Design of Novel Mechanically Resistant and Biodegradable Multichannel Platforms for the Treatment of Peripheral Nerve Injuries.

Peripheral nerve injury is one of the most debilitating pathologies that severely impair patients' life. Although many efforts have been made to advance in the treatment of such a complex disorder, successful strategies to ensure full recovery are still scarce. The aim of the present work was to develop flexible and mechanically resistant platforms intended to act as a support and guide for neural cells during the regeneration process of peripheral nerve injury. For this purpose, poly(lactic-co-glycolic acid) (PLGA)/poly(d,l-lactic acid) (PDLLA)/poly(ethylene glycol) 400 (PEG)-multichannel-based scaffolds (MCs) were prepared through a multistep process involving electrospun microfibers coated with a polymer blend solution and used as a sacrificial mold. In particular, scaffolds characterized by random (MCR) and aligned (MCA) multichannel were obtained. A design of experiments approach (DoE) was employed to identify a scaffold-optimized composition. MCs were characterized for morphological and mechanical properties, suturability, degradability, cell colonization, and in vivo safety. A new biodegradable, biocompatible, and safe microscale multichannel scaffold was developed as the result of an easy multistep procedure.

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.

Association of Alpha Tocopherol and Ag Sulfadiazine Chitosan Oleate Nanocarriers in Bioactive Dressings Supporting Platelet Lysate Application to Skin Wounds.

Chitosan oleate was previously proposed to encapsulate in nanocarriers some poorly soluble molecules aimed to wound therapy, such as the anti-infective silver sulfadiazine, and the antioxidant α tocopherol. Because nanocarriers need a suitable formulation to be administered to wounds, in the present paper, these previously developed nanocarriers were loaded into freeze dried dressings based on chitosan glutamate. These were proposed as bioactive dressings aimed to support the application to wounds of platelet lysate, a hemoderivative rich in growth factors. The dressings were characterized for hydration capacity, morphological aspect, and rheological and mechanical behavior. Although chitosan oleate nanocarriers clearly decreased the mechanical properties of dressings, these remained compatible with handling and application to wounds. Preliminary studies in vitro on fibroblast cell cultures demonstrated good compatibility of platelet lysate with nanocarriers and bioactive dressings. An in vivo study on a murine wound model showed an accelerating wound healing effect for the bioactive dressing and its suitability as support of the platelet lysate application to wounds.

Sustainable whey proteins-nanostructured zinc oxide-based films for the treatment of chronic wounds: New insights from biopharmaceutical studies.

Chronic wounds represent silent epidemic affecting a large portion of the world population, especially the elders; in this context, the development of advanced bioactive dressings is imperative to accelerate wound healing process, while contrasting or preventing infections. The aim of the present work was to provide a deep characterization of the functional and biopharmaceutical properties of a sustainable thin and flexible films, composed of whey proteins alone (WPI) and added with nanostructured zinc oxide (WPZ) and intended for the management of chronic wounds. The potential of whey proteins-based films as wound dressings has been confirmed by their wettability, hydration properties, elastic behavior upon hydration, biodegradation propensity and, when added with nanostructured zinc oxide, antibacterial efficacy against both Gram-positive and Gram-negative pathogens, i.e. Staphylococcus aureus and Escherichia coli. In-vitro experiments, performed on normal human dermal fibroblasts, confirmed film cytocompatibility, also revealing the possible role of Zn2+ ions in promoting fibroblast proliferation. Finally, in-vivo studies on rat model confirmed film suitability to act as wound dressing, since able to ensure a regular healing process while providing effective protection from infections. In particular, both films WPI and WPZ are responsible for the formation in the wound bed of a continuous collagen layer similar to that of healthy skin.

Porous Functionally Graded Scaffold prepared by a single-step freeze-drying process. A bioinspired approach for wound care.

Nowadays, chronic wounds are the major cause of morbidity worldwide and the healthcare costs related to wound care are a billion-dollar issue; chronic wounds involve a non-healing process that makes necessary the application of advanced wound dressings to promote skin integrity recovery. Functionally Graded Scaffolds (FGSs) are currently driving interest as promising candidates in mimicking the skin tissue environment and, thus, in enhancing a faster and more effective wound healing process. Aim of the present work was to design and develop a porous FGS based on κ-carrageenan (κCG) for the management of chronic skin wounds; a freeze-drying process was optimized to obtain in a single-step a three-layered FGS characterized by a pore size gradient functional to mimic the structure of native skin tissue. In addition to κCG, arginine and whey protein isolate were used as multifunctional agents for FGS preparation; these substances can not only intervene in some stages of wound healing but are able to establish non-covalent interactions with κCG, which were responsible for the production of layers with different pore size, water content capability and mechanical properties. Cell migration, adhesion and proliferation within the FGS structure were evaluated in vitro on fibroblasts and FGS wound healing potential was also studied in vivo on a murine model.

Chondroitin sulfate and caseinophosphopeptides doped polyurethane-based highly porous 3D scaffolds for tendon-to-bone regeneration.

Tendon disorders are common injuries, which can be greatly debilitating as they are often accompanied by great pain and inflammation. Moreover, several problems are also related to the laceration of the tendon-to-bone interface (TBI), a specific region subjected to great mechanical stresses. The techniques used nowadays for the treatment of tendon and TBI injuries often involve surgery. However, one critical aspect of this procedure involves the elevated risk of fail due to the tissues weakening and the postoperative alterations of the normal joint mechanics. Synthetic polymers, such as thermoplastic polyurethane, are of special interest in the tissue engineering field as they allow the production of scaffolds with tunable elastic and mechanical properties, that could guarantee an effective support during the new tissue formation. Based on these premises, the aim of this work was the design and the development of highly porous 3D scaffolds based on thermoplastic polyurethane, and doped with chondroitin sulfate and caseinophosphopeptides, able to mimic the structural, biomechanical, and biochemical functions of the TBI. The obtained scaffolds were characterized by a homogeneous microporous structure, and by a porosity optimal for cell nutrition and migration. They were also characterized by remarkable mechanical properties, reaching values comparable to the ones of the native tendons. The scaffolds promoted the tenocyte adhesion and proliferation when caseinophosphopetides and chondroitin sulfate are present in the 3D structure. In particular, caseinophosphopeptides' optimal concentration for cell proliferation resulted 2.4 mg/mL. Finally, the systems evaluation in vivo demonstrated the scaffolds' safety, since they did not cause any inflammatory effect nor foreign body response, representing interesting platforms for the regeneration of injured TBI.

Design and development of polydioxanone scaffolds for skin tissue engineering manufactured via green process.

Fiber spinning technologies attracted a great interest since the beginning of the last century. Among these, electrospinning is a widely diffuse technique; however, it presents some drawbacks such as low fiber yield, high energy demand and the use of organic solvents. On the contrary, centrifugal spinning is a more sustainable method and allows to obtain fiber using centrifugal force and melted materials. The aim of the present work was the design and the development of polydioxanone (PDO) microfibers intended for tissue engineering, using centrifugal spinning. PDO, a bioresorbable polymer currently used for sutures, was selected as low melting polyester and DES (deep eutectic solvents), either choline chloride/citric acid (ChCl/CA) or betaine/citric acid (Bet/CA) 1:1 M ratio, were used to improve PDO spinnability. Physical mixtures of DES and PDO were prepared using different weight ratios. These were then poured into the spinneret and melted at 140 °C for 5 min. After the complete melting, the blends were spun for 1 min at 700 rpm. The fibers were characterized for physico chemical properties (morphology; dimensions; chemical structure; thermal behavior; mechanical properties). Moreover, the preclinical investigation was performed in vitro (biocompatibility, adhesion and proliferation of fibroblasts) and in vivo (murine burn/excisional model to assess safety and efficacy). The multidisciplinary approach allowed to obtain an extensive characterization to develop PDO based microfibers as medical device for implant to treat full thickness skin wounds.

Mycelium-based biomaterials as smart devices for skin wound healing.

Introduction: Recently, mycelia of Ganoderma lucidum and Pleurotus ostreatus, edible fungi, have been characterized in vitro as self-growing biomaterials for tissue engineering since they are constituted of interconnected fibrous networks resembling the dermal collagen structure. Aim: This work aims to investigate the biopharmaceutical properties of G. lucidum and P. ostreatus mycelia to prove their safety and effectiveness in tissue engineering as dermal substitutes. Methods: The mycelial materials were characterized using a multidisciplinary approach, including physicochemical properties (morphology, thermal behavior, surface charge, and isoelectric point). Moreover, preclinical properties such as gene expression and in vitro wound healing assay have been evaluated using fibroblasts. Finally, these naturally-grown substrates were applied in vivo using a murine burn/excisional wound model. Conclusions: Both G. lucidum and P. ostreatus mycelia are biocompatible and able to safely and effectively enhance tissue repair in vivo in our preclinical model.

Cerium Oxide and Chondroitin Sulfate Doped Polyurethane Scaffold to Bridge Tendons.

Tendon disorders are common medical conditions, which can be greatly debilitating as they are often accompanied by great pain and inflammation. The techniques used nowadays for the treatment of chronic tendon injuries often involve surgery. However, one critical aspect of this procedure involves the scar tissue, characterized by mechanical properties that vary from healthy tissue, rendering the tendons inclined to reinjury or rupture. Synthetic polymers, such as thermoplastic polyurethane, are of special interest in the tissue engineering field as they allow the production of scaffolds with controlled elastic and mechanical properties, which could guarantee an effective support during the new tissue formation. The aim of this work was the design and the development of tubular nanofibrous scaffolds based on thermoplastic polyurethane and enriched with cerium oxide nanoparticles and chondroitin sulfate. The scaffolds were characterized by remarkable mechanical properties, especially when tubular aligned, reaching values comparable to the ones of the native tendons. A weight loss test was performed, suggesting a degradation in prolonged times. In particular, the scaffolds maintained their morphology and also remarkable mechanical properties after 12 weeks of degradation. The scaffolds promoted the cell adhesion and proliferation, in particular when in aligned conformation. Finally, the systems in vivo did not cause any inflammatory effect, representing interesting platforms for the regeneration of injured tendons.

Formulation development of collagen/chitosan-based porous scaffolds for skin wounds repair and regeneration.

Herein we developed a hydrogel based porous cross-linked scaffold intended for the treatment of chronic skin ulcers. It is made of collagen, the most abundant protein of mammals ECM, and chitosan, a natural polysaccharide endowed with numerous positive cues for wound repair. Different cross-linking methods, namely UV irradiation with the addition of glucose, addition of tannic acid as cross-linking agent and ultrasonication, were employed to prepare a cross-linked hydrogel with a highly interconnected 3D internal structure. The variables considered critical to obtain a suitable system for the envisaged application are the composition of hydrogels, especially the concentration of chitosan, and the concentration ratio between chitosan and collagen. Stable systems, characterized by high porosity, were obtained thanks to the use of freeze-drying process. To assess the influence of the above-mentioned variables on scaffold mechanical properties, a Design of Experiments (DoE) approach was exploited, which resulted in the identification of the best hydrogel composition. In vitro and in vivo assays on a fibroblast model cell line and on a murine model, respectively, demonstrated scaffold biocompatibility, biomimicry, and safety.

Nanoemulsions of Clove Oil Stabilized with Chitosan Oleate-Antioxidant and Wound-Healing Activity.

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.

Polysaccharide-protein microparticles based-scaffolds to recover soft tissue loss in mild periodontitis.

Periodontal regeneration is extremely limited and unpredictable due to structural complications, as it requires the simultaneous restoration of different tissues, including cementum, gingiva, bone, and periodontal ligament. In this work, spray-dried microparticles based on green materials (polysaccharides - gums - and a protein - silk fibroin) are proposed to be implanted in the periodontal pocket as 3D scaffolds during non-surgical treatments, to prevent the progression of periodontal disease and to promote the healing in mild periodontitis. Arabic or xanthan gum have been associated to silk fibroin, extracted from Bombyx mori cocoons, and loaded with lysozyme due to its antibacterial properties. The microparticles were prepared by spray-drying and cross-linked by water vapor annealing, inducing the amorphous to semi-crystalline transition of the protein component. The microparticles were characterized in terms of their chemico-physical features (SEM, size distribution, structural characterization - FTIR and SAXS, hydration and degradation properties) and preclinical properties (lysozyme release, antibacterial properties, mucoadhesion, in vitro cells adhesion and proliferation and in vivo safety on a murine incisional wound model). The encouraging preclinical results highlighted that these three-dimensional (3D) microparticles could provide a biocompatible platform able to prevent periodontitis progression and to promote the healing of soft tissues in mild periodontitis.

Maltodextrin-amino acids electrospun scaffolds cross-linked with Maillard-type reaction for skin tissue engineering.

The goal of this work is the design and the development of scaffolds based on maltodextrin (MD) to recover chronic lesions. MD was mixed with arginine/lysine/polylysine and the electrospinning was successfully used to prepare scaffolds with uniform and continuous nanofibers having regular shape and smooth surface. A thermal treatment was applied to obtain insoluble scaffolds in aqueous environment, taking the advantage of amino acids-polysaccharide conjugates formed via Maillard-type reaction. The morphological analysis showed that the scaffolds had nanofibrous structures, and that the cross-linking by heating did not significantly change the nanofibers' dimensions and did not alter the system stability. Moreover, the heating process caused a reduction of free amino group and proportionally increased scaffold cross-linking degree. The scaffolds were elastic and resistant to break, and possessed negative zeta potential in physiological fluids. These were characterized by direct antioxidant properties and Fe2+ chelation capability (indirect antioxidant properties). Moreover, the scaffolds were cytocompatible towards fibroblasts and monocytes-derived macrophages, and did not show any significant pro-inflammatory activity. Finally, those proved to accelerate the recovery of the burn/excisional wounds. Considering all the features, MD-poly/amino acids scaffolds could be considered as promising medical devices for the treatment of chronic wounds.

Smart nano-in-microparticles to tackle bacterial infections in skin tissue engineering.

Chronic wounds (resulting from underlying disease, metabolic disorders, infections, trauma, and even tumours) pose significant health problems. In this work, microparticles, based on polysaccharides (maltodextrin or dextran) and amino acids, and doped with antibacterial nanoparticles (CuO or ZnO NPs) are designed. Smart nano-in-microparticles with a hierarchical 3D structure are developed. The ultimate goal aims at an innovative platform to achieve skin repair and to manage skin colonization by avoiding infection that could delay and even impair the healing process. The microparticles are prepared by spray-drying and cross-linked by heating, to obtain insoluble scaffolds able to facilitate cell proliferation in the wound bed. The nano-in-microparticles are characterized using a multidisciplinary approach: chemico-physical properties (SEM, SEM-EDX, size distribution, swelling and degradation properties, structural characterization - FTIR, XRPD, SAXS - mechanical properties, surface zeta potential) and preclinical properties (in vitro biocompatibility and whole-blood clotting properties, release studies and antimicrobial properties, and in vivo safety and efficacy on murine burn/excisional wound model) were assessed. The hierarchical 3D nano-in microparticles demonstrate to promote skin tissue repair in a preclinical study, indicating that this platform deserves particular attention and further investigation will promote the prototypes translation to clinics.

Depression and antidepressants: molecular and cellular aspects.

Clinical depression is viewed as a physical and psychic disease process having a neuropathological basis, although a clear understanding of its ethiopathology is still missing. The observation that depressive symptoms are influenced by pharmacological manipulation of monoamines led to the hypothesis that depression results from reduced availability or functional deficiency of monoaminergic transmitters in some cerebral regions. However, there are limitations to current monoamine theories related to mood disorders. Recently, a growing body of experimental data has showed that other classes of endogenous compounds, such as neuropeptides and amino acids, may play a significant role in the pathophysiology of affective disorders. With the development of neuroscience, neuronal networks and intracellular pathways have been identified and characterized, describing the existence of the interaction between monoamines and receptors in turn able to modulate the expression of intracellular proteins and neurotrophic factors, suggesting that depression/antidepressants may be intermingled with neurogenesis/neurodegenerative processes.

Insulin-loaded nanoparticles based on N-trimethyl chitosan: in vitro (Caco-2 model) and ex vivo (excised rat jejunum, duodenum, and ileum) evaluation of penetration enhancement properties.

The aim of this paper was to evaluate the penetration enhancement properties of nanoparticles (NP) based on N-trimethyl chitosan (TMC 35% quaternization degree) loaded with insulin. The permeation performances of TMC NP were compared with those of chitosan (CS) NP and also with TMC and CS solutions. To estimate the mechanism of penetration enhancement, two different approaches have been taken into account: an in vitro study (Caco-2 cells) and an ex vivo study (excised rat duodenum, jejunum, and ileum). Insulin-loaded CS and TMC NP had dimensions of about 250 nm and had high yield and high encapsulation efficiency. The in vitro study evidenced that TMC and CS were able to enhance insulin permeation to the same extent. Penetration enhancement properties of TMC NP seem to be prevalently related to endocytosis while the widening of tight junctions appeared more important as mechanism in the case of CS NP. The ex vivo study put in evidence the role of mucus layer and of its microclimate pH. In duodenum (pH 5-5.5), CS and TMC solutions were more effective than NP while TMC NP were more efficient towards jejunum tissue (pH 6-6.5) for their high mucoadhesive potential. Confocal laser scanning microscopy study supported the hypothesis that penetration enhancement due to TMC NP was mainly due to internalization/endocytosis into duodenum and jejunum epithelial cells. The good penetration enhancement properties (permeation and penetration/internalization) make TMC NP suitable carriers for oral administration of insulin.

Cannabinoids, Inner Ear, Hearing, and Tinnitus: A Neuroimmunological Perspective.

Cannabis has been used for centuries for recreational and therapeutic purposes. Whereas, the recreative uses are based on the psychotropic effect of some of its compounds, its therapeutic effects range over a wide spectrum of actions, most of which target the brain or the immune system. Several studies have found cannabinoid receptors in the auditory system, both at peripheral and central levels, thus raising the interest in cannabinoid signaling in hearing, and especially in tinnitus, which is affected also by anxiety, memory, and attention circuits where cannabinoid effects are well described. Available studies on animal models of tinnitus suggest that cannabinoids are not likely to be helpful in tinnitus treatment and could even be harmful. However, the pharmacology of cannabinoids is very complex, and most studies focused on neural CB1R-based responses. Cannabinoid effects on the immune system (where CB2Rs predominate) are increasingly recognized as essential in understanding nervous system pathological responses, and data on immune cannabinoid targets have emerged in the auditory system as well. In addition, nonclassical cannabinoid targets (such as TRP channels) appear to play an important role in the auditory system as well. This review will focus on neuroimmunological mechanisms for cannabinoid effects and their possible use as protective and therapeutic agents in the ear and auditory system, especially in tinnitus.

Chitosan/glycosaminoglycan scaffolds for skin reparation.

Burns and chronic wounds, often related to chronic diseases (as diabetes and cancer), are challenging lesions, difficult to heal. The prompt and full reconstitution of a functional skin is at the basis of the development of biopolymer-based scaffolds, representing a 3D substrate mimicking the dermal extracellular matrix. Aim of the work was to develop scaffolds intended for skin regeneration, according to: fabrication by electrospinning from aqueous polysaccharide solutions; prompt and easy treatment to obtain scaffolds insoluble in aqueous fluids; best performance in supporting wound healing. Three formulations were tested, based on chitosan (CH) and pullulan (P), associated with glycosaminoglycans (chondroitin sulfate - CS or hyaluronic acid - HA). A multidisciplinary approach has been used: chemico-physical characterization and preclinical evaluation allowed to obtain integrated information. This supports that CS gives distinctive properties and optimal features to the scaffold structure for promoting cell proliferation leading tissue reparation towards a complete skin restore.

Glioblastoma under Siege: An Overview of Current Therapeutic Strategies.

Glioblastoma is known to be one of the most lethal and untreatable human tumors. Surgery and radiotherapy in combination with classical alkylating agents such as temozolomide offer little hope to escape a poor prognosis. For these reasons, enormous efforts are currently devoted to refine in vivo and in vitro models with the specific goal of finding new molecular aberrant pathways, suitable to be targeted by a variety of therapeutic approaches, including novel pharmaceutical formulations and immunotherapy strategies. In this review, we will first discuss current molecular classification based on genomic and transcriptomic criteria. Also, the state of the art in current clinical practice for glioblastoma therapy in the light of the recent molecular classification, together with ongoing phases II and III clinical trials, will be described. Finally, new pharmaceutical formulations such as nanoparticles and viral vectors, together with new strategies entailing the use of monoclonal antibodies, vaccines and immunotherapy agents, such as checkpoint inhibitors, will also be discussed.

Essential oil-loaded lipid nanoparticles for wound healing.

Chronic wounds and severe burns are diseases responsible for severe morbidity and even death. Wound repair is a crucial process and tissue regeneration enhancement and infection prevention are key factors to minimize pain, discomfort, and scar formation. The aim of this work was the development of lipid nanoparticles (solid lipid nanoparticles and nanostructured lipid carriers [NLC]), to be loaded with eucalyptus or rosemary essential oils and to be used, as medical devices, to enhance healing of skin wounds. Lipid nanoparticles were based on natural lipids: cocoa butter, as solid lipid, and olive oil or sesame oil, as liquid lipids. Lecithin was chosen as surfactant to stabilize nanoparticles and to prevent their aggregation. The systems were prepared by high shear homogenization followed by ultrasound application. Nanoparticles were characterized for physical-chemical properties, bioadhesion, cytocompatibility, in vitro proliferation enhancement, and wound healing properties toward normal human dermal fibroblasts. Antimicrobial activity of nanoparticles was evaluated against two reference microbial strains, one of Staphylococcus aureus, the other of Streptococcus pyogenes. Finally, the capability of nanoparticles to promote wound healing in vivo was evaluated on a rat burn model. NLC based on olive oil and loaded with eucalyptus oil showed appropriate physical-chemical properties, good bioadhesion, cytocompatibility, in vitro proliferation enhancement, and wound healing properties toward fibroblasts, associated to antimicrobial properties. Moreover, the in vivo results evidenced the capability of these NLC to enhance the healing process. Olive oil, which is characterized by a high content of oleic acid, proved to exert a synergic effect with eucalyptus oil with respect to antimicrobial activity and wound repair promotion.