Ataluren improves myelopoiesis and neutrophil chemotaxis by restoring ribosome biogenesis and reducing p53 levels in Shwachman-Diamond syndrome cells.

Shwachman-Diamond syndrome (SDS) is characterized by neutropenia, exocrine pancreatic insufficiency and skeletal abnormalities. SDS bone marrow haematopoietic progenitors show increased apoptosis and impairment in granulocytic differentiation. Loss of Shwachman-Bodian-Diamond syndrome (SBDS) expression results in reduced eukaryotic 80S ribosome maturation. Biallelic mutations in the SBDS gene are found in ~90% of SDS patients, ~55% of whom carry the c.183-184TA>CT nonsense mutation. Several translational readthrough-inducing drugs aimed at suppressing nonsense mutations have been developed. One of these, ataluren, has received approval in Europe for the treatment of Duchenne muscular dystrophy. We previously showed that ataluren can restore full-length SBDS protein synthesis in SDS-derived bone marrow cells. Here, we extend our preclinical study to assess the functional restoration of SBDS capabilities in vitro and ex vivo. Ataluren improved 80S ribosome assembly and total protein synthesis in SDS-derived cells, restored myelopoiesis in myeloid progenitors, improved neutrophil chemotaxis in vitro and reduced neutrophil dysplastic markers ex vivo. Ataluren also restored full-length SBDS synthesis in primary osteoblasts, suggesting that its beneficial role may go beyond the myeloid compartment. Altogether, our results strengthened the rationale for a Phase I/II clinical trial of ataluren in SDS patients who harbour the nonsense mutation.

pH-sensing hybrid hydrogels for non-invasive metabolism monitoring in tumor spheroids.

The constant increase in cancer incidence and mortality pushes biomedical research towards the development of in vitro 3D systems able to faithfully reproduce and effectively probe the tumor microenvironment. Cancer cells interact with this complex and dynamic architecture, leading to peculiar tumor-associated phenomena, such as acidic pH conditions, rigid extracellular matrix, altered vasculature, hypoxic condition. Acidification of extracellular pH, in particular, is a well-known feature of solid tumors, correlated to cancer initiation, progression, and resistance to therapies. Monitoring local pH variations, non-invasively, during cancer growth and in response to drug treatment becomes extremely important for understanding cancer mechanisms. Here, we describe a simple and reliable pH-sensing hybrid system, based on a thermoresponsive hydrogel embedding optical pH sensors, that we specifically apply for non-invasive and accurate metabolism monitoring in colorectal cancer (CRC) spheroids. First, the physico-chemical properties of the hybrid sensing platform, in terms of stability, rheological and mechanical properties, morphology and pH sensitivity, were fully characterized. Then, the proton gradient distribution in the spheroids proximity, in the presence or absence of drug treatment, was quantified over time by time lapse confocal light scanning microscopy and automated segmentation pipeline, highlighting the effects of the drug treatment in the extracellular pH. In particular, in the treated CRC spheroids the acidification of the microenvironment resulted faster and more pronounced over time. Moreover, a pH gradient distribution was detected in the untreated spheroids, with more acidic values in proximity of the spheroids, resembling the cell metabolic features observed in vivo in the tumor microenvironment. These findings promise to shed light on mechanisms of regulation of proton exchanges by cellular metabolism being essential for the study of solid tumors in 3D in vitro models and the development of personalized medicine approaches.

A thermo-sensitive chitosan/pectin hydrogel for long-term tumor spheroid culture.

Hydrogels represent a key element in the development of in vitro tumor models, by mimicking the typical 3D tumor architecture in a physicochemical manner and allowing the study of tumor mechanisms. Here we developed a thermo-sensitive, natural polymer-based hydrogel, where chitosan and pectin were mixed and, after a weak base-induced chitosan gelation, a stable semi-Interpenetrating Polymer Network formed. This resulted thermo-responsive at 37 °C, injectable at room temperature, stable up to 6 weeks in vitro, permeable to small/medium-sized molecules (3 to 70 kDa) and suitable for cell-encapsulation. Tunable mechanical and permeability properties were obtained by varying the polymer content. Optimized formulations successfully supported the formation and growth of human colorectal cancer spheroids up to 44 days of culture. The spheroid dimension and density were influenced by the semi-IPN stiffness and permeability. These encouraging results would allow the implementation of faithful tumor models for the study and development of personalized oncological treatments.

Thermosensitive chitosan-based hydrogels supporting motor neuron-like NSC-34 cell differentiation.

Motor neuron diseases are neurodegenerative diseases that predominantly affect the neuromuscular system. To date, there are no valid therapeutic treatments for such diseases, and the classical experimental models fail in faithfully reproducing the pathological mechanisms behind them. In this regard, the use of three-dimensional (3D) culture systems, which more closely reproduce the native in vivo environment, can be a promising approach. Hydrogel-based systems are among the most used materials to reproduce the extracellular matrix, featuring an intrinsic similarity with its physiological characteristics. In this study, we developed a thermosensitive chitosan-based hydrogel combined with ß-glycerophosphate (ßGP) and sodium hydrogen carbonate (SHC), which give the system optimal mechanical properties and injectability, inducing the hydrogel sol-gel transition at 37 °C. An ad hoc protocol for the preparation of the hydrogel was established in order to obtain a highly homogeneous system, leading to reproducible physicochemical characteristics and easy cell encapsulation. All formulations supported the viability of a neuroblastoma/spinal cord hybrid cell line (NSC-34) beyond two weeks of culture and enabled cell differentiation towards a motor neuron-like morphology, characterized by the presence of extended neurites. Based on our results, these hydrogels represent excellent candidates for establishing 3D in vitro models of motor neuron diseases.

A microfabricated multi-compartment device for neuron and Schwann cell differentiation.

Understanding the complex communication between different cell populations and their interaction with the microenvironment in the central and peripheral nervous systems is fundamental in neuroscience research. The development of appropriate in vitro approaches and tools, able to selectively analyze and/or probe specific cells and cell portions (e.g., axons and cell bodies in neurons), driving their differentiation into specific cell phenotypes, has become therefore crucial in this direction. Here we report a multi-compartment microfluidic device where up to three different cell populations can be cultured in a fluidically independent circuit. The device allows cell migration across the compartments and their differentiation. We showed that an accurate choice of the device geometrical features and cell culture parameters allows to (1) maximize cell adhesion and proliferation of neuron-like human cells (SH-SY5Y cells), (2) control the inter-compartment cell migration of neuron and Schwann cells, (3) perform long-term cell culture studies in which both SH-SY5Y cells and primary rat Schwann cells can be differentiated towards specific phenotypes. These results can lead to a plethora of in vitro co-culture studies in the neuroscience research field, where tuning and investigating cell-cell and cell-microenvironment interactions are essential.

PC12 neuron-like cell response to electrospun poly( 3-hydroxybutyrate) substrates.

In the last decade, the importance of topographic properties of extracellular environments has been shown to be essential to addressing cell response, especially when replacing damaged tissues with functional constructs obtained in vitro. In the current study, densely packed sub-micron poly(3-hydroxybutyrate) (PHB) fibres were electrospun with random and parallel orientations. PC12 pheochromocytoma cells that mimic central dopaminergic neurons and represent a model for neuronal differentiation were cultured on collagen-coated fibres to evaluate cell response dependence on substrate topography. Cell adhesion, viability and proliferation, as well as dopamine production were evaluated after three days since seeding. Cell differentiation was examined in terms of neurite number, orientation and length 6 days after administration of nerve growth factor (NGF). Results showed that proliferating PC12 cells secreted a higher quantity of dopamine on fibres with respect to control cultures and as a result, a possible use of PHB fibres was considered for cell transplantation in the central nervous system when local production of dopamine is impaired. Differentiated PC12 cells were characterized by highly aligned and longer neurites on parallel PHB fibres with respect to random fibres, thereby demonstrating the suitability of parallel PHB fibres for further studies in peripheral nervous system regeneration.

Recent advances in application of biosensors in tissue engineering.

Biosensors research is a fast growing field in which tens of thousands of papers have been published over the years, and the industry is now worth billions of dollars. The biosensor products have found their applications in numerous industries including food and beverages, agricultural, environmental, medical diagnostics, and pharmaceutical industries and many more. Even though numerous biosensors have been developed for detection of proteins, peptides, enzymes, and numerous other biomolecules for diverse applications, their applications in tissue engineering have remained limited. In recent years, there has been a growing interest in application of novel biosensors in cell culture and tissue engineering, for example, real-time detection of small molecules such as glucose, lactose, and H2O2 as well as serum proteins of large molecular size, such as albumin and alpha-fetoprotein, and inflammatory cytokines, such as IFN-g and TNF-α. In this review, we provide an overview of the recent advancements in biosensors for tissue engineering applications.

Organs-on-a-chip: a new tool for drug discovery.

INTRODUCTION: The development of emerging in vitro tissue culture platforms can be useful for predicting human response to new compounds, which has been traditionally challenging in the field of drug discovery. Recently, several in vitro tissue-like microsystems, also known as 'organs-on-a-chip', have emerged to provide new tools for better evaluating the effects of various chemicals on human tissue. AREAS COVERED: The aim of this article is to provide an overview of the organs-on-a-chip systems that have been recently developed. First, the authors introduce single-organ platforms, focusing on the most studied organs such as liver, heart, blood vessels and lung. Later, the authors briefly describe tumor-on-a-chip platforms and highlight their application for testing anti-cancer drugs. Finally, the article reports a few examples of other organs integrated in microfluidic chips along with preliminary multiple-organs-on-a-chip examples. The article also highlights key fabrication points as well as the main application areas of these devices. EXPERT OPINION: This field is still at an early stage and major challenges need to be addressed prior to the embracement of these technologies by the pharmaceutical industry. To produce predictive drug screening platforms, several organs have to be integrated into a single microfluidic system representative of a humanoid. The routine production of metabolic biomarkers of the organ constructs, as well as their physical environment, have to be monitored prior to and during the delivery of compounds of interest to be able to translate the findings into useful discoveries.

Composite electrospun nanofibers for influencing stem cell fate.

The design of new bioactive materials, provided with "instructive properties" and able to regulate stem cell behavior, is the goal for several research groups involved in tissue engineering. This new function, commonly reserved for growth factors, can lead to the development of a new class of implantable scaffolds, useful for accelerating tissue regeneration in a controlled manner. In this scenario, the likely most versatile and effective tools for the realization of such scaffolds are based on nano- and microtechnology. Here, we show how exploiting the electrostatic spinning (ES) technique for producing a nanofibrillar composite structure, by mimicking topographically the extracellular matrix environment, can influence the fate of human bone marrow mesenchymal stem cells, inducing osteogenic differentiation in the absence of chemical treatments or cellular reprogramming. Basic cues on the choice of the materials and useful experimental instructions for realizing composite nanofibrous scaffolds will be given as well as fundamental tips.

Dental applications of nanostructured bioactive glass and its composites.

To improve treatments of bone or dental trauma and diseases such as osteoporosis, cancer, and infections, scientists who perform basic research are collaborating with clinicians to design and test new biomaterials for the regeneration of lost or injured tissue. Developed some 40 years ago, bioactive glass (BG) has recently become one of the most promising biomaterials, a consequence of discoveries that its unusual properties elicit specific biological responses inside the body. Among these important properties are the capability of BG to form strong interfaces with both hard and soft tissues, and its release of ions upon dissolution. Recent developments in nanotechnology have introduced opportunities for materials sciences to advance dental and bone therapies. For example, the applications for BG expand as it becomes possible to finely control structures and physicochemical properties of materials at the molecular level. Here, we review how the properties of these materials have been enhanced by the advent of nanotechnology, and how these developments are producing promising results in hard-tissue regeneration and development of innovative BG-based drug delivery systems.

Osteoinduction of human mesenchymal stem cells by bioactive composite scaffolds without supplemental osteogenic growth factors.

The development of a new family of implantable bioinspired materials is a focal point of bone tissue engineering. Implant surfaces that better mimic the natural bone extracellular matrix, a naturally nano-composite tissue, can stimulate stem cell differentiation towards osteogenic lineages in the absence of specific chemical treatments. Herein we describe a bioactive composite nanofibrous scaffold, composed of poly-caprolactone (PCL) and nano-sized hydroxyapatite (HA) or beta-tricalcium phosphate (TCP), which was able to support the growth of human bone marrow mesenchymal stem cells (hMSCs) and guide their osteogenic differentiation at the same time. Morphological and physical/chemical investigations were carried out by scanning, transmission electron microscopy, Fourier-transform infrared (FTIR) spectroscopy, mechanical and wettability analysis. Upon culturing hMSCs on composite nanofibers, we found that the incorporation of either HA or TCP into the PCL nanofibers did not affect cell viability, meanwhile the presence of the mineral phase increases the activity of alkaline phosphatase (ALP), an early marker of bone formation, and mRNA expression levels of osteoblast-related genes, such as the Runt-related transcription factor 2 (Runx-2) and bone sialoprotein (BSP), in total absence of osteogenic supplements. These results suggest that both the nanofibrous structure and the chemical composition of the scaffolds play a role in regulating the osteogenic differentiation of hMSCs.

Rapid nested-PCR for tyrosinase gene detection on chip.

The availability of non-invasive, fast and sensitive technologies for detection of circulating cancer cells is still a critical need of clinical oncology, particularly for diagnosis of aggressive and highly metastatic tumors, like malignant melanoma. Here we present the first nested polymerase chain reaction process carried out by a microfabricated, hybrid plastic-glass microfluidic chip on the tyrosinase gene, a predictive marker for melanoma diagnosis. The device is a hybrid system consisting of a glass microchannel embedded in an elastomeric matrix, and operating in flow-oscillating modality on a droplet of biological sample. The convection heat transfer and the temperature distribution inside the carrier fluid in the device are investigated. The oil responds to temperature changes with a characteristic time around 53 s, and exhibits three different thermal gradients along the capillary, with temperature variations below 4°C in correspondence of heater electrodes. The sample heating/cooling rates in the chip are as high as 16°C/s, allowing rapid processes. The nested polymerase chain reaction process is performed in less than 50 min, namely more than four times faster than in a standard thermocycler. The rapidity of the analysis method, combined with the simple and low-cost fabrication, reduced sample evaporation, and flexibility of the overall microfluidic platform, make it promising for the detection of events of tumor spreading.