The Genes-Stemness-Secretome Interplay in Malignant Pleural Mesothelioma: Molecular Dynamics and Clinical Hints.

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.

A New Human Platelet Lysate for Mesenchymal Stem Cell Production Compliant with Good Manufacturing Practice Conditions Preserves the Chemical Characteristics and Biological Activity of Lyo-Secretome Isolated by Ultrafiltration.

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.

Biohybrid Bovine Bone Matrix for Controlled Release of Mesenchymal Stem/Stromal Cell Lyosecretome: A Device for Bone Regeneration.

SmartBone® (SB) is a biohybrid bone substitute advantageously proposed as a class III medical device for bone regeneration in reconstructive surgeries (oral, maxillofacial, orthopedic, and oncology). In the present study, a new strategy to improve SB osteoinductivity was developed. SB scaffolds were loaded with lyosecretome, a freeze-dried formulation of mesenchymal stem cell (MSC)-secretome, containing proteins and extracellular vesicles (EVs). Lyosecretome-loaded SB scaffolds (SBlyo) were prepared using an absorption method. A burst release of proteins and EVs (38% and 50% after 30 min, respectively) was observed, and then proteins were released more slowly with respect to EVs, most likely because they more strongly adsorbed onto the SB surface. In vitro tests were conducted using adipose tissue-derived stromal vascular fraction (SVF) plated on SB or SBlyo. After 14 days, significant cell proliferation improvement was observed on SBlyo with respect to SB, where cells filled the cavities between the native trabeculae. On SB, on the other hand, the process was still present, but tissue formation was less organized at 60 days. On both scaffolds, cells differentiated into osteoblasts and were able to mineralize after 60 days. Nonetheless, SBlyo showed a higher expression of osteoblast markers and a higher quantity of newly formed trabeculae than SB alone. The quantification analysis of the newly formed mineralized tissue and the immunohistochemical studies demonstrated that SBlyo induces bone formation more effectively. This osteoinductive effect is likely due to the osteogenic factors present in the lyosecretome, such as fibronectin, alpha-2-macroglobulin, apolipoprotein A, and TGF-ß.

Freeze-Dried Secretome (Lyosecretome) from Mesenchymal Stem/Stromal Cells Promotes the Osteoinductive and Osteoconductive Properties of Titanium Cages.

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.

Characterization and Antioxidant Activity of Essential Oil of Four Sympatric Orchid Species.

The volatile fractions from fresh inflorescences of naturally growing orchids Anacamptis coriophora (L.) R. M. Bateman, Pridgeon & M. W. Chase subsp. fragrans (Pollini), Anacamptis pyramidalis (L.) R. Ophrys holosericea (Burm.) Greuter and Serapias vomeracea (Burm. f.) B. were isolated by steam distillation and analyzed by GC/FID and GC/MS. Saturated hydrocarbons were quantified as the major constituents of the volatile fraction (47.87-81.57% of the total essential oil), of which long-chain monounsaturated hydrocarbons accounted from 9.20% to 32.04% of the total essential oil. Double bond position in linear alkenes was highlighted by dimethyl disulfide derivatization and MS fragmentation. Aldehydes (from 3.45 to 18.18% of the total essential oil), alcohols (from 0.19% to 13.48%), terpenes (from 0.98 to 2.50%) and acids (0.30 to 2.57%) were also detected. These volatiles compounds may represent a particular feature of these plant species, playing a critical role in the interaction with pollinators. DPPH assay evaluating the antioxidant activity of the essential oils was carried out, showing a dose-dependent antioxidant activity.

Silk nanoparticles: from inert supports to bioactive natural carriers for drug delivery.

Silk proteins have been studied and employed for the production of drug delivery (nano)systems. They show excellent biocompatibility, controllable biodegradability and non-immunogenicity and, if needed, their properties can be modulated by blending with other polymers. Silk fibroin (SF), which forms the inner core of silk, is a (bio)material officially recognized by the Food and Drug Administration for human applications. Conversely, the potential of silk sericin (SS), which forms the external shell of silk, could still be considered under evaluation. At the best of our knowledge, nanoparticles based on silk sericin "alone" cannot be produced, due to its physicochemical instability influenced by extreme pH, high water solubility and temperature; for these reasons, it almost always needs to be combined with other polymers for the development of drug delivery systems. In this review, we focused on silk proteins as bioactive natural carriers, since they show not only optimal features as inert excipients, but also remarkable intrinsic biological activities. SF has anti-inflammatory properties, while SS presents antioxidant, anti-tyrosine, anti-aging, anti-elastase and anti-bacterial features. Here, we give an overview on SF or SS silk-based nanosystems, with particular attention on the production techniques.

Chitosan for improved encapsulation of thyme aqueous extract in alginate-based microparticles.

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.

Prediction of the mechanical response of a 3D (bio)printed hybrid scaffold for improving bone tissue regeneration by structural finite element analysis.

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.

cRGD-Functionalized Silk Fibroin Nanoparticles: A Strategy for Cancer Treatment with a Potent Unselective Naphthalene Diimide Derivative.

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 (cRGDs) to provide active targeting toward glioma cell lines that abundantly express ανß3 and ανß5 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 (d50 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 cRGDs 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 cRGD-silk fibroin nanoparticles' active site-specific targeting of tumors, paving the way for further in vivo efficacy tests.

Silk Fibroin Bioink for 3D Printing in Tissue Regeneration: Controlled Release of MSC extracellular Vesicles.

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 (ß-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.

Mesenchymal stem cell secretome and extracellular vesicles for neurodegenerative diseases: Risk-benefit profile and next steps for the market access.

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.

Sericin/crocetin micro/nanoparticles for nucleus pulposus cells regeneration: An "active" drug delivery system.

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.

Spray-Dried Powder Containing Cannabigerol: A New Extemporaneous Emulgel for Topical Administration.

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-ß-cyclodextrin (MßCD), 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.

Trojan-horse silk fibroin nanocarriers loaded with a re-call antigen to redirect immunity against cancer.

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.

Design and development of a hepatic lyo-dECM powder as a biomimetic component for 3D-printable hybrid hydrogels.

Bioprinting offers new opportunities to obtain reliable 3Din vitromodels 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 definea priorithe 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.

Three-Dimensional Bioprinted Controlled Release Scaffold Containing Mesenchymal Stem/Stromal Lyosecretome for Bone Regeneration: Sterile Manufacturing and In Vitro Biological Efficacy.

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.

Solid Lipid Microparticles by Spray Congealing of Water/Oil Emulsion: An Effective/Versatile Loading Strategy for a Highly Soluble Drug.

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.

3D Bioprinted Scaffolds Containing Mesenchymal Stem/Stromal Lyosecretome: Next Generation Controlled Release Device for Bone Regenerative Medicine.

Three-dimensional printing of poly(ε-caprolactone) (PCL) is a consolidated scaffold manufacturing technique for bone regenerative medicine. Simultaneously, the mesenchymal stem/stromal cell (MSC) secretome is osteoinductive, promoting scaffold colonization by cells, proliferation, and differentiation. The present paper combines 3D-printed PCL scaffolds with lyosecretome, a freeze-dried formulation of MSC secretome, containing proteins and extracellular vesicles (EVs). We designed a lyosecretome 3D-printed scaffold by two loading strategies: (i) MSC secretome adsorption on 3D-printed scaffold and (ii) coprinting of PCL with an alginate-based hydrogel containing MSC secretome (at two alginate concentrations, i.e., 6% or 10% w/v). A fast release of proteins and EVs (a burst of 75% after 30 min) was observed from scaffolds obtained by absorption loading, while coprinting of PCL and hydrogel, encapsulating lyosecretome, allowed a homogeneous loading of protein and EVs and a controlled slow release. For both loading modes, protein and EV release was governed by diffusion as revealed by the kinetic release study. The secretome's diffusion is influenced by alginate, its concentration, or its cross-linking modes with protamine due to the higher steric hindrance of the polymer chains. Moreover, it is possible to further slow down protein and EV release by changing the scaffold shape from parallelepiped to cylindrical. In conclusion, it is possible to control the release kinetics of proteins and EVs by changing the composition of the alginate hydrogel, the scaffold's shape, and hydrogel cross-linking. Such scaffold prototypes for bone regenerative medicine are now available for further testing of safety and efficacy.

Crocetin as New Cross-Linker for Bioactive Sericin Nanoparticles.

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.

Mesenchymal Stromal Cell Secretome for Post-COVID-19 Pulmonary Fibrosis: A New Therapy to Treat the Long-Term Lung Sequelae?

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.