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Background: The design of an appropriate microenvironment for stem cell differentiation constitutes a multitask mission and a critical step toward the clinical application of tissue substitutes. With the aim of producing a bioactive material for orthopedic applications, a transforming growth factor-? (TGF- ?1)/hydroxyapatite (HA) association within an alginate-based scaffold was investigated. The bioactive scaffold was carefully designed to offer specific biochemical cues for an efficient and selective cell differentiation toward the bony and chondral lineages. Methods: Highly porous alginate scaffolds were fabricated from a mixture of calcium cross-linked alginates by means of a freeze-drying technique. In the chondral layer, the TGF in citric acid was mixed with an alginate/ alginate-sulfate solution. In the bony layer, HA granules were added as bioactive signal, to offer an osteoinductive surface to the cells. Optical and scanning electron microscopy analyses were performed to assess the macromicro architecture of the biphasic scaffold. Different mechanical tests were conducted to evaluate the elastic modulus of the grafts. For the biological validation of the developed prototype, mesenchymal stem cells were loaded onto the samples; cellular adhesion, proliferation and in vivo biocompatibility were evaluated. Results and conclusions: The results successfully demonstrated the efficacy of the designed osteochondral graft, which combined interesting functional properties and biomechanical performances, thus becoming a promising candidate for osteochondral tissue-engineering applications.

Atomic force microscopy (AFM) proved to be able to obtain high-resolution three-dimensional images of single-membrane proteins, isolated, crystallized, or included in reconstructed model membranes. The extension of this technique to native systems, such as the protein immersed in a cell membrane, needs a careful manipulation of the biological sample to meet the experimental constraints for high-resolution AFM imaging. In this article, a general protocol for sample preparation is presented, based on the mechanical stretch of the cell membrane. The effectiveness for AFM imaging has been tested on the basis of an integrated optical and AFM approach and the proposed method has been applied to cells expressing cystic fibrosis transmembrane conductance regulator, a channel involved in cystic fibrosis, showing the possibility to identify and analyze single proteins in the plasma membrane.

Single-cell force spectroscopy is an emerging technique in the field of biomedicine because it has proved to be a unique tool to obtain mechanical and functional information on living cells, with force resolution up to single molecular bonds. This technique was applied to the study of the cytoskeleton organisation of neuroblastoma cells, a life-threatening cancer typically developing during childhood, and the results were interpreted on the basis of reference experiments on human embryonic kidney cell line. An intimate connection emerges among cellular state, cytoskeleton organisation and experimental outcome that can be potentially exploited towards a new method for cancer stadiation of neuroblastoma cells.

The need for investigating the properties of new materials at nanoscale level continuously pushes the development of higher resolution measurement instruments. In this context, a promising dynamic atomic force microscopy setup, where the cantilever gets excited by a nonlinear feedback loop, has been recently introduced. In the first part of the paper the application of this working mode to imaging is experimentally investigated, showing the effectiveness of this novel approach. Furthermore, the presence of a variable saturation in the nonlinear loop is exploited to design a specific algorithm that dynamically adapts the cantilever free oscillation amplitude to sudden variations of the sample profile. In imaging applications this additional control action significantly reduces the tip-sample interaction force yet maintaining high image quality, thus resulting in a suitable setup for better preserving the state of soft and damageable samples such as biological specimens.

Riassunto. Sono più di dieci anni ormai che, con alterne vicende, “giochiamo” con il LEGO escogitando nuovi esperimenti per invogliare studenti d’ogni ordine e grado e semplici curiosi ad avvicinarsi alla scienza in modo divertente. Con il contributo del MIUR `e adesso disponibile un sito internet dedicato a questa attività di diffusione della cultura scientifica e tecnica attraverso il gioco. Abstract. More than ten years ago we started to “play” with LEGO to set up optical experiments for students of every age and amateur scientists. Since then, now and again, in spare time, we design new experiments. Now thanks to a financial support from MIUR a web site is available devoted to the diffusion of scientific and technical culture through ludic activity.