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Human adipose-derived mesenchymal stem/stromal cells (Ad-MSCs) have great potential for bone tissue engineering. Cryogels, mimicking the three-dimensional structure of spongy bone, represent ideal carriers for these cells. We developed poly(2-hydroxyethyl methacrylate) cryogels, containing hydroxyapatite to mimic inorganic bone matrix. Cryogels were additionally supplemented with different types of proteins, namely collagen (Coll), platelet rich plasma (PRP), immune cells-conditioned medium (CM), and RGD peptides (RGD). The different protein components did not affect scaffolds’ porosity or water-uptake capacity, but altered pore size and stiffness. Stiffness was highest in scaffolds with PRP (82.3 kPa), followed by Coll (55.3 kPa), CM (45.6 kPa), and RGD (32.8 kPa). Scaffolds with PRP, CM, and Coll had the largest pore diameters (~60 µm). Ad MSCs were osteogenically differentiated on these scafffolds for 14 days. Cell attachment and survival rates were comparable for all four scaffolds. Runx2 and osteocalcin levels only increased in Ad-MSCs on Coll, PRP and CM cryogels. Osterix levels increased slightly in Ad-MSCs differentiated on Coll and PRP cryogels. With differentiation alkaline phosphatase activity decreased under all four conditions. In summary, besides Coll cryogel our PRP cryogel constitutes as an especially suitable carrier for bone tissue engineering. This is of special interest, as this scaffold can be generated with patients’ PRP.
A series of novel biomedical TPCUs with different percentages of hard segment and a silicone component in the soft segment were synthesized in a multi stage one-pot method. The kinetic profiles of the urethane formation in TPCU-based copolymer systems were monitored by rheological, in line FTIR spectroscopic (React IR) and real-time calorimetric (RC1) methods. This process-analytically monitored multi step synthesis was successfully used to optimize the production of medical-grade TPCU elastomers on preparative scale (in lots of several kg) with controlled molecular structure and mechanical properties. Various surface and bulk analytical methods as well as systematic studies of the mechanic response of the elastomer end-products towards compression and tensile loading were used to estimate the bio-stability of the prepared TPCUs in vitro after 3 months. The tests suggested that high bio-stability of all polyurethane formulations using accelerating in vitro test can be attributed to the synthetic design as well as to the specific techniques used for specimen preparation, namely: (1) the annealing for reducing residual polymer surface stress and preventing IES, (2) stabilization of the morphology by long time storage of the specimens after processing before being immersed in the test liquids, (3) purification by extraction to remove the shot chain oligomers which are the most susceptible to degradation. All mechanical tests were performed on cylindrical and circular disc specimens for modelling the thickness of the meniscus implants under application-relevant stress conditions.
A wide variety of cell types exhibit substrate topography-based behavior, also known as contact guidance. However, the precise cellular mechanisms underlying this process are still unknown. In this study, we investigated contact guidance by studying the reaction of human endothelial cells (ECs) to well-defined microgroove topographies, both during and after initial cell spreading. As the cytoskeleton plays a major role in cellular adaptation to topographical features, two methods were used to perturb cytoskeletal structures. Inhibition of actomyosin contractility with the chemical inhibitor blebbistatatin demonstrated that initial contact guidance events are independent of traction force generation. However, cell alignment to the grooved substrate was altered at later time points, suggesting an initial ‘passive’ phase of contact guidance, followed by a contractility-dependent ‘active’ phase that relies on mechanosensitive feedback. The actin cytoskeleton was also perturbed in an indirect manner by culturing cells upside down, resulting in decreased levels of contact guidance and suggesting that a possible loss of contact between the actin cytoskeleton and the substrate could lead to cytoskeleton impairment. The process of contact guidance at the microscale was found to be primarily lamellipodia driven, as no bias in filopodia extension was observed on micron-scale grooves.
Intermediate filament reorganization dynamically influences cancer cell alignment and migration
(2017)
The interactions between a cancer cell and its extracellular matrix (ECM) have been the focus of an increasing amount of investigation. The role of the intermediate filament keratin in cancer has also been coming into focus of late, but more research is needed to understand how this piece fits in the puzzle of cytoskeleton-mediated invasion and metastasis. In Panc-1 invasive pancreatic cancer cells, keratin phosphorylation in conjunction with actin inhibition was found to be sufficient to reduce cell area below either treatment alone. We then analyzed intersecting keratin and actin fibers in the cytoskeleton of cyclically stretched cells and found no directional correlation. The role of keratin organization in Panc-1 cellular morphological adaptation and directed migration was then analyzed by culturing cells on cyclically stretched polydimethylsiloxane (PDMS) substrates, nanoscale grates, and rigid pillars. In general, the reorganization of the keratin cytoskeleton allows the cell to become more ‘mobile’- exhibiting faster and more directed migration and orientation in response to external stimuli. By combining keratin network perturbation with a variety of physical ECM signals, we demonstrate the interconnected nature of the architecture inside the cell and the scaffolding outside of it, and highlight the key elements facilitating cancer cell-ECM interactions.
Soft lithography, a tool widely applied in biology and life sciences with numerous applications, uses the soft molding of photolithography-generated master structures by polymers. The central part of a photolithography set-up is a mask-aligner mostly based on a high-pressure mercury lamp as an ultraviolet (UV) light source. This type of light source requires a high level of maintenance and shows a decreasing intensity over its lifetime, influencing the lithography outcome. In this paper, we present a low-cost, bench-top photolithography tool based on ninety-eight 375 nm light-emitting diodes (LEDs). With approx. 10 W, our presented lithography set-up requires only a fraction of the energy of a conventional lamp, the LEDs have a guaranteed lifetime of 1000 h, which becomes noticeable by at least 2.5 to 15 times more exposure cycles compared to a standard light source and with costs less than 850 C it is very affordable. Such a set-up is not only attractive to small academic and industrial fabrication facilities who want to enable work with the technology of photolithography and cannot afford a conventional set-up, but also microfluidic teaching laboratories and microfluidic research and development laboratories, in general, could benefit from this cost-effective alternative. With our self-built photolithography system, we were able to produce structures from 6 μm to 50 μm in height and 10 μm to 200 μm in width. As an optional feature, we present a scaled-down laminar flow hood to enable a dust-free working environment for the photolithography process.
Adapting characteristics of biomaterials specifically for in vitro and in vivo applications is becoming increasingly important in order to control interactions between material and biological systems. These complex interactions are influenced by surface properties like chemical composition, charge, mechanical and topographic attributes. In many cases it is not useful or even not possible to alter the base material but changing surface, to improve biocompatibility or to make surfaces bioactive, may be achieved by thin coatings. An already established method is the coating with polyelectrolyte multilayers (PEM). To adjust adhesion, proliferation and improve vitality of certain cell types, we modified the roughness of PEM coatings. We included different types nanoparticles (NP’s) in different concentrations into PEM coatings for controlling surface roughness. Surface properties were characterized and the reaction of 3 different cell types on these coatings was tested.
The extracellular environment of vascular cells in vivo is complex in its chemical composition, physical properties, and architecture. Consequently, it has been a great challenge to study vascular cell responses in vitro, either to understand their interaction with their native environment or to investigate their interaction with artificial structures such as implant surfaces. New procedures and techniques from materials science to fabricate bio-scaffolds and surfaces have enabled novel studies of vascular cell responses under well-defined, controllable culture conditions. These advancements are paving the way for a deeper understanding of vascular cell biology and materials–cell interaction. Here, we review previous work focusing on the interaction of vascular smooth muscle cells (SMCs) and endothelial cells (ECs) with materials having micro- and nanostructured surfaces. We summarize fabrication techniques for surface topographies, materials, geometries, biochemical functionalization, and mechanical properties of such materials. Furthermore, various studies on vascular cell behavior and their biological responses to micro- and nanostructured surfaces are reviewed. Emphasis is given to studies of cell morphology and motility, cell proliferation, the cytoskeleton and cell-matrix adhesions, and signal transduction pathways of vascular cells. We finalize with a short outlook on potential interesting future studies.
Programmable nano-bio interfaces driven by tuneable vertically configured nanostructures have recently emerged as a powerful tool for cellular manipulations and interrogations. Such interfaces have strong potential for ground-breaking advances, particularly in cellular nanobiotechnology and mechanobiology. However, the opaque nature of many nanostructured surfaces makes non-destructive, live-cell characterization of cellular behavior on vertically aligned nanostructures challenging to observe. Here, a new nanofabrication route is proposed that enables harvesting of vertically aligned silicon (Si) nanowires and their subsequent transfer onto an optically transparent substrate, with high efficiency and without artefacts. We demonstrate the potential of this route for efficient live-cell phase contrast imaging and subsequent characterization of cells growing on vertically aligned Si nanowires. This approach provides the first opportunity to understand dynamic cellular responses to a cell-nanowire interface, and thus has the potential to inform the design of future nanoscale cellular manipulation technologies.
In Neurofibromatosis 1 (NF1) germ line loss of function mutations result in reduction of cellular neurofibromin content (NF1+/−, NF1 haploinsufficiency). The Ras-GAP neurofibromin is a very large cytoplasmic protein (2818 AA, 319 kDa) involved in the RAS-MAPK pathway. Aside from regulation of proliferation, it is involved in mechanosensoric of cells. We investigated neurofibromin replacement in cultured human fibroblasts showing reduced amount of neurofibromin. Full length neurofibromin was produced recombinantly in insect cells and purified. Protein transduction into cultured fibroblasts was performed employing cell penetrating peptides along with photochemical internalization. This combination of transduction strategies ensures the intracellular uptake and the translocation to the cytoplasm of neurofibromin. The transduced neurofibromin is functional, indicated by functional rescue of reduced mechanosensoric blindness and reduced RasGAP activity in cultured fibroblasts of NF1 patients or normal fibroblasts treated by NF1 siRNA. Our study shows that recombinant neurofibromin is able to revert cellular effects of NF1 haploinsuffiency in vitro, indicating a use of protein transduction into cells as a potential treatment strategy for the monogenic disease NF1.
Poly(dimethylsiloxane) can be covalently coated with ultrathin NCO-sP(EO-stat-PO) hydrogel layers which permit covalent binding of cell adhesive moieties, while minimizing unspecific cell adhesion on non-functionalized areas. We applied long term uniaxial cyclic tensile strain (CTS) and revealed (a) the preservation of protein and cell-repellent properties of the NCO-sP(EO-stat-PO) coating and (b) the stability and bioactivity of a covalently bound fibronectin (FN) line pattern. We studied the adhesion of human dermal fibroblast (HDFs) on non-modified NCO-sP(EO-stat-PO) coatings and on the FN. HDFs adhered to FN and oriented their cell bodies and actin fibers along the FN lines independently of the direction of CTS. This mechanical long term stability of the bioactive, patterned surface allows unraveling biomechanical stimuli for cellular signaling and behavior to understand physiological and pathological cell phenomenon. Additionally, it allows for the application in wound healing assays, tissue engineering, and implant development demanding spatial control over specific cell adhesion.