20 research outputs found
Nanomechanical and topographical imaging of living cells by Atomic Force Microscopy with colloidal probes
Atomic Force Microscopy (AFM) has a great potential as a tool to characterize
mechanical and morphological properties of living cells; these properties have
been shown to correlate with cells' fate and patho-physiological state in view
of the development of novel early-diagnostic strategies. Although several
reports have described experimental and technical approaches for the
characterization of cell elasticity by means of AFM, a robust and commonly
accepted methodology is still lacking. Here we show that micrometric spherical
probes (also known as colloidal probes) are well suited for performing a
combined topographic and mechanical analysis of living cells, with spatial
resolution suitable for a complete and accurate mapping of cell morphological
and elastic properties, and superior reliability and accuracy in the mechanical
measurements with respect to conventional and widely used sharp AFM tips. We
address a number of issues concerning the nanomechanical analysis, including
the applicability of contact mechanical models and the impact of a constrained
contact geometry on the measured elastic modulus (the finite-thickness effect).
We have tested our protocol by imaging living PC12 and MDA-MB-231 cells, in
order to demonstrate the importance of the correction of the finite-thickness
effect and the change in cell elasticity induced by the action of a
cytoskeleton-targeting drug.Comment: 51 pages, 12 figures, 3 table
3D Bioprinting of Human Adipose-Derived Stem Cells and Their Tenogenic Differentiation in Clinical-Grade Medium
Defining the best combination of cells and biomaterials is a key challenge for the development of tendon tissue engineering (TE) strategies. Adipose-derived stem cells (ASCs) are ideal candidates for this purpose. In addition, controlled cell-based products adherent to good manufacturing practice (GMP) are required for their clinical scale-up. With this aim, in this study, ASC 3D bioprinting and GMP-compliant tenogenic differentiation were investigated. In detail, primary human ASCs were embedded within a nanofibrillar-cellulose/alginate bioink and 3D-bioprinted into multi-layered square-grid matrices. Bioink viscoelastic properties and scaffold ultrastructural morphology were analyzed by rheology and scanning electron microscopy (SEM). The optimal cell concentration for printing among 3, 6 and 9 × 106 ASC/mL was evaluated in terms of cell viability. ASC morphology was characterized by SEM and F-actin immunostaining. Tenogenic differentiation ability was then evaluated in terms of cell viability, morphology and expression of scleraxis and collagen type III by biochemical induction using BMP-12, TGF-β3, CTGF and ascorbic acid supplementation (TENO). Pro-inflammatory cytokine release was also assessed. Bioprinted ASCs showed high viability and survival and exhibited a tenocyte-like phenotype after biochemical induction, with no inflammatory response to the bioink. In conclusion, we report a first proof of concept for the clinical scale-up of ASC 3D bioprinting for tendon TE
Conversion of nanoscale topographical information of cluster-assembled zirconia surfaces into mechanotransductive events promotes neuronal differentiation
Additional file 4: Table S1. Proteomic data for upregulated proteins. Proteins upregulated (compared to flat-Zr) or present only in cells grown on ns-Zr15. Adhesome proteins and proteins with roles in mechanobiological processes are marked in dark and light grey, respectively
Standardized nanomechanical atomic force microscopy procedure (SNAP) for measuring soft and biological samples
We present a procedure that allows a reliable determination of the elastic (Young's) modulus of soft samples, including living cells, by atomic force microscopy (AFM). The standardized nanomechanical AFM procedure (SNAP) ensures the precise adjustment of the AFM optical lever system, a prerequisite for all kinds of force spectroscopy methods, to obtain reliable values independent of the instrument, laboratory and operator. Measurements of soft hydrogel samples with a well-defined elastic modulus using different AFMs revealed that the uncertainties in the determination of the deflection sensitivity and subsequently cantilever's spring constant were the main sources of error. SNAP eliminates those errors by calculating the correct deflection sensitivity based on spring constants determined with a vibrometer. The procedure was validated within a large network of European laboratories by measuring the elastic properties of gels and living cells, showing that its application reduces the variability in elastic moduli of hydrogels down to 1%, and increased the consistency of living cells elasticity measurements by a factor of two. The high reproducibility of elasticity measurements provided by SNAP could improve significantly the applicability of cell mechanics as a quantitative marker to discriminate between cell types and conditions
Relevance of lunar periodicity in human spontaneous abortions
The effect of the moon on human reproduction has been scarcely investigated and
with controversial results. The present analysis describes a significant effect
of extreme perigeal lunar positions on the number of hospitalized spontaneous
abortions (n = 1,329) recorded at two university clinics and a public hospital at
Padova, Italy, during the years 2000-2003. Spectral analysis evidenced a 205-day
period which appears to be correlated with the 206-day periodicity in extreme
lunar distances. Circa-septan and circa-annual periodicities were also observed.
Peak significances were determined by a Monte Carlo approach (circa-septan and
205-day periodicities: p < 0.001; circa-annual periodicity: p < 0.05). Our study
indicates that the occurrence of human abortions displays suggestive
periodicities that may be of relevance for gynecological and obstetrical
practice
The Gut-Brain-Immune Axis in Autism Spectrum Disorders: A State-of-Art Report
The interest elicited by the large microbial population colonizing the human gut has ancient origins and has gone through a long evolution during history. However, it is only in the last decades that the introduction of high-throughput technologies has allowed to broaden this research field and to disentangle the numerous implications that gut microbiota has in health and disease. This comprehensive ecosystem, constituted mainly by bacteria but also by fungi, parasites, and viruses, is proven to be involved in several physiological and pathological processes that transcend the intestinal homeostasis and are deeply intertwined with apparently unrelated body systems, such as the immune and the nervous ones. In this regard, a novel speculation is the relationship between the intestinal microbial flora and the pathogenesis of some neurological and neurodevelopmental disorders, including the clinical entities defined under the umbrella term of autism spectrum disorders. The bidirectional interplay has led researchers to coin the term gut-brain-immune system axis, subverting the theory of the brain as an immune-privileged site and underscoring the importance of this reciprocal influence already from fetal life and especially during the pre- and post-natal neurodevelopmental process. This revolutionary theory has also unveiled the possibility to modify the gut microbiota as a way to treat and even to prevent different kinds of pathologies. In this sense, some attempts have been made, ranging from probiotic administration to fecal microbiota transplantation, with promising results that need further elaboration. This state-of-art report will describe the main aspects regarding the human gut microbiome and its specific role in the pathogenesis of autism and its related disorders, with a final discussion on the therapeutic and preventive strategies aiming at creating a healthy intestinal microbial environment, as well as their safety and ethical implications
Cutting-Edge Delivery Systems and Adjuvants in Tolerogenic Vaccines: A Review
Conventional therapies for immune-mediated diseases, including autoimmune disorders, transplant reactions, and allergies, have undergone a radical evolution in the last few decades; however, they are still not specific enough to avoid widespread immunosuppression. The idea that vaccine usage could be extended beyond its traditional immunogenic function by encompassing the ability of vaccines to induce antigen-specific tolerance may revolutionize preventive and therapeutic strategies in several clinical fields that deal with immune-mediated disorders. This approach has been supported by improved data relating to the several mechanisms involved in controlling unwanted immune responses and allowing peripheral tolerance. Given these premises, several approaches have been developed to induce peripheral tolerance against the antigens that are involved in the pathological immune response, including allergens, autoantigens, and alloantigens. Technological innovations, such as nucleic acid manipulation and the advent of micro- and nanoparticles, have further supported these novel preventive and therapeutic approaches. This review focuses on the main strategies used in the development of tolerogenic vaccines, including the technological issues used in their design and the role of "inverse adjuvants". Even though most studies are still limited to the preclinical field, the enthusiasm generated by their results has prompted some initial clinical trials, and they show great promise for the future management of immune-mediated pathological conditions