Academic literature on the topic 'Nanotopographical cue'

Contact guidance is a powerful topographical cue that induces persistent directional cell migration. Healthy tissue stroma is characterized by a meshwork of wavy extracellular matrix (ECM) fiber bundles, whereas metastasis-prone stroma exhibit less wavy, more linear fibers. The latter topography correlates with poor prognosis, whereas more wavy bundles correlate with benign tumors. We designed nanotopographic ECM-coated substrates that mimic collagen fibril waveforms seen in tumors and healthy tissues to determine how these nanotopographies may regulate cancer cell polarization and migration machineries. Cell polarization and directional migration were inhibited by fibril-like wave substrates above a threshold amplitude. Although polarity signals and actin nucleation factors were required for polarization and migration on low-amplitude wave substrates, they did not localize to cell leading edges. Instead, these factors localized to wave peaks, creating multiple “cryptic leading edges” within cells. On high-amplitude wave substrates, retrograde flow from large cryptic leading edges depolarized stress fibers and focal adhesions and inhibited cell migration. On low-amplitude wave substrates, actomyosin contractility overrode the small cryptic leading edges and drove stress fiber and focal adhesion orientation along the wave axis to mediate directional migration. Cancer cells of different intrinsic contractility depolarized at different wave amplitudes, and cell polarization response to wavy substrates could be tuned by manipulating contractility. We propose that ECM fibril waveforms with sufficiently high amplitude around tumors may serve as “cell polarization barriers,” decreasing directional migration of tumor cells, which could be overcome by up-regulation of tumor cell contractility.

Le cellule β-pancreatiche svolgono un ruolo fondamentale nell’omeostasi glucidica, essendo le uniche cellule del corpo in grado di produrre e secernere insulina. Alterazioni della loro funzionalità o del loro numero determinano lo sviluppo del diabete mellito, un gruppo di patologie ad eziologia eterogenea caratterizzate da iperglicemia. L’approccio più efficace per trattare i pazienti diabetici dovrebbe consistere nel ripristinare la funzionalità o la massa β-cellulare tramite terapie rigenerative o sostitutive. Tra le terapie sostitutive particolarmente promettenti vi sono quelle che utilizzano cellule staminali embrionali o pluripotenti. Tuttavia, i protocolli ad oggi disponibili per promuovere il loro differenziamento in vitro non sono del tutto efficaci nel generare cellule β completamente mature. Le cellule β, infatti, per essere funzionali ed efficienti necessitano di uno specifico microambiente che fornisca molteplici stimoli chimici e fisici. Particolarmente studiati sono gli stimoli chimici, mentre poco conosciuti sono gli effetti delle proprietà fisiche della matrice extracellulare. Lo scopo del nostro studio è stato, quindi, quello di valutare l’impatto della nanotopografia sul differenziamento e la funzionalità β-cellulare e caratterizzarne i meccanismi molecolari coinvolti. Per riprodurre le proprietà nanotopografiche della matrice extracellulare sono stati utilizzati substrati di zirconio ingegnerizzati su scala nanometrica. Nel nostro lavoro abbiamo dimostrato che le cellule β percepiscono la nanotopografia della matrice e rispondono attraverso una riorganizzazione del citoscheletro di actina e dell’architettura nucleare. Tale processo, noto come meccanotrasduzione, permette il mantenimento delle isole di Langerhans per lungo tempo in cultura, preservando il differenziamento e la funzionalità delle cellule β. I nostri dati, inoltre, dimostrano che la nanostruttura induce una modifica della dinamicità, morfologia e funzionalità mitocondriale favorendo un cambiamento del metabolismo β-cellulare. Anche il processo mitomorfico sembra essere direttamente promosso dalla riorganizzazione del citoscheletro e dalla modifica delle interazioni dei mitocondri con altri organelli citoplasmatici. Nell’insieme, i nostri dati dimostrano che le cellule β percepiscono e rispondono alla nanotopografia attivando un processo meccanotransduttivo che promuove la sopravvivenza e la funzionalità β-cellulare. L’utilizzo di substrati ingegnerizzati che riproducono le proprietà biofisiche del microambiente rappresenta un ottimo strumento per approfondire le conoscenze sui meccanismi molecolari alla base della meccanotransduzione. Queste conoscenze saranno utili per potenziare le terapie rigenerative e per promuovere il differenziamento di cellule staminali in vitro, migliorando così anche l’efficacia delle terapie sostitutive.
Pancreatic β-cells, the only cells within the body able to secrete large amount of insulin, play a crucial role in the control of glucose homeostasis and alteration of their function and mass leads to diabetes pathogenesis, a group of pathologies characterized by severe hyperglycemia. Therefore, preserving the remaining β-cell function and replacing the β-cell mass represent the most promising strategies to treat diabetes. Embryonic and pluripotent stem cells hold great promise in generating β-cells for novel therapeutic discoveries in diabetes mellitus. However, their differentiation in vitro is still inefficient, and functional studies reveal that most of these β-like cells still fail to fully mirror the adult β-cell physiology. For their proper growth and functioning, β-cells require a very specific environment, the islet niche, which provides a myriad of chemical and physical signals. While the nature and effects of chemical stimuli have been widely characterized, less is known about the mechanical signals. Therefore, aim of the proposed research was to investigate the contribution of nanotopographical cues on β-cell differentiation and function and to characterize the molecular mechanisms involved. To mimic the nanotopography of the extracellular matrix, cluster-assembled zirconia substrates with tailored roughness were employed. We demonstrated that β-cells perceive nanoscale features and convert these stimuli into mechanotransductive processes which modulate the cellular behavior, via remodeling of the actin cytoskeleton and nuclear architecture. These changes are also paralleled by modulation of mitochondrial dynamics, morphology, and function, favoring a metabolic switch of the cells. The mitomorphosis is driven by substrate-induced reorganization of the cytoskeleton and modification of the mitochondria interplay with other organelles. In conclusion, our data suggest that β-cells sense and respond to nanoscale features by activating a mechanotransductive pathway that promotes β-cell survival and function. By engineering microenvironments mirroring the biophysical niche properties it is possible to elucidate the β-cell mechanotransductive-regulatory mechanisms and to harness them for the promotion of β-cell differentiation capacity. This hopefully will allow us to improve the efficacy of β-cell transplantation therapies and to identify a core set of signaling pathways useful for accelerating regenerative strategies for diabetes treatment.

This thesis is focused on understanding the fundamental physical interaction occurring, when a material interacts with a biological fluid containing protein molecules and cells. The interaction of proteins with defined surface chemistry and nanotopography is of major interest in the field of biomaterials. Despite the degree of research undertaken to study this interface, there is still a lack of understanding of how the protein layer evolves with respect to surface parameters, and further how cells condition the surface. By understanding these processes, advanced biomaterial coatings may be developed that allow control over specific cell responses to direct healing processes. Colourimetric and fluorometric assays were carried out to assess protein-surface affinity and amount of protein adsorption. Infrared spectroscopy was used to quantify protein conformational changes incurred upon adsorption on defined nanoscale surfaces presenting a range of chemical functional groups. Model experiments were performed using bovine albumin and fibrinogen adsorbing onto surfaces presenting defined surface chemistry: OH, COOH, NH2, and CH3. Surface curvature on the nanoscale was used to model topography on the same length scale as protein molecules. Silica colloidal dispersions were prepared in batches,11-215 nm diameters allowing chemical modification whilst keeping nanotopography constant. 3T3 fibroblasts were cultured over a library of surfaces presenting a spectrum of batches chemical functionality and nanostructure. Changes in cell attachment, morphology, migration and proliferation were examined. Media was removed at two different time points of 30 minutes and 24 hours, and examined to identify changes in fibroblast secreted proteins. Liquid chromatography was used to separate the cell culture media after incubation with cells over various chemically functionalised surfaces. Electrospray ionisation (ESI) and matrix assisted laser desorption (MALDI) mass spectrometry were used to identify changes in media with respect to the varying surfaces used and over time. The studies presented in this thesis give a better understanding of the interaction between silica nanoparticles and protein molecules, including conformational changes that occur when protein adsorbs on the nanoparticle; the effect of surface nanotopography and defined chemistry on protein adsorption is examined with respect to both chemical functionality and nanotopography. Clear differences were observed in the amount of protein adsorbed and its structural presentation when bound. The strength of the interaction, described through isotherm fitting, gave insight into the mechanism of competitive protein binding. Surface curvature on the nanoscale was also found to act synergistically with surface chemistry to dictate the dynamic accumulation of protein at the surface interface. In the later chapter discussion is given in terms of cell-surface interaction. Experimental evidence is shown for different mass spectroscopic analysis of reduced complexity media following initial cell-surface interaction and that after 24 hours. From this it is postulated that cell secretions are effected through interaction with the surface, with these changes being significant even after 30 minutes of cell culture with the defined surfaces. These changes are specific to the presented surface as they do not alter with respect to longer culture periods, but media are clearly different collected from cells cultured on different surfaces. This research will help solve challenges facing materials science, understand biological responses to surroundings and help in the development and advance of medical devices, drug delivery, therapeutics and diagnostics.