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Background: Obesity is considered a major public health issue in most developed countries nowadays. This paper provides an overview of current population data available in Spain and the approach to develop preventive strategies in the country.Methods: Review of population data available is based on individually measured weight and height as well as determinants. On this basis, the approach used in the country to develop preventive strategies is discussed.Results: According to the DORICA study, the prevalence of obesity (BMI >or=30 kg m-2) is 15.5% in Spanish adults aged 25-60 years (13.2% in men and 17.5% in women). Obesity rates are higher among women aged 45 years and older, low social class, living in semi-urban places. Population estimates for the prevalence of obesity in Spanish children and young people based on the enKid study are 13.9% for the whole group. In this study, overweight and obesity is related to absence of breastfeeding, low consumption of fruit and vegetables, high consumption of cakes, buns, softdrinks and butchery products, low physical activity levels and a positive association with time spent watching TV. In 2005, the Spanish Ministry of Health jointly with the Spanish Agency for Food Safety and Nutrition launched the multifaceted NAOS strategy for nutrition, physical activity and the prevention of obesity. The important role of the family and the school setting as well as the responsibility of the Health Administration and Pediatric Care in the prevention of obesity is highlighted in the document. The need for environmental actions is recognised. The PERSEO programme, a multicomponent school-based intervention project is part of the strategy currently in place.Conclusion: Obesity is a public health issue in Spain. A national multifaceted strategy was launched to counteract the problem. Environmental and policy actions are a priority. Young children and their families are among the main target groups. [ABSTRACT FROM AUTHOR]

Objective: To estimate the prevalence of the metabolic syndrome (MS) in a population of patients with overweight and obesity of the A Coruña and Granada health areas, using the definitions of the Third Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (ATP III) and of the International Diabetes Federation (IDF).Patients and Methods: During a period extending from 1996 to 2003, only those patients attending endocrinology outpatient clinics for whom all the anthropometric and biochemical parameters used to define the MS, both according to the ATP IIII and the IDF, were available were selected. The final study sample consisted of 285 patients, 198 females (69.5%) and 87 males (30.5%).Results: The prevalence of the MS was 29.8% when the ATP III definition was applied, and 41.1% according to the IDF criteria. Prevalence by sex was 32.2% in men and 28.8% in women according to the ATP III, and 42.5% and 40.4%, respectively, according to the IDF.Conclusions: In a patient population with overweight or obesity, the prevalence of the MS is higher when the IDF criteria, instead of the ATP III criteria, are used. These findings may have significant implications when it comes to addressing early diagnosis of cardiovascular disease and diabetes mellitus in these patients, in order to perform therapeutic measures at the initial stages and thereby reduce metabolic and cardiovascular complications. [ABSTRACT FROM AUTHOR]

Silk fibroin nanoparticles (SFNs) have been widely investigated for drug delivery, but their clinical application still faces technical (large-scale and GMP-compliant manufacturing), economic (cost-effectiveness in comparison to other polymer-based nanoparticles), and biological (biodistribution assessments) challenges. To address biodistribution challenge, we provide a straightforward desolvation method (in acetone) to produce homogeneous SFNs incorporating increasing amounts of Fe 2 O 3 (SFNs-Fe), detectable by Magnetic Resonance Imaging (MRI), and loaded with curcumin as a model lipophilic drug. SFNs-Fe were characterized by a homogeneous distribution of the combined materials and showed an actual Fe 2 O 3 loading close to the theoretical one. The amount of Fe 2 O 3 incorporated affected the physical-chemical properties of SFNs-Fe, such as polymer matrix compactness, mean diameter and drug release mechanism. All formulations were cytocompatible; curcumin encapsulation mitigated its cytotoxicity, and iron oxide incorporation did not impact cell metabolic activity but affected cellular uptake in vitro. SFNs-Fe proved optimal for biodistribution studies, as MRI showed significant nanoparticle retention at the administration site, supporting their potential for locoregional cancer therapy. Finally, technical and economic challenges in SFN production were overcome using a GMP-compliant microfluidic scalable technology, which optimized preparation to produce smaller particle sizes compared to manual methods and reduced acetone usage, thus offering environmental and economic benefits. Moreover, enabling large-scale production of GMP-like SFNs, this represents a considerable step forward for their application in the clinic., Competing Interests: Declaration of competing interest Sara Perteghellaa and Maria Luisa Torre are co-founders and members of the company's advisory board Pharmaexceed S.r.l. Marzio Sorlini is the CEO of Pharmaexceed S.r.l. This company had no role in the design of the study, in the collection, analysis, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Ionotropic gelation is a low-cost, easy and green microencapsulation technique. However, the encapsulation of highly soluble compounds is challenging because of the wide loss of material into the external water phase by passive diffusion and the consequent low encapsulation efficiency. In this work an important increase of encapsulation efficiency for Thymus vulgaris L. aqueous extract in alginate-based microparticles has been obtained. A formulation with the proper thyme extract/alginate ratio (30:70) was used as reference and then optimized by adding different co-carrier excipients. Microparticles obtained by dropping a solution containing thyme extract and alginate into a chitosan/calcium-chloride/acid acetic solution lead to a high encapsulation efficiency (70.43 ± 5.28 %). After drying, microparticles had a particle size of 1096 ± 72 μm, 20.087 ± 1.487 % of extract content, 6.2 % of residual water, and showed a complete release of thyme extract within one hour. Combining alginate and chitosan as polymeric co-carrier was a valuable option for efficiently encapsulating an aqueous extract by ionotropic gelation., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Segale L., Giovannelli L. are co-founders and members of the advisory board of the company APTSol S.r.l., Milanesi A. is member of advisory board of the company APTSol S.r.l. Torre M.L. is co-founder and member of the advisory board of the company Pharmaexceed. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

A protocol for the encapsulation in sodium alginate of granulosa cells in primary culture and coculture of oocyte-cumulus complexes is reported. Sodium alginate forms strong gels when jellified with barium ions, allowing the self-organization of cells into a 3D structure. This method of encapsulation is simple and cheap, allowing the culture of cells in a three-dimensional fashion., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cannabigerol (CBG), a cannabinoid from Cannabis sativa L., recently attracted noteworthy attention for its dermatological applications, mainly due to its anti-inflammatory, antioxidant, and antimicrobial effectiveness similar to those of cannabidiol (CBD). In this work, based on results from studies of in vitro permeation through biomimetic membranes performed with CBG and CBD in the presence and in the absence of a randomly substituted methyl-β-cyclodextrin (MβCD), a new CBG extemporaneous emulgel (oil-in-gel emulsion) formulation was developed by spray-drying. The powder (SDE) can be easily reconstituted with purified water, leading to a product with chemical-physical and technological characteristics that are comparable to those of the starting emulgels (E). Thermogravimetric analysis (TGA), attenuated total reflection-Fourier transformed infrared spectroscopy (ATR-FTIR), x-ray powder diffraction (XRPD), and high-performance liquid chromatography (HPLC) analyses demonstrated that the spray-drying treatment did not alter the chemical properties of CBG. This product can represent a metered-dosage form for the localized treatment of cutaneous afflictions such as acne and psoriasis.

Bioprinting offers new opportunities to obtain reliable 3D in vitro models of the liver for testing new drugs and studying pathophysiological mechanisms, thanks to its main feature in controlling the spatial deposition of cell-laden hydrogels. In this context, decellularized extracellular matrix (dECM)-based hydrogels have caught more and more attention over the last years because of their characteristic to closely mimic the tissue-specific microenvironment from a biological point of view. In this work, we describe a new concept of designing dECM-based hydrogels; in particular, we set up an alternative and more practical protocol to develop a hepatic lyophilized dECM (lyo-dECM) powder as an 'off-the-shelf' and free soluble product to be incorporated as a biomimetic component in the design of 3D-printable hybrid hydrogels. To this aim, the powder was first characterized in terms of cytocompatibility on human and porcine mesenchymal stem cells (MSCs), and the optimal powder concentration (i.e. 3.75 mg ml -1 ) to use in the hydrogel formulation was identified. Moreover, its non-immunogenicity and capacity to reactivate the elastase enzyme potency was proved. Afterward, as a proof-of-concept, the powder was added to a sodium alginate/gelatin blend, and the so-defined multi-component hydrogel was studied from a rheological point of view, demonstrating that adding the lyo-dECM powder at the selected concentration did not alter the viscoelastic properties of the original material. Then, a printing assessment was performed with the support of computational simulations, which were useful to define a priori the hydrogel printing parameters as window of printability and its post-printing mechanical collapse. Finally, the proposed multi-component hydrogel was bioprinted with cells inside, and its post-printing cell viability for up to 7 d was successfully demonstrated., (Creative Commons Attribution license.)

Neurodegenerative diseases represent a growing burden on healthcare systems worldwide. Mesenchymal stem cells (MSCs) have shown promise as a potential therapy due to their neuroregenerative, neuroprotective, and immunomodulatory properties, which are, however, linked to the bioactive substances they release, collectively known as secretome. This paper provides an overview of the most recent research on the safety and efficacy of MSC-derived secretome and extracellular vesicles (EVs) in clinical (if available) and preclinical models of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, acute ischemic stroke, and spinal cord injury. The article explores the biologically active substances within MSC-secretome/EVs, the mechanisms responsible for the observed therapeutic effects, and the strategies that may be used to optimize MSC-secretome/EVs production based on specific therapeutic needs. The review concludes with a critical discussion of current clinical trials and a perspective on potential future directions in translating MSC-secretome and EVs into the clinic, specifically regarding how to address the challenges associated with their pharmaceutical manufacturing, including scalability, batch-to-batch consistency, adherence to Good Manufacturing Practices (GMP) guidelines, formulation, and storage, along with quality controls, access to the market and relative costs, value for money and impact on total expenditure., Competing Interests: Maria Luisa Torre is a co-founder and member of the advisory board of Pharmaexceed S.r.l., (© 2023 The Authors.)

Scaffolds for bone tissue engineering should be osteoinductive, osteoconductive, biocompatible, biodegradable, and, at the same time, exhibit proper mechanical properties. The present study investigated the mechanical properties of a coprinted hybrid scaffold made of polycaprolactone (PCL) and an alginate-based hydrogel, which was conceived to possess a double function of in vivo bio-integration (due to the ability of the hydrogel to release lyosecretome, a freeze-dried formulation of mesenchymal stem cell secretome with osteoinductive and osteoconductive properties) and withstanding loads (due to the presence of polycaprolactone, which provides mechanical resistance). To this end, an in-silico study was conducted to predict mechanical properties. Structural finite element analysis (FEA) of the hybrid scaffold under compression was performed to compare the numerical results with the corresponding experimental data. The impact of alginate inclusion and infill patterns on scaffold stiffness was investigated. Results show an increase in mechanical properties by changing the scaffold infill pattern (linear: 145.38±28.90 vs. honeycomb: 278.96±50.19, mean and standard deviation, n = 8), while alginate inclusion does not always impact the mechanical performance of the hybrid scaffold (stiffness: 145.38±28.90 vs. 195.42±38.68 N/mm, with vs without hydrogel inclusion, respectively). This is confirmed by FEA analysis, in which a good correspondence between experimental and numerical stiffness is shown (142±28.94 vs. 117.18, respectively, linear scaffold with hydrogel inclusion). In conclusion, the computational framework is a valid tool for predicting the mechanical performance of scaffolds and is promising for future clinical applications in the maxillofacial field., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Background: Diabetes is one of the most prevalent chronic diseases worldwide, and innovative patient support programmes can help and inform patients about their disease and improve their quality of life. The purpose of this study was to evaluate the effect of the T-Coach programme in terms of improvement of disease knowledge, self-management and adherence to treatment in a real-world setting in Spain between July 2016 and October 2018., Methods: We analyzed data from the T-Coach programme, a telephone platform that gives support to patients with type 2 diabetes mellitus treated with insulin glargine 300 U/ml (Gla-300). Support was provided by diabetes care nurses. Patients followed their treatment and aimed to achieve fasting blood glucose targets through diabetes education., Results: A total of 479 patients were included in the programme. The mean (SD) dose of Gla-300 was 28.5 (16.3) U at baseline and 31.8 (16.1) U, 31.4 (16.4) U and 32.2 (16.3) U, respectively, at 3, 6 and 12 months. A satisfaction survey was completed by 240 (50.1%) patients, who, on average, were very highly satisfied with the programme, general assistance provided, recommendations received, and calls from nurses., Conclusions: T-Coach could be an effective tool to help patients achieve their optimal dose of Gla-300 insulin and manage their blood glucose levels. It could also act as an effective support for diabetes education., Competing Interests: Disclosure and potential conflicts of interest: VB has received consulting fees, honoraria for lectures, and support for attending meetings and/or travel from Abbott, AstraZeneca, Boehringer Ingelheim, Eli Lilly, Esteve, Janssen, Merck, Mundipharma, Novartis, Novo Nordisk, Roche and Sanofi. CM has received honoraria for lectures and participated on the advisory board for Novo Nordisk, Eli Lilly, Boehringer Ingelheim, Abbott, Sanofi, AstraZeneca, MSD and Amgen. BGA has received honoraria for lectures and support for attending meetings and/or travel from Novo Nordisk, Eli Lilly, Abbott, Fresenius and Nutricia. MLdlT has received grants and contracts from Eli Lilly as well as honoraria for lectures and support for attending meetings and/or travel from Novo Nordisk, Eli Lilly, Novartis, Sanofi, MSD, Boehringer Ingelheim, AstraZeneca, Esteve, Abbott, Amgen and Mundipharma. DB has received honoraria for lectures and support for attending meetings and/or travel and participated on the advisory board for Sanofi, Novo Nordisk, Abbott, Eli Lilly, Boehringer Ingelheim, Persan Pharma and Menarini. DB has also participated in a leadership role on boards or committees for SEEDO (Sociedade Española de Obesidad) and SEEN (Sociedad Española de Endocrinología y Nutrición). AMG, JMG and JLFM declare that they have no conflicts of interest. The International Committee of Medical Journal Editors (ICMJE) Potential Conflicts of Interests form for the authors is available for download at: https://www.drugsincontext.com/wp-content/uploads/2023/04/dic.2023-1-1-COI.pdf, (Copyright © 2023 Bellido V, Morales C, Muñoz Garach A, García Almeida JM, Fernández Morera JL, González Aguilera B, López de la Torre M, Bellido D.)

The existence of more than thirty stress-strain equations, including those proposed by the government regulations in many countries, seems to indicate that additional, unifying, and at the same time generalizing research is necessary for this subject. Many expressions can be found to set or determine the initial modulus of elasticity of concrete, i.e., the modulus of elasticity of concrete when no load has been applied to it. This work proposes a complete generalization of the equations based on scalar damage models, applicable to all types of concrete tested under uniaxial compression with any constant rate of stress or strain, although in no case can it be considered a constitutive model. We prefer to discuss an equation that models the shape of the stress-strain curve. Thus, the shape of this curve is studied here in the same way a forensic scientist would, which is why we could see this work as an autopsy carried out on the test specimen through the trace left in the plane σ - ε by the straining process up until its inevitable outcome. That is to say, we believe in a purely phenomenological approach. The results are compared with the data obtained experimentally by analyzing test specimens made using various mixed portions of cement, water, and aggregates.

Developing drug delivery systems to target cytotoxic drugs directly into tumor cells is still a compelling need with regard to reducing side effects and improving the efficacy of cancer chemotherapy. In this work, silk fibroin nanoparticles (SFNs) have been designed to load a previously described cytotoxic compound (NDI-1) that disrupts the cell cycle by specifically interacting with non-canonical secondary structures of DNA. SFNs were then functionalized on their surface with cyclic pentapeptides incorporating the Arg-Gly-Asp sequence ( c RGDs) to provide active targeting toward glioma cell lines that abundantly express ανβ3 and ανβ5 integrin receptors. Cytotoxicity and selective targeting were assessed by in vitro tests on human glioma cell lines U373 (highly-expressing integrin subunits) and D384 cell lines (low-expressing integrin subunits in comparison to U373). SFNs were of nanometric size (d 50 less than 100 nm), round shaped with a smooth surface, and with a negative surface charge; overall, these characteristics made them very likely to be taken up by cells. The active NDI-1 was loaded into SFNs with high encapsulation efficiency and was not released before the internalization and degradation by cells. Functionalization with c RGDs provided selectivity in cell uptake and thus cytotoxicity, with a significantly higher cytotoxic effect of NDI-1 delivered by cRGD-SFNs on U373 cells than on D384 cells. This manuscript provides an in vitro proof-of-concept of c RGD-silk fibroin nanoparticles' active site-specific targeting of tumors, paving the way for further in vivo efficacy tests.

Introduction: Initiation and progression of intervertebral disk degeneration are linked to oxidative stress, with reactive oxygen species being a key factor. Therefore, as a potentially novel approach able to regenerate the damaged intervertebral disk, this work aimed to prepare an "active per sé" drug delivery system by combining sericin and crocetin: both are bioactive compounds with antioxidant, anti-inflammatory, immunomodulant and regenerative properties. Methods: In detail, sericin nanoparticles were prepared using crocetin as a cross-linker; then, the nanoparticle dispersions were dried by spray drying as it is (NP), with an excess of sericin (NPS) or crocin/crocetin (NPMix), obtaining three microparticle formulations. Results and Discussion: Before drying, the nanoparticles were nanometric (about 250 nm), with a negative surface charge, and appeared spherical and smooth. Following the drying process, spherical and smooth microparticles were obtained, with a mean diameter of about 1.7-2.30 μm. NPMix was the most active in antioxidant and anti-tyrosinase activities, likely due to the excess of crocin/crocetin, while NPS had the best anti-elastase activity, likely due to sericin in excess. Furthermore, all the formulations could prevent oxidative stress damage on nucleus pulposus cells, with NPMix being the best. Overall, the intrinsic anti-tyrosinase and anti-elastase activities and the ability to protect from oxidative stress-induced damages justify future investigations of these "active per sé" formulations in treating or preventing intervertebral disk degeneration., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bari, Perteghella, Rassu, Gavini, Petretto, Bonferoni, Giunchedi and Torre.)

MPM has a uniquely poor somatic mutational landscape, mainly driven by environmental selective pressure. This feature has dramatically limited the development of effective treatment. However, genomic events are known to be associated with MPM progression, and specific genetic signatures emerge from the exceptional crosstalk between neoplastic cells and matrix components, among which one main area of focus is hypoxia. Here we discuss the novel therapeutic strategies focused on the exploitation of MPM genetic asset and its interconnection with the surrounding hypoxic microenvironment as well as transcript products and microvesicles representing both an insight into the pathogenesis and promising actionable targets.

Sodium alginate (SA)-based hydrogels are often employed as bioink for three-dimensional (3D) scaffold bioprinting. They offer a suitable environment for cell proliferation and differentiation during tissue regeneration and also control the release of growth factors and mesenchymal stem cell secretome, which is useful for scaffold biointegration. However, such hydrogels show poor mechanical properties, fast-release kinetics, and low biological performance, hampering their successful clinical application. In this work, silk fibroin (SF), a protein with excellent biomechanical properties frequently used for controlled drug release, was blended with SA to obtain improved bioink and scaffold properties. Firstly, we produced a printable SA solution containing SF capable of the conformational change from Silk I (random coil) to Silk II (β-sheet): this transition is a fundamental condition to improve the scaffold's mechanical properties. Then, the SA-SF blends' printability and shape fidelity were demonstrated, and mechanical characterization of the printed hydrogels was performed: SF significantly increased compressive elastic modulus, while no influence on tensile response was detected. Finally, the release profile of Lyosecretome-a freeze-dried formulation of MSC-secretome containing extracellular vesicles (EV)-from scaffolds was determined: SF not only dramatically slowed the EV release rate, but also modified the kinetics and mechanism release with respect to the baseline of SA hydrogel. Overall, these results lay the foundation for the development of SA-SF bioinks with modulable mechanical and EV-release properties, and their application in 3D scaffold printing.

Background: The current challenge for immunotherapies is to generate effective antitumor immunity. Since tumor immune escape mechanisms do not impact pre-existing and consolidated immune responses, we tested the hypothesis of redirecting a pregenerated immunity to cancer: to recall a non-tumor antigen response against the tumor, silk fibroin nanoparticles (SFNs) have been selected as 'Trojan-horse' carriers, promoting the antigen uptake by the tumor cells., Methods: SFNs have been loaded with either ovalbumin (OVA) or CpG oligonucleotide (CpG) as antigen or adjuvant, respectively. In vitro uptake of SFNs by tumor (B16/F10 melanoma and MB49 bladder cancer) or dendritic cells, as well as the presence of OVA-specific T cells in splenic and tumor-infiltrating lymphocytes, were assessed by cytometric analyses. Proof-of-concept of in vivo efficacy was achieved in an OVA-hyperimmune B16/F10 murine melanoma model: SFNs-OVA or SFNs-CpG were injected, separately or in association, into the subcutaneous peritumoral area. Cancer dimensions/survival time were monitored, while, at the molecular level, system biology approaches based on graph theory and experimental proteomic data were performed., Results: SFNs were efficiently in vitro uptaken by cancer and dendritic cells. In vivo peritumor administration of SFNs-OVA redirected OVA-specific cytotoxic T cells intratumorally. Proteomics and systems biology showed that peritumoral treatment with either SFNs-OVA or SFNs-CpG dramatically modified tumor microenvironment with respect to the control (CTR), mainly involving functional modules and hubs related to angiogenesis, inflammatory mediators, immune function, T complex and serpins expression, redox homeostasis, and energetic metabolism. Both SFNs-OVA and SFNs-CpG significantly delayed melanoma growth/survival time, and their effect was additive., Conclusions: Both SFNs-OVA and SFNs-CpG induce effective anticancer response through complementary mechanisms and show the efficacy of an innovative active immunotherapy approach based on the redirection of pre-existing immunity against cancer cells. This approach could be universally applied for solid cancer treatments if translated into the clinic using re-call antigens of childhood vaccination., Competing Interests: Competing interests: Data presented in this manuscript pertain to the Italian Patent Application N. 102019000008658 ‘Immunizzazione antitumorale mediata da nanoparticelle e basata su un’immunità preesistente’ filed on June 11, 2019, granted on April 21, 2021, and the international patent application PCT/IB2020/055456 ‘Nanoparticles for use in the redirection against the tumour of a non-tumour-specific immune response, based on a pre-existing immunity’, Publication number WO/2020/250153, filed on June 10, 2020.The authors who are co-inventors in these patents are EB, FF, DF, SP, MLT and GF., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Spray congealing technique was exploited to produce solid lipid microparticles (SLMp) loaded with a highly water-soluble drug (metoclopramide hydrochloride) dissolved in the aqueous phase of a water in oil (W/O) emulsion. The use of an emulsion as starting material for a spray congealing treatment is not so frequent. Moreover, for this application, a W/O emulsion with a drug dissolved in water is a totally novel path. A ternary diagram was built to optimize the emulsion composition, a factorial design was used to identify the factors affecting the properties of the microparticles and a Design of Experiment strategy was applied to define the impact of process conditions and formulation variables on the SLMp properties. SLMp were characterized by particle size distribution, morphology, residual moisture, drug content, release behavior, FT-IR analysis and XRPD. The obtained microparticles presented a spherical shape, particle size distribution between 54-98 µm depending on atomizing pressure used during the production step and 2-5% residual moisture 4 days after the preparation. XRPD analysis revealed that lipid polymorphic transition alfa-beta occurs depending on the presence of water. In vitro drug release tests highlighted that all the formulations had a reduced release rate compared to the drug alone. These results suggest that spray congealing of a W/O emulsion could be proposed as a good strategy to obtain SLMp with a high loading of a hydrophilic drug and able to control its release rate.

Background: Congenital ichthyosis (CI) is a heterogeneous group of genetic disorders characterized by generalized dry skin, scaling and hyperkeratosis, often associated to erythroderma. They are rare diseases, with overall incidence of 6.7 in 100,000. Clinical manifestations are due to mutations in genes mostly involved in skin barrier formation. Based on clinical presentation, CI is distinguished in non-syndromic and syndromic forms. To date, mutations of more than 50 genes have been associated to different types of CI., Cases Presentation: We report on three Italian unrelated newborns showing clinical signs compatible with different forms of CI of variable severity, namely Harlequin ichtyosis (HI), epidermolytic ichtyosis (EI) and autosomal recessive ichtyosis with hypotrichosis (ARIH). Target next generation sequencing (NGS) analysis identified three novel mutations of the ABCA12, KRT1 and ST14 genes, respectively associated to such congenital ichtyoses, not reported in literature. Genomic investigation allowed to provide the more appropriate management to each patient, based on an individualized approach., Conclusions: Our report highlights the wide genetic heterogeneity and phenotypic variability of CI. It expands the current knowledge on such diseases, widening their genomic database, and providing a better clinical characterization. Furthermore, it underlines the clinical relevance of NGS, which is essential to address the management of patients. Indeed, it may guide towards the most adequate approach, preventing clinical obstinacy for subjects with more severe forms and unfavorable outcomes (together with the support, in such situations, of bioethicists included within the multidisciplinary care team), as well as reassuring families in those with milder course and favorable evolution., (© 2022. The Author(s).)

Recently, 3D-printed scaffolds for the controlled release of mesenchymal stem cell (MSC) freeze-dried secretome (Lyosecretome) have been proposed to enhance scaffold osteoinduction and osteoconduction; coprinting of poly(ε-caprolactone) (PCL) with alginate hydrogels allows adequate mechanical strength to be combined with the modulable kinetics of the active principle release. This study represents the feasibility study for the sterile production of coprinted scaffolds and the proof of concept for their in vitro biological efficacy. Sterile scaffolds were obtained, and Lyosecretome enhanced their colonization by MSCs, sustaining differentiation towards the bone line in an osteogenic medium. Indeed, after 14 days, the amount of mineralized matrix detected by alizarin red was significantly higher for the Lyosecretome scaffolds. The amount of osteocalcin, a specific bone matrix protein, was significantly higher at all the times considered (14 and 28 days) for the Lyosecretome scaffolds. Confocal microscopy further confirmed such results, demonstrating improved osteogenesis with the Lyosecretome scaffolds after 14 and 28 days. Overall, these results prove the role of MSC secretome, coprinted in PCL/alginate scaffolds, in inducing bone regeneration; sterile scaffolds containing MSC secretome are now available for in vivo pre-clinical tests of bone regeneration.

Recently, we proposed a Good Manufacturing Practice (GMP)-compliant production process for freeze-dried mesenchymal stem cell (MSC)-secretome (lyo-secretome): after serum starvation, the cell supernatant was collected, and the secretome was concentrated by ultrafiltration and freeze-dried, obtaining a standardized ready-to-use and stable powder. In this work, we modified the type of human platelet lysate (HPL) used as an MSC culture supplement during the lyo-secretome production process: the aim was to verify whether this change had an impact on product quality and also whether this new procedure could be validated according to GMP, proving the process robustness. MSCs were cultured with two HPLs: the standard previously validated one (HPL-E) and the new one (HPL-S). From the same pool of platelets, two batches of HPL were obtained: HPL-E (by repeated freezing and thawing cycles) and HPL-S (by adding Ca-gluconate to form a clot and its subsequent mechanical wringing). Bone marrow MSCs from three donors were separately cultured with the two HPLs until the third passage and then employed to produce lyo-secretome. The following indicators were selected to evaluate the process performance: (i) the lyo-secretome quantitative composition (in lipids and proteins), (ii) the EVs size distribution, and (iii) anti-elastase and (iv) immunomodulant activity as potency tests. The new HPL supplementation for MSCs culture induced only a few minimal changes in protein/lipid content and EVs size distribution; despite this, it did not significantly influence biological activity. The donor intrinsic MSCs variability in secretome secretion instead strongly affected the quality of the finished product and could be mitigated by concentrating the final product to reach a determined protein (and lipid) concentration. In conclusion, the modification of the type of HPL in the MSCs culture during lyo-secretome production induces only minimal changes in the composition but not in the potency, and therefore, the new procedure can be validated according to GMP.

In recent years, mesenchymal stromal cells (MSCs) have shown promise as a therapy in treating musculoskeletal diseases, and it is currently believed that their therapeutic effect is mainly related to the release of proteins and extracellular vesicles (EVs), known as secretome. In this work, three batches of canine MSC-secretome were prepared by standardized processes according to the current standard ISO9001 and formulated as a freeze-dried powder named Lyosecretome. The final products were characterized in protein and lipid content, EV size distribution and tested to ensure the microbiological safety required for intraarticular injection. Lyosecretome induced the proliferation of adipose tissue-derived canine MSCs, tenocytes, and chondrocytes in a dose-dependent manner and showed anti-elastase activity, reaching 85% of inhibitory activity at a 20 mg/mL concentration. Finally, to evaluate the safety of the preparation, three patients affected by bilateral knee or elbow osteoarthritis were treated with two intra-articular injections (t = 0 and t = 40 days) of the allogeneic Lyosecretome (20 mg corresponding 2 × 10 6 cell equivalents) resuspended in hyaluronic acid in one joint and placebo (mannitol resuspended in hyaluronic acid) in the other joint. To establish the safety of the treatment, the follow-up included a questionnaire addressed to the owner and orthopaedic examinations to assess lameness grade, pain score, functional disability score and range of motion up to day 80 post-treatment. Overall, the collected data suggest that intra-articular injection of allogeneic Lyosecretome is safe and does not induce a clinically significant local or systemic adverse response.

Surgical resection is the gold standard for the treatment of many kinds of tumor, but its success depends on the early diagnosis and the absence of metastases. However, many deep-seated tumors (liver, pancreas, for example) are often unresectable at the time of diagnosis. Chemotherapies and radiotherapies are a second line for cancer treatment. The "enhanced permeability and retention" (EPR) effect is believed to play a fundamental role in the passive uptake of drug-loaded nanocarriers, for example polymeric nanoparticles, in deep-seated tumors. However, criticisms of the EPR effect were recently raised, particularly in advanced human cancers: obstructed blood vessels and suppressed blood flow determine a heterogeneity of the EPR effect, with negative consequences on nanocarrier accumulation, retention, and intratumoral distribution. Therefore, to improve the nanomedicine uptake, there is a strong need for "EPR enhancers". Electrochemotherapy represents an important tool for the treatment of deep-seated tumors, usually combined with the systemic (intravenous) administration of anticancer drugs, such as bleomycin or cisplatin. A possible new strategy, worthy of investigation, could be the use of this technique as an "EPR enhancer" of a target tumor, combined with the intratumoral administration of drug-loaded nanoparticles. This is a general overview of the rational basis for which EP could be envisaged as an "EPR enhancer" in nanomedicine.

Tissue repair and regeneration is an interdisciplinary field focusing on developing bioactive substitutes aimed at restoring pristine functions of damaged, diseased tissues. Biomaterials, intended as those materials compatible with living tissues after in vivo administration, play a pivotal role in this area and they have been successfully studied and developed for several years. Namely, the researches focus on improving bio-inert biomaterials that well integrate in living tissues with no or minimal tissue response, or bioactive materials that influence biological response, stimulating new tissue re-growth. This review aims to gather and introduce, in the context of Italian scientific community, cutting-edge advancements in biomaterial science applied to tissue repair and regeneration. After introducing tissue repair and regeneration, the review focuses on biodegradable and biocompatible biomaterials such as collagen, polysaccharides, silk proteins, polyesters and their derivatives, characterized by the most promising outputs in biomedical science. Attention is pointed out also to those biomaterials exerting peculiar activities, e.g., antibacterial. The regulatory frame applied to pre-clinical and early clinical studies is also outlined by distinguishing between Advanced Therapy Medicinal Products and Medical Devices.

Titanium is one of the most frequently used materials in bone regeneration due to its good biocompatibility, excellent mechanical properties, and great osteogenic performance. However, osseointegration with host tissue is often not definite, which may cause implant failure at times. The present study investigates the capacity of the mesenchymal stem cell (MSC)-secretome, formulated as a ready-to-use and freeze-dried medicinal product (the Lyosecretome), to promote the osteoinductive and osteoconductive properties of titanium cages. In vitro tests were conducted using adipose tissue-derived MSCs seeded on titanium cages with or without Lyosecretome. After 14 days, in the presence of Lyosecretome, significant cell proliferation improvement was observed. Scanning electron microscopy revealed the cytocompatibility of titanium cages: the seeded MSCs showed a spread morphology and an initial formation of filopodia. After 7 days, in the presence of Lyosecretome, more frequent and complex cellular processes forming bridges across the porous surface of the scaffold were revealed. Also, after 14 and 28 days of culturing in osteogenic medium, the amount of mineralized matrix detected by alizarin red was significantly higher when Lyosecretome was used. Finally, improved osteogenesis with Lyosecretome was confirmed by confocal analysis after 28 and 56 days of treatment, and demonstrating the production by osteoblast-differentiated MSCs of osteocalcin, a specific bone matrix protein.

Producing mesenchymal stem cell (MSC)-secretome for dose escalation studies and clinical practice requires scalable and good manufacturing practice (GMP)-compliant production procedures and formulation into a standardized medicinal product. Starting from a method that combines ultrafiltration and freeze-drying to transform MSC-secretome into a pharmaceutical product, the lyosecretome, this work aims to: (i) optimize the lyosecretome formulation; (ii) investigate sources of variability that can affect the robustness of the manufacturing process; (iii) modify the ultrafiltration step to obtain a more standardized final product. Design of experiments and principal component analysis of the data were used to study the influence of batch production, lyophilization, mannitol (M)/sucrose (S) binary mixture, selected as cryoprotectant excipients, and the total amount of excipients on the extracellular vesicles (EV) particle size, the protein and lipid content and the in vitro anti-elastase. The different excipients ratios did not affect residual moisture or EV particle size; simultaneously, proteins and lipids were better preserved in the freeze-dried product using the maximum total concentration of excipients (1.5% w / v ) with a M:S ratio of about 60% w / w . The anti-elastase activity was instead better preserved using 0.5% w / w of M as excipient. The secretome batch showed to be the primary source of variability; therefore, the manufacturing process has been modified and then validated: the final product is now concentrated to reach a specific protein (and lipid) concentration instead of cell equivalent concentration. The new standardization approach led to a final product with more reproducible quali-quantitative composition and higher biological activity.

Background/objective: The optimal prognostic value of testosterone following androgen deprivation therapy (ADT) is controversial. We studied the effect of serum testosterone levels on clinical outcome in localized prostate cancer (PCa) treated with ADT and high-dose radiotherapy (HRT)., Patients and Methods: The DART01/05 trial randomized 355 men with intermediate and high-risk PCa to 4 months of ADT plus HRT (STADT, N = 178) or the same treatment followed by 24 months of ADT (LTADT, N = 177). This study included patients treated with LTADT who had at least 3 determinations of testosterone during ADT (N = 154). Patients were stratified into 3 subgroups by testosterone level: minimum <20 ng/dL; median 20-49 ng/dL; and maximum ≥50 ng/dL. Kaplan-Meyer and Cox regression analysis were used for overall survival (OS) and Fine & Gray regression model for metastasis free survival (MFS), biochemical disease-free survival (bDFS) and time to TT recovery., Results: There were no statistically significant differences in 10-year bDFS, MFS, or OS between the <20 ng/mL and 20-49 ng/dL subgroups. Multivariate analysis showed that a median testosterone ≥50 ng/dL was significantly associated with a decrease in bDFS (HR: 6.58, 95%CI 1.28-33.76, p = 0.03). Time to testosterone recovery after ADT did not correlate with bDFS, MFS, or OS and was not significantly associated with any of the testosterone subgroups., Conclusions: Our results do not support the concept that additional serum testosterone suppression below 20 ng/dL is associated with better outcomes than 20-49 ng/dL. Time to testosterone recovery after ADT and HRT did not impact clinical failure., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

In the last decades, it has been demonstrated that the regenerative therapeutic efficacy of mesenchymal stromal cells is primarily due to the secretion of soluble factors and extracellular vesicles, collectively known as secretome. In this context, our work described the preparation and characterization of a freeze-dried secretome (Lyosecretome) from adipose tissue-derived mesenchymal stromal cells for the therapy of equine musculoskeletal disorder. An intraarticular injectable pharmaceutical powder has been formulated, and the technological process has been validated in an authorized facility for veterinary clinical-use medicinal production. Critical parameters for quality control and batch release have been identified regarding (i) physicochemical properties; (ii) extracellular vesicle morphology, size distribution, and surface biomarker; (iii) protein and lipid content; (iv) requirements for injectable pharmaceutical dosage forms such as sterility, bacterial endotoxin, and Mycoplasma; and (v) in vitro potency tests, as anti-elastase activity and proliferative activity on musculoskeletal cell lines (tenocytes and chondrocytes) and mesenchymal stromal cells. Finally, proteins putatively responsible for the biological effects have been identified by Lyosecretome proteomic investigation: IL10RA, MXRA5, RARRES2, and ANXA1 modulate the inflammatory process RARRES2, NOD1, SERPINE1, and SERPINB9 with antibacterial activity. The work provides a proof-of-concept for the manufacturing of clinical-grade equine freeze-dried secretome, and prototypes are now available for safety and efficacy clinical trials in the treatment of equine musculoskeletal diseases.

To date, more than 100 million people worldwide have recovered from COVID-19. Unfortunately, although the virus is eradicated in such patients, fibrotic irreversible interstitial lung disease (pulmonary fibrosis, PF) is clinically evident. Given the vast numbers of individuals affected, it is urgent to design a strategy to prevent a second wave of late mortality associated with COVID-19 PF as a long-term consequence of such a devastating pandemic. Available antifibrotic therapies, namely nintedanib and pirfenidone, might have a role in attenuating profibrotic pathways in SARS-CoV-2 infection but are not economically sustainable by national health systems and have critical adverse effects. It is our opinion that the mesenchymal stem cell secretome could offer a new therapeutic approach in treating COVID-19 fibrotic lungs through its anti-inflammatory and antifibrotic factors.

The nose-to-brain delivery route is used to bypass the blood-brain barrier and deliver drugs directly into the brain. Over the years, significant signs of progress have been made in developing nano-drug delivery systems to address the very low drug transfer levels seen with conventional formulations (e.g., nasal solutions). In this paper, sericin nanoparticles were prepared using crocetin as a new bioactive natural cross-linker (NPc) and compared to sericin nanoparticles prepared with glutaraldehyde (NPg). The mean diameter of NPc and NPg was about 248 and 225 nm, respectively, and suitable for nose-to-brain delivery. The morphological investigation revealed that NPc are spherical-like particles with a smooth surface, whereas NPg seem small and rough. NPc remained stable at 4 °C for 28 days, and when freeze-dried with 0.1% w/v of trehalose, the aggregation was prevented. The use of crocetin as a natural cross-linker significantly improved the in vitro ROS-scavenging ability of NPc with respect to NPg. Both formulations were cytocompatible at all the concentrations tested on human fibroblasts and Caco-2 cells and protected them against oxidative stress damage. In detail, for NPc, the concentration of 400 µg/mL resulted in the most promising to maintain the cell metabolic activity of fibroblasts higher than 90%. Overall, the results reported in this paper support the employment of NPc as a nose-to-brain drug delivery system, as the brain targeting of antioxidants is a potential tool for the therapy of neurological diseases.