In this work, co-extrusion Direct Ink Writing 3D printing technology was used to produce 316L-sphene (CaTiSiO5) core-shell scaffolds, with the main objective of developing a new generation of bone substitutes capable of mimicking the mechanical behavior of natural bone under compressive load. The presence of a ductile core in the struts of bioceramics scaffolds increases the strain energy density, preventing brittle fracture and enabling a more graceful failure. The proposed CO2-free synthesis route for sphene, without decomposition byproducts, preserves the intrinsic ductility of the stainless-steel core while maintaining a level of porosity suitable for promoting the bioactive properties of the bioceramic shell. Compression tests on the 3D-printed bio-scaffolds demonstrated that the incorporation of a ductile metallic core alters the fracture behavior under compressive loading, resulting in a fourfold increase in strain energy density compared with fully ceramic scaffolds, while preserving a high porosity (similar to 70%). The in-vitro studies confirmed the bioactive behavior of both sphene and 316L-sphene scaffolds, as evidenced by the formation of calcium phosphate phases and apatite-like precipitates after immersion in SBF. ICP-MS analyses revealed very low metal ion release (Fe < 0.3 ppm; Ti < 20 ppb; Cr < 10 ppb; Ni < 5 ppb; Mn < 100 ppb). Cytotoxicity tests showed cell viability consistently above the 70% threshold defined by ISO 10993-5, confirming the absence of cytotoxic effects. These results represent a first step toward the development of a new generation of composite bio-scaffolds, in which the combination of materials with both structural and functional properties may enable the fabrication of patient-specific implants. Further studies will require the optimization of the core-shell interface strength, the study of their mechanical behavior under dynamic loading conditions, and the assessment of the effect of scaffold architecture on their bioactive response.

BackgroungMelanoma remains one of the most lethal cancers due to its high metastatic capacity and resistance to current therapies, emphasizing the need for novel antineoplastic agents, including bioactive molecules from marine organisms, as potential sources of anticancer compound.AimsThis study aimed to investigate, for the first time, the anticancer potential of Hallachrome (HC), an anthraquinone derived from the marine polychaete Halla parthenopeia, in A375 melanoma cells.MethodsA375 melanoma cells were treated with HC in vitro to evaluate its impact on cell proliferation, migration, and morphology. Proteomic analysis was carried out to identify differentially expressed proteins in order to elucidate the biological processes modulated by the treatment. Mitochondrial morphology and function were analyzed by confocal microscopy and oxygen consumption rate, respectively, while mitochondrial membrane potential, intracellular reactive oxygen species (ROS) levels and GSH/GSSG ratio were evaluated by fluorescence-based assays.ResultsHC treatment significantly reduced melanoma cell proliferation and migration in vitro, accompanied by morphological alteration and apoptosis death. Proteomic profiling revealed differential expression of several proteins involved, for example, in membrane and mitochondria organization. Moreover, HC treatment induced severe mitochondrial dysfunction, characterized by ATP depletion, mitochondrial membrane depolarization, fragmentation of the mitochondrial network, altered intracellular ROS level and GSH/GSSG ratio.DiscussionThese findings suggest that HC inhibits melanoma cell survival by disrupting mitochondrial homeostasis and inducing energetic stress. Given the pivotal role of mitochondria in cancer progression, the ability of HC to interfere with mitochondrial functions supports its potential as a promising anticancer agent.

Ectopic calcification (EC) is a still untreatable hallmark of genetic and acquired diseases. Recent findings suggested that minocycline (Mi) retains several biological impacts beyond its antibiotic function, including anti-inflammatory and antioxidant effects, modulation of the nucleus-mitochondria crosstalk, inhibition of the stress-induced over-activation of the poly(ADP-ribose) polymerase-1 (PARP-1) involved in DNA repair and possibly in EC. Interestingly, some of these pathways are involved in the pathogenesis of Pseudoxanthoma elasticum (PXE), a genetic disease considered a paradigm of EC. Notably, data have been provided indicating an association between anti-calcifying effects of Mi and PARP-1 inhibition, although the involved pathways remain unclear. Therefore, we have investigated through integrated approaches which molecular pathways can be involved in the proposed anti-calcifying potential of Mi. This study shows that Mi inhibits mineralization acting both in the extracellular (i.e., acellular model of coacervated elastin fibrils) and in the intracellular (i.e., cultured PXE dermal fibroblasts) environments. Results support and broaden the calcification inhibitory capability of Mi suggesting that, also in PARP-1 independent in vitro models of calcification, mineral deposition can be inhibited through the calcium chelating properties of the drug and the action on mitochondria mainly preserving Δψm although oxidative stress is not affected.

In the cardiovascular system, elastic fibres exert a fundamental role providing the long-range elasticity required for physiological functions. Elastic fibres are complex in composition and structure containing, in addition to elastin, a wide range of matrix components, such as microfibrillar proteins, calcium-binding proteins and glycosaminoglycans. Changes in composition and/or structure can affect the biomechanics of the tissue as well as the intrinsic affinity of elastin for Ca2+ ions. Mineralization of elastic fibres can occur in genetic as well as in age-related chronic diseases. In cardiovascular diseases, for instance, calcification represents an integral part of the pathogenetic process, although the regulatory mechanisms are not completely understood. Therefore, a focus is given on elastin synthesis and assembly, on elastic fibre components and on elastin degradation. Moreover, the role and the impact of altered composition and supramolecular organization of elastic fibres are described in the context of the calcified cardiovascular system. Finally, some in vitro and in vivo models of elastic fibres calcification are presented and discussed.

BACKGROUND:Despite their life-saving potential, tissue engineering approaches for the treatment of extensive tracheal and bronchial defects still face significant limitations. A major challenge is the inability to regenerate a functional airway epithelium containing the appropriate amount of stem cells required for long-term tissue renewal following transplantation of the bioengineered graft. In this scenario, extensive cell culture characterization, validation assays and quality controls are needed to guide each step of the regeneration process. METHODS:Stem cell depletion is often due to suboptimal culture conditions, therefore we tested the ability of a clinical-grade culture system to support the safe and efficient in vitro expansion and differentiation of primary human tracheal and bronchial epithelial cells. Single-cell clonal analysis was used to unravel the heterogeneity of airway basal cells and to understand tissue-specific regeneration and differentiation mechanisms. Functional assays were used to investigate the wound healing ability and tightness of the regenerated epithelium under the selected culture conditions. RESULTS:Primary tracheobronchial epithelial cells showed an impressive proliferative potential, allowing the regeneration of a mature and functional epithelium without immortalisation events. Analysis at the single cell level allowed the identification of the subpopulation of basal cells endowed with in vitro self-renewal, distinguishing them from transient amplifying cells. This approach has further defined the hierarchy of cellular differentiation and its correlation with regenerative and differentiation potential. CONCLUSIONS:Our results show that primary airway epithelial cell cultures can maintain stem cells together with their differentiation lineages in vitro. Airway cells can be safely and effectively used in autologous tissue engineering approaches when cultured under appropriate and well-standardised conditions. In addition to the validation assays proposed for the development of new advanced therapy products, this study outlines possible quality controls to enhance therapeutic success and maximise patient safety in future clinical applications.

Purpose:To investigate the choroid in patients affected by pseudoxanthoma elasticum (PXE)-related retinopathy using the choroidal vascularity index (CVI). Methods:PXE patients and controls were recruited at the Eye Clinic in Florence. High-resolution imaging optical coherence tomography (OCT) scans (12 × 9 mm) of 32 PXE patients and 20 age-matched controls were examined. Images were binarized using the ImageJ software, and subfoveal choroidal thickness (SFCT), luminal area (LA), stromal area (SA), total choroidal area (TCA), and CVI were measured. Results:Sixty-four eyes of 32 PXE patients (mean age 45.65 ± 16.12; range 14-69) and 40 eyes of 20 controls (mean age 47.3 ± 13.7; range 18-71) were included in the study. SFCT was significantly lower in PXE patients compared to controls. The LA, SA, and TCA of the PXE patients were significantly reduced in comparison with those obtained for controls (p = 0.012, p < 0.001, and p = 0.001, respectively). On the contrary, the CVI did not significantly differ between patients and controls (p = 0.744). In young subjects, differences regarding SFCT, LA, SA, TCA, and CVI were not detected between PXE patients and healthy controls (p = 0.170, p = 0.990, p = 0.264, p = 0.351, and p = 0.487, respectively). Conclusion:In PXE-related retinopathy, choroidal impairment appears progressive with age, and there is a simultaneous, proportional impairment of both the stromal and vascular components of the choroid.

B cells have emerged as central players in the tumor microenvironment (TME) of non-small cell lung cancer (NSCLC). However, although there is clear evidence for their involvement in cancer immunity, scanty data exist on the characterization of B cell phenotypes, bioenergetic profiles and possible interactions with T cells in the context of NSCLC. In this study, using polychromatic flow cytometry, mass cytometry, and spatial transcriptomics we explored the intricate landscape of B cell phenotypes, bioenergetics, and their interaction with T cells in NSCLC. Our analysis revealed that TME contains diverse B cell clusters, including VISTA+ Bregs, with distinct metabolic and functional profiles. Target liquid chromatography-tandem mass spectrometry confirmed the expression of VISTA on B cells. VISTA+ Bregs displayed high metabolic demand and were able to produce different cytokines, including interleukin (IL)-10, transforming growth factor (TGF)-β, IL-6, tumor necrosis factor (TNF), and granulocyte–macrophage colony-stimulating factor (GM-CSF). Spatial analysis showed colocalization of B cells with CD4+/CD8+ T lymphocytes in TME. The computational analysis of intercellular communications that links ligands to target genes, performed by NicheNet, predicted B-T interactions via VISTA-PSGL-1 axis. Colocalization analyses revealed that PSGL-1 T cells and VISTA+ B cells are adjacent in the TME. Notably, tumor infiltrating CD8+ T cells expressing PSGL-1 exhibited enhanced metabolism and cytotoxicity. In NSCLC patients, prediction analysis performed by PENCIL revealed the presence of an association between PSGL-1+CD8+ T cells and VISTA+ Bregs with lung recurrence. Our findings suggest a potential interaction between Bregs and T cells through the VISTA-PSGL-1 axis, that could influence NSCLC recurrence.

Pseudoxanthoma elasticum (PXE) is a rare disease characterized by ectopic calcification, however, despite the widely spread effect of pro/anti-calcifying systemic factors associated with this genetic metabolic condition, it is not known why elastic fibers in the same patient are mainly fragmented or highly mineralized in clinically unaffected (CUS) and affected (CAS) skin, respectively. Cellular morphology and secretome are investigated in vitro in CUS and CAS fibroblasts. Here we show that, compared to CUS, CAS fibroblasts exhibit: a) differently distributed and organized focal adhesions and stress fibers; b) modified cell-matrix interactions (i.e., collagen gel retraction); c) imbalance between matrix metalloproteinases and tissue inhibitor of metalloproteinases; d) differentially expressed pro- and anti-calcifying proteoglycans and elastic-fibers associated glycoproteins. These data emphasize that in the development of pathologic mineral deposition fibroblasts play an active role altering the stability of elastic fibers and of the extracellular matrix milieu creating a local microenvironment guiding the level of matrix remodeling at an extent that may lead to degradation (in CUS) or to degradation and calcification (in CAS) of the elastic component. In conclusion, this study contributes to a better understanding of the mechanisms of the mineral deposition that can be also associated with several inherited or age-related diseases (e.g., diabetes, atherosclerosis, chronic kidney diseases).

Fibroblasts are typical mesenchymal cells widely distributed throughout the human body where they (1) synthesise and maintain the extracellular matrix, ensuring the structural role of soft connective tissues; (2) secrete cytokines and growth factors; (3) communicate with each other and with other cell types, acting as signalling source for stem cell niches; and (4) are involved in tissue remodelling, wound healing, fibrosis, and cancer. This review focuses on the developmental heterogeneity of dermal fibroblasts, on their ability to sense changes in biomechanical properties of the surrounding extracellular matrix, and on their role in aging, in skin repair, in pathologic conditions and in tumour development. Moreover, we describe the use of fibroblasts in different models (e.g., in vivo animal models and in vitro systems from 2D to 6D cultures) for tissue bioengineering and the informative potential of high-throughput assays for the study of fibroblasts under different disease contexts for personalized healthcare and regenerative medicine applications.

IntroductionOne of the hallmarks of Parkinsońs Disease (PD) is oxidative distress, leading to mitochondrial dysfunction and neurodegeneration. Insulin-like growth factor II (IGF-II) has been proven to have antioxidant and neuroprotective effects in some neurodegenerative diseases, including PD. Consequently, there isgrowing interest in understanding the different mechanisms involved in the neuroprotective effect of this hormone.ObjectivesTo clarify the mechanism of action of IGF-II involved in the protective effect of this hormone.MethodsThe present study was carried out on a cellular model PD based on the incubation of dopaminergic cells (SN4741) in a culture with the toxic 1-methyl-4-phenylpyridinium (MPP+), in the presence of IGF-II. This model undertakes proteomic analyses in order to understand which molecular cell pathways might be involved in the neuroprotective effect of IGF-II. The most important proteins found in the proteomic study were tested by Western blot, colorimetric enzymatic activity assay and immunocytochemistry. Along with the proteomic study, mitochondrial morphology and function were also studied by transmission electron microscopy and oxygen consumption rate. The cell cycle was also analysed using 7AAd/BrdU staining, and flow cytometry.ResultsThe results obtained indicate that MPP+, MPP++IGF-II treatment and IGF-II, when compared to control, modified the expression of 197, 246 proteins and 207 respectively. Some of these proteins were found to be involved in mitochondrial structure and function, and cell cycle regulation. Including IGF-II in the incubation medium prevents the cell damage induced by MPP+, recovering mitochondrial function and cell cycle dysregulation, and thereby decreasing apoptosis.ConclusionIGF-II improves mitochondrial dynamics by promoting the association of Mitofilin with mitochondria, regaining function and redox homeostasis. It also rebalances the cell cycle, reducing the amount of apoptosis and cell death by the regulation of transcription factors, such as Checkpoint kinase 1.

IntroductionSystemic sclerosis (SSc) is a connective tissue disease at the interface between inflammation and autoimmunity progressively leading to diffuse microvascular and fibrotic involvement of the skin and of multiple internal organs. Approximately, 20-40% of SSc patients suffer from cutaneous calcinosis, a debilitating manifestation due to calcium salt deposition in soft connective tissues, causing pain, ulceration, infection, and deformities, responsible of severe functional limitations. Pathomechanisms are poorly understood as well as markers/molecules capable to predict the risk of patients to develop calcinosis.MethodsAn observational study was performed in 51 female patients, 25 with and 26 without calcinosis to compare clinical and laboratory parameters and to evaluate pro- and anti-calcifying circulating markers and the in vitro serum calcification potential (T50). Moreover, calcinosis samples were analyzed to characterize their mineral composition.Results and discussionData demonstrate statistically significant differences in the prevalence of clinical manifestations and ACA and Scl70 autoantibodies in SSc patient with calcinosis compared to those without calcinosis. In SSc patients with calcinosis, serum levels of BMP-4 are higher, fetuin A might be regarded as a potential circulating prognostic marker and a negative correlation was observed between T50 and the global score of clinical manifestations, suggesting a potential predictive role of pro- and anti-calcifying molecules in SSc patients. Furthermore, calcinosis samples were characterized by the co-existence of phosphate and carbonate minerals with different stability and solubility. Further investigations on circulating markers in larger patient cohorts, especially at the early stages and throughout the natural course of the disease, may clarify their pathogenetic role in the SSc-related cutaneous calcinosis.

Bioactive glasses (BGs) have emerged as invaluable resources for bone tissue engineering due to their remarkable properties such as bioactivity, resorbability, cell compatibility, and osteoconductivity. However, these materials exhibit certain limitations when subjected to high temperatures, for their tendency to crystallize, thus leading to diminished bioactivity, reduced mechanical strength, and altered dissolution kinetics. One promising approach to counteract this problem is to reduce the alkaline element content in BGs while simultaneously adding strontium and magnesium. Building on previous studies of Bio_MS, a recently developed experimental formulation, we investigated the contributions of strontium and magnesium to the thermal, mechanical, and biological properties of various bioactive glasses, including commercially available options. Differential thermal analysis, heating microscopy, X-ray diffractometry, environmental scanning electron microscopy, measurement of the Young's modulus, simulated body fluid testing, cytotoxicity tests, cell viability, growth, adhesion and morphology were assessed through an integrated approach and compared for a complete evaluation of BGs, and of doped BGs, also undergoing thermal treatments. The results demonstrated improved thermal, mechanical and biological behaviors of the magnesium-strontium-doped BGs, thus paving the way for the development of BGs with enhanced biomedical perspectives.

ABSTRACT The clinical results of tissue engineering approaches aimed at treating extensive tracheal or bronchial defects highlighted some impactful limitations, among which stand out the inability to regenerate a functional and self-renewing airway epithelium onto the bioengineered graft. This outcome is often caused by culture conditions unable to sustain the long-term proliferative and differentiative potential of primary airway epithelial cells, resulting in progressive stem cell exhaustion. Here, we examined the capacity of a clinical-grade culture system to safely and efficiently support the in vitro expansion of primary human tracheal and bronchial epithelial cells, regenerating a functional and fully differentiated airway epithelium. Moreover, we identified and characterized airway epithelial stem cells, unravelling the mechanisms that underlie tracheobronchial epithelium renewal. Finally, we suggested possible parameters to be employed as quality controls during critical stages of the tissue engineering process, thereby maximizing the likelihood of success of the treatment and prioritizing patients’ safety, crucial prerequisites before embarking on other clinical applications. GRAPHICAL ABSTRACT

Neutrophils are the most abundant myeloid cells in the blood and are a considerable immunological component of the tumor microenvironment. However, their functional importance has often been ignored, as they have always been considered a mono-dimensional population of terminally differentiated, short-living cells. During the last decade, the use of cutting-edge, single-cell technologies has revolutionized the classical view of these cells, unmasking their phenotypic and functional heterogeneity. In this review, we summarize the emerging concepts in the field of neutrophils in cancer, by reviewing the recent literature on the heterogeneity of both circulating neutrophils and tumor-associated neutrophils, as well as their possible significance in tumor prognosis and resistance to immune checkpoint inhibitors.

Animal models are currently used in several fields of biomedical research as useful alternatives to human-based studies. However, the obtained results do not always effectively translate into clinical applications, due to interspecies anatomical and physiological differences. Detailed comparability studies are therefore required to verify whether the selected animal species could be a representative model for the disease or for cellular process under investigation. This has proven to be fundamental to obtaining reliable data from preclinical studies. Among the different species, swine is deemed an excellent animal model in many fields of biological research, and has been largely used in respiratory medicine, considering the high homology between human and swine airways. In the context of in vitro studies, the validation of porcine airway epithelial cells as an alternative to human epithelial cells is crucial. In this paper, porcine and human tracheal and bronchial epithelial cells are compared in terms of in vivo tissue architecture and in vitro cell behaviour under standard and airlifted conditions, analyzing the regenerative, proliferative and differentiative potentials of these cells. We report multiple analogies between the two species, validating the employment of porcine airway epithelial cells for most in vitro preclinical studies, although with some limitations due to species-related divergences.

Abstract Animal models are currently used in several fields of biomedical research as useful alternatives to human-based studies. However, the obtained results do not always effectively translate into clinical applications, due to interspecies anatomical and physiological differences. Detailed comparability studies are therefore required to verify whether the selected animal species could be a representative model for the disease or for cellular process under investigation. This has proven to be fundamental to obtaining reliable data from preclinical studies. Among the different species, swine is deemed an excellent animal model in many fields of biological research, and they have been largely used in respiratory medicine, considering the high homology between human and swine airways. In the context of in vitro studies, the validation of porcine airway epithelial cells as an alternative to human epithelial cells is crucial. In this paper, porcine and human tracheal and bronchial epithelial cells are compared in terms of in vivo tissue architecture and in vitro cell behaviour under standard and airlifted conditions, analyzing the regenerative, proliferative and differentiative potentials of these cells. We report multiple analogies between the two species, validating the employment of porcine airway epithelial cells for most in vitro preclinical studies, although with some limitations due to species-related divergences.

Introduction: A regular physical training is known to contribute to preserve muscle mass and strength, maintaining structure and function of neural and vascular compartments and preventing muscle insulin resistance and inflammation. However, physical activity is progressively reduced during aging causing mobility limitations and poor quality of life. Although physical exercise for rehabilitation purposes (e.g., after fractures or cardiovascular events) or simply aiming to counteract the development of sarcopenia is frequently advised by physicians, nevertheless few data are available on the targets and the global effects on the muscle organ of adapted exercise especially if started at old age.Methods: To contribute answering this question for medical translational purposes, the proteomic profile of the gastrocnemius muscle was analyzed in 24-month-old mice undergoing adapted physical training on a treadmill for 12 weeks or kept under a sedentary lifestyle condition. Proteomic data were implemented by morphological and morphometrical ultrastructural evaluations.Results and Discussion: Data demonstrate that muscles can respond to adapted physical training started at old age, positively modulating their morphology and the proteomic profile fostering protective and saving mechanisms either involving the extracellular compartment as well as muscle cell components and pathways (i.e., mitochondrial processes, cytoplasmic translation pathways, chaperone-dependent protein refolding, regulation of skeletal muscle contraction). Therefore, this study provides important insights on the targets of adapted physical training, which can be regarded as suitable benchmarks for future in vivo studies further exploring the effects of this type of physical activity by functional/metabolic approaches.

Elastin represents the structural component of the extracellular matrix providing elastic recoil to tissues such as skin, blood vessels and lungs. Elastogenic cells secrete soluble tropoelastin monomers into the extracellular space where these monomers associate with other matrix proteins (e.g., microfibrils and glycoproteins) and are crosslinked by lysyl oxidase to form insoluble fibres. Once elastic fibres are formed, they are very stable, highly resistant to degradation and have an almost negligible turnover. However, there are circumstances, mainly related to inflammatory conditions, where increased proteolytic degradation of elastic fibres may lead to consequences of major clinical relevance. In severely affected COVID-19 patients, for instance, the massive recruitment and activation of neutrophils is responsible for the profuse release of elastases and other proteolytic enzymes which cause the irreversible degradation of elastic fibres. Within the lungs, destruction of the elastic network may lead to the permanent impairment of pulmonary function, thus suggesting that elastases can be a promising target to preserve the elastic component in COVID-19 patients. Moreover, intrinsic and extrinsic factors additionally contributing to damaging the elastic component and to increasing the spread and severity of SARS-CoV-2 infection are reviewed.