FIB patterning of stainless steel for the development of nano-structured stent surfaces for cardiovascular applications

Stent implantation is a percutaneous interventional procedure that mitigates vessel stenosis, providing mechanical support within the artery and as such a very valuable tool in the fight against coronary artery disease. However, stenting causes physical damage to the arterial wall. It is well accepted that a valuable route to reduce in-stent re-stenosis can be based on promoting cell response to nano-structured stainless steel (SS) surfaces such as by patterning nano-pits in SS. In this regard patterning by focused ion beam (FIB) milling offers several advantages for flexible prototyping. On the other hand FIB patterning of polycrystalline metals is greatly influenced by channelling effects and redeposition. Correlative microscopy methods present an opportunity to study such effects comprehensively and derive structure–property understanding that is important for developing improved patterning. In this chapter we present a FIB patterning protocol for nano-structuring features (concaves) ordered in rectangular arrays on pre-polished 316L stainless steel surfaces. An investigation based on correlative microscopy approach of the size, shape and depth of the developed arrays in relation to the crystal orientation of the underlying SS domains is presented. The correlative microscopy protocol is based on cross-correlation of top-view scanning electron microscopy, electron backscattering diffraction, atomic force microscopy and cross-sectional (serial) sectioning. Various FIB tests were performed, aiming at improved productivity by preserving nano-size accuracy of the patterned process. The optimal FIB patterning conditions for achieving reasonably high throughput (patterned rate of about 0.03 mm2/h) and nano-size accuracy in dimensions and shapes of the features are discussed as well

Investigation of Polyurea-Crosslinked Silica Aerogels as a Neuronal Scaffold: A Pilot Study

BACKGROUND: Polymer crosslinked aerogels are an attractive class of materials for future implant applications particularly as a biomaterial for the support of nerve growth. The low density and nano-porous structure of this material combined with large surface area, high mechanical strength, and tunable surface properties, make aerogels materials with a high potential in aiding repair of injuries of the peripheral nervous system. however, the interaction of neurons with aerogels remains to be investigated. METHODOLOGY: In this work the attachment and growth of neurons on clear polyurea crosslinked silica aerogels (PCSA) coated with: poly-L-lysine, basement membrane extract (BME), and laminin1 was investigated by means of optical and scanning electron microscopy. After comparing the attachment and growth capability of neurons on these different coatings, laminin1 and BME were chosen for nerve cell attachment and growth on PCSA surfaces. The behavior of neurons on treated petri dish surfaces was used as the control and behavior of neurons on treated PCSA discs was compared against it. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that: 1) untreated PCSA surfaces do not support attachment and growth of nerve cells, 2) a thin application of laminin1 layer onto the PCSA discs adhered well to the PCSA surface while also supporting growth and differentiation of neurons as evidenced by the number of processes extended and b3-tubulin expression, 3) three dimensional porous structure of PCSA remains intact after fixing protocols necessary for preservation of biological samples and 4) laminin1 coating proved to be the most effective method for attaching neurons to the desired regions on PCSA discs. This work provides the basis for potential use of PCSA as a biomaterial scaffold for neural regeneration

Impact of surface adhesion and sample heterogeneity on the multiscale mechanical characterisation of soft biomaterials

Funding from the Engineering and Physical Sciences Research Council (EP/L505602/1) is gratefully acknowledged

Mapping the Complex Morphology of Cell Interactions with Nanowire Substrates Using FIB-SEM

Using high resolution focused ion beam scanning electron microscopy (FIB-SEM) we study the details of cell-nanostructure interactions using serial block face imaging. 3T3 Fibroblast cellular monolayers are cultured on flat glass as a control surface and on two types of nanostructured scaffold substrates made from silicon black (Nanograss) with low- and high nanowire density. After culturing for 72 hours the cells were fixed, heavy metal stained, embedded in resin, and processed with FIB-SEM block face imaging without removing the substrate. The sample preparation procedure, image acquisition and image post-processing were specifically optimised for cellular monolayers cultured on nanostructured substrates. Cells display a wide range of interactions with the nanostructures depending on the surface morphology, but also greatly varying from one cell to another on the same substrate, illustrating a wide phenotypic variability. Depending on the substrate and cell, we observe that cells could for instance: break the nanowires and engulf them, flatten the nanowires or simply reside on top of them. Given the complexity of interactions, we have categorised our observations and created an overview map. The results demonstrate that detailed nanoscale resolution images are required to begin understanding the wide variety of individual cells' interactions with a structured substrate. The map will provide a framework for light microscopy studies of such interactions indicating what modes of interactions must be considered

Biophysical mechanisms of single-cell interactions with microtopographical cues

Biophysical cues encoded in the extracellular matrix (ECM) are increasingly being explored to control cell behavior in tissue engineering applications. Recently, we showed that cell adhesion to microtopographical structures (“micropegs”) can suppress proliferation in a manner that may be blunted by inhibiting cellular contractility, suggesting that this effect is related to altered cell-scaffold mechanotransduction. We now directly investigate this possibility at the microscale through a combination of live-cell imaging, single-cell mechanics methods, and analysis of gene expression. Using time-lapse imaging, we show that when cells break adhesive contacts with micropegs, they form F-actin-filled tethers that extend and then rupture at a maximum, critical length that is greater than trailing-edge tethers observed on topographically flat substrates. This critical tether length depends on myosin activation, with inhibition of Rho-associated kinase abolishing topography-dependent differences in tether length. Using cellular de-adhesion and atomic force microscopy indentation measurements, we show that the micropegs enhance cell-scaffold adhesive interactions without changing whole-cell elasticity. Moreover, micropeg adhesion increases expression of specific mechanotransductive genes, including RhoA GTPase and myosin heavy chain II, and, in myoblasts, the functional marker connexin 43. Together, our data support a model in which microtopographical cues alter the local mechanical microenvironment of cells by modulating adhesion and adhesion-dependent mechanotransductive signaling

Osteoinduction of Human Mesenchymal Stem Cells by Bioactive Composite Scaffolds without Supplemental Osteogenic Growth Factors

The development of a new family of implantable bioinspired materials is a focal point of bone tissue engineering. Implant surfaces that better mimic the natural bone extracellular matrix, a naturally nano-composite tissue, can stimulate stem cell differentiation towards osteogenic lineages in the absence of specific chemical treatments. Herein we describe a bioactive composite nanofibrous scaffold, composed of poly-caprolactone (PCL) and nano-sized hydroxyapatite (HA) or beta-tricalcium phosphate (TCP), which was able to support the growth of human bone marrow mesenchymal stem cells (hMSCs) and guide their osteogenic differentiation at the same time. Morphological and physical/chemical investigations were carried out by scanning, transmission electron microscopy, Fourier-transform infrared (FTIR) spectroscopy, mechanical and wettability analysis. Upon culturing hMSCs on composite nanofibers, we found that the incorporation of either HA or TCP into the PCL nanofibers did not affect cell viability, meanwhile the presence of the mineral phase increases the activity of alkaline phosphatase (ALP), an early marker of bone formation, and mRNA expression levels of osteoblast-related genes, such as the Runt-related transcription factor 2 (Runx-2) and bone sialoprotein (BSP), in total absence of osteogenic supplements. These results suggest that both the nanofibrous structure and the chemical composition of the scaffolds play a role in regulating the osteogenic differentiation of hMSCs

Molecular Organization of Integrin-Based Adhesion Complexes in Mouse Embryonic Stem Cells

10.1021/acsbiomaterials.8b01124ACS Biomaterials Science and Engineering583828-384

Rabbit Surgery Protocol for End-to-End and End-to-Side Vascular Graft Anastomosis

10.1007/978-1-0716-1708-3_15Methods Mol Biol2375177-18

Correlation and Comparison of Cortical and Hippocampal Neural Progenitor Morphology and Differentiation through the Use of Micro- and Nano-Topographies

10.3390/jfb8030035Journal of functional biomaterials83E3

Nanoscale Architecture of the Cortical Actin Cytoskeleton in Embryonic Stem Cells

10.1016/j.celrep.2019.06.089Cell Reports2851251-1267.e