In conclusion, absence of mechanical load in simulated microgravity has a negative effect on initial osteoblastogenesis, and nanotextured surfaces can partly reverse such a process. Copyright

N-glycan mediated adhesion strengthening during pathogen-receptor binding revealed by cell-cell force spectroscopy

Glycan-protein lateral interactions have gained increased attention as important modulators of receptor function, by regulating surface residence time and endocytosis of membrane glycoproteins. The pathogen-recognition receptor DC-SIGN is highly expressed at the membrane of antigen-presenting dendritic cells, where it is organized in nanoclusters and binds to different viruses, bacteria and fungi. We recently demonstrated that DC-SIGN N-glycans spatially restrict receptor diffusion within the plasma membrane, favoring its internalization through clathrin-coated pits. Here, we investigated the involvement of the N-glycans of DC-SIGN expressing cells on pathogen binding strengthening when interacting with Candida fungal cells by using atomic force microscope (AFM)-assisted single cell-pathogen adhesion measurements. The use of DC-SIGN mutants lacking the N-glycans as well as blocking glycan-mediated lateral interactions strongly impaired cell stiffening during pathogen binding. Our findings demonstrate for the first time the direct involvement of the cell membrane glycans in strengthening cell-pathogen interactions. This study, therefore, puts forward a possible role for the glycocalyx as extracellular cytoskeleton contributing, possibly in connection with the intracellular actin cytoskeleton, to optimize strengthening of cell-pathogen interactions in the presence of mechanical forces.

Nanometer-grooved topography stimulates trabecular bone regeneration around a concave implant in a rat femoral medulla model

In the present study, a method was developed to reproduce two nanogrooved patterns (groove width/ridge width/depth: 150/150/50 nm and 200/800/70 nm) into cylindrical epoxy resin implants, which were subsequently coated with 20 nm of titanium. Also, implants with a conventional surface roughness (Rq=1.6 μm) were produced. After cytocompatibility analysis of the produced surfaces, implants were installed into the femoral condyle of rats for 4 and 8 weeks. The histomorphometrical analysis of bone volume in a 100 μm wide zone close to the implant surface showed that only for the 200/800 grooves the amount of bone increased significantly between 4 and 8 weeks of implantation. In addition, at the late time point only implants with the 200/800 pattern revealed a significantly higher bone volume compared to the rough controls. In conclusion, the 200/800 grooved pattern can positively influence bone volume adjacent to the implant surface, and should be evaluated and optimized in further (pre-)clinical studies.