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Manna, a well-known herbal drug has multiple traditional and pharmaceutical uses and the entire composition, sugar derivatives and polyphenols, gives rise to a very interesting bioactive complex with versatile therapeutic and benefic properties such as antioxidant and anti-inflammatory activities. The aim of this research was to investigate a F. ornus manna extract loaded in a pectin hydrogel as a synergic vehicle to evaluate the potential use of the complex for cosmetic and dermatological applications. In particular, the study set out to disclose manna properties as a wound healing agent with antimicrobial and reparative activity on infected tissues. Moreover, considering the correlation between antioxidant activity and antiaging potential, the extract was investigated in regard to the anti-elastase activity and skin whitening potential. The total phenolic content of each extract was also determined and a safe profile by in vitro cytotoxicity studies was verified. The hydrogel complex, containing the manna extract and pectin as the gelling agent, exhibited suitable properties in terms of pH (from 5.50 to 6.80), rheological behavior and ability of preserving the antioxidant activity of the manna exudate (around 10%). All the peculiarities that make the pectin hydrogels ideal systems for skin disease, as wound dressings and for antiaging cosmetic formulations.

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.

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.

Berberine (BBR) is known for its antitumor activity and photosensitizer properties in anti-cancer photodynamic therapy (PDT), and it has previously been favorably assayed against glioblastoma multiforme (GBM)-derived cells. In this work, two BBR hydrophobic salts, dodecyl sulfate (S) and laurate (L), have been encapsulated in PLGA-based nanoparticles (NPs), chitosan-coated by the addition of chitosan oleate in the preparation. NPs were also further functionalized with folic acid. All the BBR-loaded NPs were efficiently internalized into T98G GBM established cells, and internalization increased in the presence of folic acid. However, the highest mitochondrial co-localization percentages were obtained with BBR-S NPs without folic acid content. In the T98G cells, BBR-S NPs appeared to be the most efficient in inducing cytotoxicity events and were therefore selected to assess the effect of photodynamic stimulation (PDT). As a result, PDT potentiated the viability reduction for the BBR-S NPs at all the studied concentrations, and a roughly 50% reduction of viability was obtained. No significant cytotoxic effect on normal rat primary astrocytes was observed. In GBM cells, a significant increase in early and late apoptotic events was scored by BBR NPs, with a further increase following the PDT scheme. Furthermore, a significantly increased depolarization of mitochondria was highlighted following BBR-S NPs’ internalization and mostly after PDT stimulation, compared to untreated and PDT-only treated cells. In conclusion, these results highlighted the efficacy of the BBR-NPs-based strategy coupled with photoactivation approaches to induce favorable cytotoxic effects in GBM cells.

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.

Clove oil (CO) is a powerful antioxidant essential oil (EO) with anti-inflammatory, anesthetic, and anti-infective properties. It can be therefore considered a good candidate for wound-healing applications, especially for chronic or diabetic wounds or burns, where the balance of reactive oxygen species (ROS) production and detoxification is altered. However, EOs require suitable formulations to be efficiently administered in moist wound environments. Chitosan hydrophobically modified by an ionic interaction with oleic acid (chitosan oleate, CSO) was used in the present work to stabilize CO nanoemulsions (NEs). The dimensions of the NE were maintained at around 300 nm as the volume distribution for up to six months, and the CO content did not decrease to under 80% over 4 months, confirming the good stabilizing properties of CSO. The antioxidant properties of the CO NE were evaluated in vitro by a 2,2-diphenil-2-picrylhydrazyl hydrate (DPPH) assay, and in fibroblast cell lines by electron paramagnetic resonance (EPR) using α-phenyl-N-tert-butyl nitrone (PBN) as a spin trap; a protective effect was obtained comparable to that obtained with α-tocopherol treatment. In a murine burn model, the ability of CO formulations to favor macroscopic wound closure was evidenced, and a histological analysis revealed a positive effect of the CO NE on the reparation of the lesion after 18 days. Samples of wounds at 7 days were subjected to a histological analysis and parallel dosage of lipid peroxidation by means of a thiobarbituric acid-reactive substances (TBARS) assay, confirming the antioxidant and anti-inflammatory activity of the CO NE.

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.

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 × 106 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.

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.

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.

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.

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.

Regenerative medicine aims to restore the normal function of diseased or damaged cells, tissues, and organs using a set of different approaches, including cell-based therapies. In the veterinary field, regenerative medicine is strongly related to the use of mesenchymal stromal cells (MSCs), which belong to the body repair system and are defined as multipotent progenitor cells, able to self-replicate and to differentiate into different cell types. This review aims to take stock of what is known about the MSCs and their use in the veterinary medicine focusing on clinical reports on dogs and horses in musculoskeletal diseases, a research field extensively reported in the literature data. Finally, a perspective regarding the use of the secretome and/or extracellular vesicles (EVs) in the veterinary field to replace parental MSCs is provided. The pharmaceuticalization of EVs is wished due to the realization of a Good Manufacturing Practice (GMP product suitable for clinical trials.

From the end of 2019, the world population has been faced the spread of the novel coronavirus SARS-CoV-2 responsible for COVID-19 infection. In approximately 14% of the patients affected by the novel coronavirus, the infection progresses with the development of pneumonia that requires mechanical ventilation. At the moment, there is no specific antiviral treatment recommended for the COVID-19 pandemic and the therapeutic strategies to deal with the infection are only supportive. In our opinion, mesenchymal stem cell secretome could offer a new therapeutic approach in treating COVID-19 pneumonia, due to the broad pharmacological effects it shows, including anti-inflammatory, immunomodulatory, regenerative, pro-angiogenic and anti-fibrotic properties.

Mesenchymal stem/stromal cells (MSCs) are a therapeutic target to promote tissue regeneration, mainly when oxidative stress-mediated damage is involved in disease pathogenesis. Here, slow-release silk sericin nanoparticles (SNPs) loaded with natural antioxidant polyphenols were developed to sustain regeneration by tissue-resident MSCs. SNPs were prepared by exploiting a self-assembly method with poloxamer and were loaded with proanthocyanidins (P), quercetin (Q) or epigallocatechin gallate (E). SNPs, with a diameter less than 150 nm, were able to encapsulate both hydrophilic (P and E) and hydrophobic (Q) drugs. A slow and controlled release was obtained from SNPs for all the actives in PBS, while in EtOH, Q and E showed a burst release but P did not. Kinetic models revealed lower diffusion of P than other biomolecules, probably due to the higher steric hindrance of P. The in vitro anti-oxidant, anti-elastase and anti-tyrosinase properties of SNPs were assessed: loading the P and E into SNPs preserved the in vitro biological activities whereas for Q, the anti-elastase activity was strongly improved. Moreover, all formulations promoted MSC metabolic activity over 72 h. Finally, SNPs exhibited a strong ability to protect MSCs from oxidative stress, which supports their potential use for regenerative purposes mediated by tissue-resident MSCs.

Standard treatments of chronic skin ulcers based on the direct application of dressings still present several limits with regard to a complete tissue regeneration. Innovative strategies in tissue engineering offer materials that can tune cell behavior and promote growth tissue favoring cell recruitment in the early stages of wound healing. A combination of Alginate (Alg), Sericin (SS) with Platelet Lysate (PL), as a freeze-dried sponge, is proposed to generate a bioactive wound dressing to care skin lesions. Biomembranes at different composition were tested for the release of platelet growth factors, cytotoxicity, protective effects against oxidative stress and cell proliferation induction. The highest level of the growth factors release occurred within 48 h, an optimized time to burst a healing process in vivo; the presence of SS differently modulated the release of the factors by interaction with the proteins composing the biomembranes. Any cytotoxicity was registered, whereas a capability to protect cells against oxidative stress and induce proliferation was observed when PL was included in the biomembrane. In a mouse skin lesion model, the biomembranes with PL promoted the healing process, inducing an accelerated and more pronounced burst of inflammation, formation of granulation tissue and new collagen deposition, leading to a more rapid skin regeneration.

Accumulating evidence shows that Mesenchymal Stem/Stromal Cells (MSCs) exert their therapeutic effects by the release of secretome, made of both soluble proteins and nano/microstructured extracellular vesicles (EVs). In this work, for the first time, we proved by a proteomic investigation that adipose-derived (AD)-MSC-secretome contains alpha-1-antitrypsin (AAT), the main elastase inhibitor in the lung, 72 other proteins involved in protease/antiprotease balance, and 46 proteins involved in the response to bacteria. By secretome fractionation, we proved that AAT is present both in the soluble fraction of secretome and aggregated and/or adsorbed on the surface of EVs, that can act as natural carriers promoting AAT in vivo stability and activity. To modulate secretome composition, AD-MSCs were cultured in different stimulating conditions, such as serum starvation or chemicals (IL-1β and/or dexamethasone) and the expression of the gene encoding for AAT was increased. By testing in vitro the anti-elastase activity of MSC-secretome, a dose-dependent effect was observed; chemical stimulation of AD-MSCs did not increase their secretome anti-elastase activity. Finally, MSC-secretome showed anti-bacterial activity on Gram-negative bacteria, especially for Klebsiella pneumoniae. These preliminary results, in addition to the already demonstrated immunomodulation, pave the way for the use of MSC-secretome in the treatment of AAT-deficiency lung diseases.

The aim of this work was to load an anticancer drug, paclitaxel (PTX), on Silk Fibroin Nanoparticles (SFNs) by using an exogenous approach. SFNs were produced, freeze-dried and then loaded with PTX. An exogenous method allowed us to reduce both drug loss and environmental impact. In order to quantify PTX loaded in SFNs, a simple and reliable method using reversed phase liquid chromatography coupled to tandem mass spectrometry (rp-UHPLC-MS/MS) was developed. This methodology was validated by the determination of spiked QC samples in three consecutive days. Good accuracy and precision of the method were obtained, while the intra-day and inter-day precisions were less than 10.3%. For PTX, the limit of quantitation (LOQ) was 5.0 ng/mL. Recovery from the matrix (SFNs-PTX pellets) was calculated (81.2% at LOQ value) as PTX was entrapped in a new matrix like the polymer silk fibroin-based. This method was successfully applied to determine the encapsulation efficiency (1.00 ± 0.19%) and the nanoparticle loading (0.12 ± 0.02% w/w). The in vitro anticancer activity of SFNs-PTX was tested against CFPAC-1 cancer cells; results demonstrated a very high cytotoxic activity of SFNs-PTX, with a dose dependent inhibition of CFPAC-1 proliferation, confirmed by the IC50 value of 3450 ± 750 ng/mL.

In this paper, a pilot production process for mesenchymal stem/stromal freeze-dried secretome was performed in a validated good manufacturing practice (GMP)-compliant cell factory. Secretome was purified from culture supernatants by ultrafiltration, added to cryoprotectant, lyophilized and characterized. We obtained a freeze-dried, “ready-off-the-shelf„ and free soluble powder containing extracellular vesicles and proteins. In the freeze-dried product, a not-aggregated population of extracellular vesicles was detected by nanoparticle tracking analysis; Fourier transform infrared spectra showed the simultaneous presence of protein and lipids, while differential scanning calorimetry demonstrated that lyophilization process successfully occurred. A proteomic characterization allowed the identification of proteins involved in immune response, response to stress, cytoskeleton and metabolism. Moreover, the product was not cytotoxic up to concentrations of 25 mg/mL (on human fibroblasts, chondrocytes and nucleus pulposus cells by MTT assay) and was blood compatible up to 150 mg/mL. Finally, at concentrations between 5 and 50 mg/mL, freeze-dried secretome showed to in vitro counteract the oxidative stress damage induced by H2O2 on nucleus pulposus cells by MTT assay.

In this methodological paper, lyophilized fibroin-coated alginate microcarriers (LFAMs) proposed as mesenchymal stem cells (MSCs) delivery systems and optimal MSCs seeding conditions for cell adhesion rate and cell arrangement, was defined by a Design of Experiment (DoE) approach. Cells were co-incubated with microcarriers in a bioreactor for different time intervals and conditions: variable stirring speed, dynamic culture intermittent or continuous, and different volumes of cells-LFAMs loaded in the bioreactor. Intermittent dynamic culture resulted as the most determinant parameter; the volume of LFAMs/cells suspension and the speed used for the dynamic culture contributed as well, whereas time was a less influencing parameter. The optimized seeding conditions were: 98 min of incubation time, 12.3 RPM of speed, and 401.5 µL volume of cells-LFAMs suspension cultured with the intermittent dynamic condition. This DoE predicted protocol was then validated on both human Adipose-derived Stem Cells (hASCs) and human Bone Marrow Stem Cells (hBMSCs), revealing a good cell adhesion rate on the surface of the carriers. In conclusion, microcarriers can be used as cell delivery systems at the target site (by injection or arthroscopic technique), to maintain MSCs and their activity at the injured site for regenerative medicine.

(1) Objective: to obtain a reproducible, robust, well-defined, and cost-affordable in vitro model of human cartilage degeneration, suitable for drug screening; (2) Methods: we proposed 3D models of engineered cartilage, considering two human chondrocyte sources (articular/nasal) and five culture methods (pellet, alginate beads, silk/alginate microcarriers, and decellularized cartilage). Engineered cartilages were treated with pro-inflammatory cytokine IL-1β to promote cartilage degradation; (3) Results: articular chondrocytes have been rejected since they exhibit low cellular doubling with respect to nasal cells, with longer culture time for cell expansion; furthermore, pellet and alginate bead cultures lead to insufficient cartilage matrix production. Decellularized cartilage resulted as good support for degeneration model, but long culture time and high cell amount are required to obtain the adequate scaffold colonization. Here, we proposed, for the first time, the combined use of decellularized cartilage, as aggrecanase substrate, with pellet, alginate beads, or silk/alginate microcarriers, as polymeric scaffolds for chondrocyte cultures. This approach enables the development of suitable models of cartilaginous pathology. The results obtained after cryopreservation also demonstrated that beads and microcarriers are able to preserve chondrocyte functionality and metabolic activity; (4) Conclusions: alginate and silk/alginate-based scaffolds can be easily produced and cryopreserved to obtain a cost-affordable and ready-to-use polymer-based product for the subsequent screening of anti-inflammatory drugs for cartilage diseases.

Hydrogels are among the most common materials used in drug delivery, as polymeric micelles are too. They, preferentially, load hydrophilic and hydrophobic drugs, respectively. In this paper, we thought to combine the favorable behaviors of both hydrogels and polymeric micelles with the specific aim of delivering hydrophilic and hydrophobic drugs for dual delivery in combination therapy, in particular for colon drug delivery. Thus, we developed a hydrogel by UV crosslinking of a methacrylated (MA) amphiphilic derivative from inulin (INU) (as known INU is specifically degraded into the colon) and vitamin E (VITE), called INVITEMA. The methacrylated micelles were physicochemically characterized and subjected to UV irradiation to form what we called the “nanogrids”. The INVITEMA nanogrids were characterized by DSC, SEM, TEM, water uptake and beclomethasone dipropionate (BDP) release. In particular, the release of the hydrophobic drug was specifically assessed to verify that it can spread along the hydrophilic portions and, therefore, effectively released. These systems can open new pharmaceutical applications for known hydrogels or micelle systems, considering that in literature only few examples are present.

Some natural compounds have recently been widely employed in wound healing applications due to their biological properties. One such compound is sericin, which is produced by Bombix mori, while active polyphenols, polysaccharides and proteins are synthetized by Chlorella vulgaris and Arthrospira platensis microalgae. Our hypothesis was that sericin, as an optimal bioactive polymeric carrier for microencapsulation process, could also improve the regenerative effect of the microalgae. A solvent-free extraction method and spray drying technique were combined to obtain five formulations, based on algal extracts (C. vulgaris and A. platensis, Chl and Art, respectively) or silk sericin (Ser) or their mixtures (Chl-Ser and Art-Ser). The spray drying was a suitable method to produce microspheres with similar dimensions, characterized by collapsed morphology with a rough surface. Art and Art-Ser showed higher antioxidant properties than other formulations. All microspheres resulted in cytocompatibility on fibroblasts until 1.25 mg/mL and promoted cell migration and the complete wound closure; this positive effect was further highlighted after treatment with Art and Art-Ser. To our surprize the combination of sericin to Art did not improve the microalgae extract efficacy, at least in our experimental conditions.