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Characterisation of porous knitted titanium for replacement of intervertebral disc nucleus pulposus
(2017)
Effective restoration of human intervertebral disc degeneration is challenged by numerous limitations of the currently available spinal fusion and arthroplasty treatment strategies. Consequently, use of artificial biomaterial implant is gaining attention as a potential therapeutic strategy. Our study is aimed at investigating and characterizing a novel knitted titanium (Ti6Al4V) implant for the replacement of nucleus pulposus to treat early stages of chronic intervertebral disc degeneration. Specific knitted geometry of the scaffold with a porosity of 67.67 ± 0.824% was used to overcome tissue integration failures. Furthermore, to improve the wear resistance without impairing original mechanical strength, electro-polishing step was employed. Electro-polishing treatment changed a surface roughness from 15.22 ± 3.28 to 4.35 ± 0.87 μm without affecting its wettability which remained at 81.03 ± 8.5°. Subsequently, cellular responses of human mesenchymal stem cells (SCP1 cell line) and human primary chondrocytes were investigated which showed positive responses in terms of adherence and viability. Surface wettability was further enhanced to super hydrophilic nature by oxygen plasma treatment, which eventually caused substantial increase in the proliferation of SCP1 cells and primary chondrocytes. Our study implies that owing to scaffolds physicochemical and biocompatible properties, it could improve the clinical performance of nucleus pulposus replacement.
Cell-cell and cell-extracellular matrix (ECM) adhesion regulates fundamental cellular functions and is crucial for cell-material contact. Adhesion is influenced by many factors like affinity and specificity of the receptor-ligand interaction or overall ligand concentration and density. To investigate molecular details of cell ECM and cadherins (cell-cell) interaction in vascular cells functional nanostructured surfaces were used Ligand-functionalized gold nanoparticles (AuNPs) with 6-8 nm diameter, are precisely immobilized on a surface and separated by non-adhesive regions so that individual integrins or cadherins can specifically interact with the ligands on the AuNPs. Using 40 nm and 90 nm distances between the AuNPs and functionalized either with peptide motifs of the extracellular matrix (RGD or REDV) or vascular endothelial cadherins (VEC), the influence of distance and ligand specificity on spreading and adhesion of endothelial cells (ECs) and smooth muscle cells (SMCs) was investigated. We demonstrate that RGD-dependent adhesion of vascular cells is similar to other cell types and that the distance dependence for integrin binding to ECM-peptides is also valid for the REDV motif. VEC-ligands decrease adhesion significantly on the tested ligand distances. These results may be helpful for future improvements in vascular tissue engineering and for development of implant surfaces.
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.
Perivascular stromal cells, including mesenchymal stem/stromal cells (MSCs), secrete paracrine factor in response to exercise training that can facilitate improvements in muscle remodeling. This study was designed to test the capacity for muscle-resident MSCs (mMSCs) isolated from young mice to release regenerative proteins in response to mechanical strain in vitro, and subsequently determine the extent to which strain-stimulated mMSCs can enhance skeletal muscle and cognitive performance in a mouse model of uncomplicated aging. Protein arrays confirmed a robust increase in protein release at 24 h following an acute bout of mechanical strain in vitro (10%, 1 Hz, 5 h) compared to non-strain controls. Aged (24 month old), C57BL/6 mice were provided bilateral intramuscular injection of saline, non strain control mMSCs, or mMSCs subjected to a single bout of mechanical strain in vitro (4 ×104). No significant changes were observed in muscle weight, myofiber size, maximal force, or satellite cell quantity at 1 or 4 wks between groups. Peripheral perfusion was significantly increased in muscle at 4 wks post-mMSC injection (p < 0.05), yet no difference was noted between control and preconditioned mMSCs. Intramuscular injection of preconditioned mMSCs increased the number of new neurons and astrocytes in the dentate gyrus of the hippocampus compared to both control groups (p < 0.05), with a trend toward an increase in water maze performance noted (p=0.07). Results from this study demonstrate that acute injection of exogenously stimulated muscle-resident stromal cells do not robustly impact aged muscle structure and function, yet increase the survival of new neurons in the hippocampus.
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.
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.
Cancer cells invade confined microchannels via a self-directed mesenchymal-to-amoeboid transition
(2019)
Cancer cell invasion through physical barriers in the extracellular matrix (ECM) requires a complex synergy of traction force against the ECM, mechanosensitive feedback, and subsequent cytoskeletal rearrangement. PDMS microchannels were used to investigate the transition from mesenchymal to amoeboid invasion in cancer cells. Migration was faster in narrow 3 μm-wide channels than in wider 10 μm channels, even in the absence of cell-binding ECM proteins. Cells permeating narrow channels exhibited blebbing and had smooth leading edge profiles, suggesting an ECM-induced transition from mesenchymal invasion to amoeboid invasion. Live cell labeling revealed a mechanosensing period in which the cell attempts mesenchymal-based migration, reorganizes its cytoskeleton, and proceeds using an amoeboid phenotype. Rho/ROCK (amoeboid) and Rac (mesenchymal) pathway inhibition revealed that amoeboid invasion through confined environments relies on both pathways in a time- and ECM dependent manner. This demonstrates that cancer cells can dynamically modify their invasion programming to navigate physically confining matrix conditions.
Polyurethane-bases block copolymers (TPCUs) are block-copolymers with systematically varied soft and hard segments. They have been suggested to serve as material for chondral implants in joint regeneration. Such applications may require the adhesion of chondrocytes to the implant surface, facilitating cell growth while keeping their phenotype. Thus, aims of this work were (1) to modify the surface of soft biostable polyurethane-based model implants (TPCU and TSiPCU) with high-molecular weight hyaluronic acid (HA) using an optimized multistep strategy of immobilization, and (2) to evaluate bioactivity of the modified TPCUs in vitro. Our results show no cytotoxic potential of the TPCUs. HAbioactive molecules (Mw =700kDa) were immobilized onto the polyurethane surface via polyethylenimine (PEI) spacers, and modifications were confirmed by several characterization methods. Tests with porcine chondrocytes indicated the potential of the TPCU-HA for inducing enhanced cell proliferation.
Stronger than they look
(2019)
In vitro, hydrogel-based ECMs for functionalizing surfaces of various material have played an essential role in mimicking native tissue matrix. Polydimethylsiloxane (PDMS) is widely used to build microfluidic or organ-on-chip devices compatible with cells due to its easy handling in cast replication. Despite such advantages, the limitation of PDMS is its hydrophobic surface property. To improve wettability of PDMS-based devices, alginate, a naturally derived polysaccharide, was covalently bound to the PDMS surface. This alginate then crosslinked further hydrogel onto the PDMS surface in desired layer thickness. Hydrogel-modified PDMS was used for coating a topography chip system and in vitro investigation of cell growth on the surfaces. Moreover, such hydrophilic hydrogel-coated PDMS is utilized in a microfluidic device to prevent unspecific absorption of organic solutions. Hence, in both exemplary studies, PDMS surface properties were modified leading to improved devices.