Infrared nanospectroscopy depth-dependent study of modern materials: morpho-chemical analysis of polyurethane/fibroin binary meshes

Infrared scattering-type scanning near-field optical microscopy (IR s-SNOM) and imaging is here exploited together with attenuated total reflection (ATR) IR imaging and scanning electron microscopy (SEM) to depict the chemical composition of fibers in hybrid electrospun meshes. The focus is on a recently developed bio-hybrid material for vascular tissue engineering applications, named Silkothane & REG;, obtained in the form of nanofibrous matrices from the processing of a silk fibroin-polyurethane (SFPU) blend via electrospinning. Morphology and chemistry of single fibers, at both surface and subsurface level, have been successfully characterized with nanoscale resolution, taking advantage of the IR s-SNOM capability to portray the nanoscale depth profile of this modern material working at diverse harmonics of the signal. The applied methodology allowed to describe the superficial characteristics of the mesh up to a depth of about 100 nm, showing that SF and PU do not tend to co-aggregate to form hybrid fibers, at least at the length scale of hundreds of nanometers, and that subdomains other than the fibrillar ones can be present. More generally, in the present contribution, the depth profiling capabilities of IR s-SNOM, so far theoretically predicted and experimentally proven only on model systems, have been corroborated on a real material in its natural conditions with respect to production, opening the room for the exploitation of IR s-SNOM as valuable technique to support the production and the engineering of nanostructured materials by the precise understanding of their chemistry at the interface with the environment

Prokineticin System Is a Pharmacological Target to Counteract Pain and Its Comorbid Mood Alterations in an Osteoarthritis Murine Model

Osteoarthritis (OA) is the most prevalent joint disease associated with chronic pain. OA pain is often accompanied by mood disorders. We addressed the role of the Prokineticin (PK) system in pain and mood alterations in a mice OA model induced with monosodium iodoacetate (MIA). The effect of a PK antagonist (PC1) was compared to that of diclofenac. C57BL/6J male mice injected with MIA in the knee joint were characterized by allodynia, motor deficits, and fatigue. Twenty-eight days after MIA, in the knee joint, we measured high mRNA of PK2 and its receptor PKR1, pro-inflammatory cytokines, and MMP13. At the same time, in the sciatic nerve and spinal cord, we found increased levels of PK2, PKR1, IL-1β, and IL-6. These changes were in the presence of high GFAP and CD11b mRNA in the sciatic nerve and GFAP in the spinal cord. OA mice were also characterized by anxiety, depression, and neuroinflammation in the prefrontal cortex and hippocampus. In both stations, we found increased pro-inflammatory cytokines. In addition, PK upregulation and reactive astrogliosis in the hippocampus and microglia reactivity in the prefrontal cortex were detected. PC1 reduced joint inflammation and neuroinflammation in PNS and CNS and counteracted OA pain and emotional disturbances

Tyr78Phe Transthyretin Mutation with Predominant Motor Neuropathy as the Initial Presentation

Transthyretin (TTR) amyloidosis, the most frequent form of hereditary amyloidosis, is caused by dominant mutations in the TTR gene. More than 100 mutations have been identified. Clinical manifestations of TTR amyloidosis are usually induced by extracellular amyloid deposition in several organs. The major neurological manifestation is motor-sensory neuropathy associated with dysautonomic impairment. Here, we describe a 63-year-old man who came to our institution due to a suspected motor neuron disease. During a 4-year follow-up period, he underwent extensive clinical examination, electromyographic studies, sural nerve biopsy and TTR gene analysis by direct sequencing. Despite the predominant motor involvement, the detailed clinical examination also showed some mild sensory and dysautonomic signs. In addition, his clinical and family history included multiorgan disorders, such as carpal tunnel syndrome, as well as conditions with cardiac, renal, eye, and hepatic involvement. The sural nerve biopsy disclosed amyloid deposition, and the sequence analysis of the TTR gene detected a heterozygous Tyr78Phe substitution. The TTR gene variant found in our patient had only been described once so far, in a French man of Italian origin presenting with late-onset peripheral neuropathy and bilateral carpal tunnel syndrome. The predominant motor involvement presented by our patient is an uncommon occurrence and demonstrates the clinical heterogeneity of TTR amyloidosis

Changes Induced by Exposure of the Human Lung to Glass Fiber–Reinforced Plastic

The inhalation of glass dusts mixed in resin, generally known as glass fiber–reinforced plastic (GRP), represents a little-studied occupational hazard. The few studies performed have highlighted nonspecific lung disorders in animals and in humans. In the present study we evaluated the alteration of the respiratory system and the pathogenic mechanisms causing the changes in a group of working men employed in different GRP processing operations and exposed to production dusts. The study was conducted on a sample of 29 male subjects whose mean age was 37 years and mean length of service 11 years. All of the subjects were submitted to a clinical check-up, basic tests, and bronchoalveolar lavage (BAL); microscopic studies and biochemical analysis were performed on the BAL fluid. Tests of respiratory function showed a large number of obstructive syndromes; scanning electron microscopy highlighted qualitative and quantitative alterations of the alveolar macrophages; and transmission electron microscopy revealed the presence of electron-dense cytoplasmatic inclusions indicating intense and active phlogosis (external inflammation). Biochemical analyses highlighted an increase in protein content associated with alterations of the lung oxidant/antioxidant homeostasis. Inhalation of GRP, independent of environmental concentration, causes alterations of the cellular and humoral components of pulmonary interstitium; these alterations are identified microscopically as acute alveolitis

Modulation of Glial Cell Functions by the Gut–Brain Axis: A Role in Neurodegenerative Disorders and Pain Transmission

Studies on host microbiota and their interactions with the central nervous system (CNS) have grown considerably in the last decade. Indeed, it has been widely demonstrated that dysregulations of the bidirectional gut–brain crosstalk are involved in the development of several pathological conditions, including chronic pain. In addition, the activation of central and peripheral glial cells is also implicated in the pathogenesis and progression of pain and other neurodegenerative disorders. Recent preclinical findings suggest that the gut microbiota plays a pivotal role in regulating glial maturation, morphology and function, possibly through the action of different microbial metabolites, including the most studied short-chain fatty acids (SCFAs). Moreover, altered microbiota composition has been reported in CNS disorders characterized by glial cell activation. In this review, we discuss recent studies showing the role of the gut microbiota and the effects of its depletion in modulating the morphology and function of glial cells (microglia and astrocytes), and we hypothesize a possible role for glia–microbiota interactions in the development and maintenance of chronic pain

In vitro dynamic culture of cell-biomaterial constructs

In the last years, the interest in bioreactors is constantly increasing in several fields of bioengineering and for various applications, thanks to the progresses in technology, engineering, stem cell biology, and material science. At different levels and for various applications bioreactors can be used in research as actuator systems (i.e., to apply complex stimuli to cells in dynamic culture), as model systems to study the development of biological tissues, or as systems to monitor the developmental parameters. Exploiting the potentiality of bioreactors, the biomedical industry invested in developing reliable and automated devices for 2D or 3D cell culture, having specific features such as modularity, scalability, and process traceability, to make bioreactors essential tools for the development of safe and reproducible biological constructs and for the study of new therapeutic drugs. Thanks to the efforts of researchers and industry, bioreactors can potentially be applied in the clinics for the development of products for Advanced Therapies, acting as automatic platforms for the economically and clinically sustainable fabrication of bioprocesses and cell products. This chapter is focused on the analysis of the translation of bioreactors from research to clinical application, providing information on the fundamental features and issues, as well as practical examples of devices design at each stage of development

Dendritic cell-endothelial cell cross-talk in angiogenesis.

Dendritic cells (DCs) are professional antigen-presenting cells that have a pivotal role in the onset and regulation of adaptive immune responses. DCs have the ability to regulate inflammation through their capacity to release cytokines and chemokines and kill pathogens, which they share with other phagocytes. Recent observations have shown that different DC subsets produce and release various pro- and anti-angiogenic mediators depending on their activation status and cytokine milieu. In particular, alternatively activated DCs exert a potent pro-angiogenic activity that is mediated by the prototypic angiogenic growth factor vascular endothelial growth factor-A (VEGF-A). In turn, pro- and anti-angiogenic mediators can affect the biology of DCs, modulating their differentiation and maturation. Finally, DCs can trans-differentiate into endothelial-like cells, possibly contributing to vasculogenesis in the adult. Thus, DCs might exert an important impact on the neovascularization process in different physiopathological conditions

Mitochondrial Fusion Proteins and Human Diseases

Mitochondria are highly dynamic, complex organelles that continuously alter their shape, ranging between two opposite processes, fission and fusion, in response to several stimuli and the metabolic demands of the cell. Alterations in mitochondrial dynamics due to mutations in proteins involved in the fusion-fission machinery represent an important pathogenic mechanism of human diseases. The most relevant proteins involved in the mitochondrial fusion process are three GTPase dynamin-like proteins: mitofusin 1 (MFN1) and 2 (MFN2), located in the outer mitochondrial membrane, and optic atrophy protein 1 (OPA1), in the inner membrane. An expanding number of degenerative disorders are associated with mutations in the genes encoding MFN2 and OPA1, including Charcot-Marie-Tooth disease type 2A and autosomal dominant optic atrophy. While these disorders can still be considered rare, defective mitochondrial dynamics seem to play a significant role in the molecular and cellular pathogenesis of more common neurodegenerative diseases, for example, Alzheimer’s and Parkinson’s diseases. This review provides an overview of the basic molecular mechanisms involved in mitochondrial fusion and focuses on the alteration in mitochondrial DNA amount resulting from impairment of mitochondrial dynamics. We also review the literature describing the main disorders associated with the disruption of mitochondrial fusion

Interstitial Perfusion Culture with Specific Soluble Factors Inhibits Type I Collagen Production from Human Osteoarthritic Chondrocytes in Clinical-Grade Collagen Sponges

<div><p>Articular cartilage has poor healing ability and cartilage injuries often evolve to osteoarthritis. Cell-based strategies aiming to engineer cartilaginous tissue through the combination of biocompatible scaffolds and articular chondrocytes represent an alternative to standard surgical techniques. In this context, perfusion bioreactors have been introduced to enhance cellular access to oxygen and nutrients, hence overcoming the limitations of static culture and improving matrix deposition. Here, we combined an optimized cocktail of soluble factors, the BIT (BMP-2, Insulin, Thyroxin), and clinical-grade collagen sponges with a bidirectional perfusion bioreactor, namely the oscillating perfusion bioreactor (OPB), to engineer <i>in vitro</i> articular cartilage by human articular chondrocytes (HACs) obtained from osteoarthritic patients. After amplification, HACs were seeded and cultivated in collagen sponges either in static or dynamic conditions. Chondrocyte phenotype and the nature of the matrix synthesized by HACs were assessed using western blotting and immunohistochemistry analyses. Finally, the stability of the cartilaginous tissue produced by HACs was evaluated <i>in vivo</i> by subcutaneous implantation in nude mice. Our results showed that perfusion improved the distribution and quality of cartilaginous matrix deposited within the sponges, compared to static conditions. Specifically, dynamic culture in the OPB, in combination with the BIT cocktail, resulted in the homogeneous production of extracellular matrix rich in type II collagen. Remarkably, the production of type I collagen, a marker of fibrous tissues, was also inhibited, indicating that the association of the OPB with the BIT cocktail limits fibrocartilage formation, favoring the reconstruction of hyaline cartilage.</p></div