Nanotechnological Research for Regenerative Medicine: The Role of Hyaluronic Acid.

Hyaluronic acid (HA) is a linear, anionic, non-sulfated glycosaminoglycan occurring in almost all body tissues and fluids of vertebrates including humans. It is a main component of the extracellular matrix and, thanks to its high water-holding capacity, plays a major role in tissue hydration and osmotic pressure maintenance, but it is also involved in cell proliferation, differentiation and migration, inflammation, immunomodulation, and angiogenesis. Based on multiple physiological effects on tissue repair and reconstruction processes, HA has found extensive application in regenerative medicine. In recent years, nanotechnological research has been applied to HA in order to improve its regenerative potential, developing nanomedical formulations containing HA as the main component of multifunctional hydrogels systems, or as core component or coating/functionalizing element of nanoconstructs. This review offers an overview of the various uses of HA in regenerative medicine aimed at designing innovative nanostructured devices to be applied in various fields such as orthopedics, dermatology, and neurology.

Molecular and Structural Alterations of Skeletal Muscle Tissue Nuclei during Aging.

Aging is accompanied by a progressive loss of skeletal muscle mass and strength. The mechanisms underlying this phenomenon are certainly multifactorial and still remain to be fully elucidated. Changes in the cell nucleus structure and function have been considered among the possible contributing causes. This review offers an overview of the current knowledge on skeletal muscle nuclei in aging, focusing on the impairment of nuclear pathways potentially involved in age-related muscle decline. In skeletal muscle two types of cells are present: fiber cells, constituting the contractile muscle mass and containing hundreds of myonuclei, and the satellite cells, i.e., the myogenic mononuclear stem cells occurring at the periphery of the fibers and responsible for muscle growth and repair. Research conducted on different experimental models and with different methodological approaches demonstrated that both the myonuclei and satellite cell nuclei of aged skeletal muscles undergo several structural and molecular alterations, affecting chromatin organization, gene expression, and transcriptional and post-transcriptional activities. These alterations play a key role in the impairment of muscle fiber homeostasis and regeneration, thus contributing to the age-related decrease in skeletal muscle mass and function.

1954-2024: 70 years of histochemical research with the European Journal of Histochemistry.

This Editorial celebrates the 70th anniversary of the European Journal of Histochemistry since its foundation as Rivista di Istochimica Normale e Patologica, and introduces a Special Collection of selected articles on the application of the histochemical approach for investigating cell biological features and processes in animals and plants, and under diseased conditions. The year 2024 is a special one for histochemists, as 100 years ago J.W. Robert Feulgen and H. Rossenbeck introduced the histochemical procedure for the specific stoichiometric staining of DNA in histological samples: to commemorate this influential publication, three papers in the present issue are devoted to the application of the Feulgen reaction at light and electron microscopy, and in cytometry.

Modifications of Blood Molecular Components after Treatment with Low Ozone Concentrations.

The ex vivo treatment of a limited volume of blood with gaseous oxygen-ozone (O2-O3) mixtures and its rapid reinfusion into the patient is a widespread medical procedure. O3 instantly reacts with the blood's antioxidant systems, disappearing before reinfusion, although the molecules formed act as messengers in the organism, inducing multiple antioxidant and anti-inflammatory responses. An appropriate dose of O3 is obviously essential to ensure both safety and therapeutic efficacy, and in recent years, the low-dose O3 concept has led to a significant reduction in the administered O3 concentrations. However, the molecular events triggered by such low concentrations in the blood still need to be fully elucidated. In this basic study, we analysed the molecular modifications induced ex vivo in sheep blood by 5 and 10 µg O3/mL O2 by means of a powerful metabolomics analysis in association with haemogas, light microscopy and bioanalytical assays. This combined approach revealed increased oxygenation and an increased antioxidant capacity in the O3-treated blood, which accorded with the literature. Moreover, original information was obtained on the impact of these low O3 concentrations on the metabolic pathways of amino acids, carbohydrates, lipids and nucleotides, with the modified metabolites being mostly involved in the preservation of the oxidant-antioxidant balance and in energy production.

Aged gastrocnemius muscle of mice positively responds to a late onset adapted physical training.

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

Ozone and procaine increase secretion of platelet-derived factors in platelet-rich plasma.

Platelet-rich plasma (PRP) is gaining more and more attention in regenerative medicine as an innovative and efficient therapeutic approach. The regenerative properties of PRP rely on the numerous bioactive molecules released by the platelets: growth factors are involved in proliferation and differentiation of endothelial cells and fibroblasts, angiogenesis and extracellular matrix formation, while cytokines are mainly involved in immune cell recruitment and inflammation modulation. Attempts are ongoing to improve the therapeutic potential of PRP by combining it with agents able to promote regenerative processes. Two interesting candidates are ozone, administered at low doses as gaseous oxygen-ozone mixtures, and procaine. In the present study, we investigated the effects induced on platelets by the in vitro treatment of PRP with ozone or procaine, or both. We combined transmission electron microscopy to obtain information on platelet modifications and bioanalytical assays to quantify the secreted factors. The results demonstrate that, although platelets were already activated by the procedure to prepare PRP, both ozone and procaine induced differential morpho-functional modifications in platelets resulting in an increased release of factors. In detail, ozone induced an increase in surface protrusions and open canalicular system dilation suggestive of a marked α-granule release, while procaine caused a decrease in surface protrusions and open canalicular system dilation but a remarkable increase in microvesicle release suggestive of high secretory activity. Consistently, nine of the thirteen platelet-derived factors analysed in the PRP serum significantly increased after treatment with ozone and/or procaine. Therefore, ozone and procaine proved to have a remarkable stimulating potential without causing any damage to platelets, probably because they act through physiological, although different, secretory pathways.

A novel in-frame deletion in MYOT causes an early adult onset distal myopathy.

Missense mutations in MYOT encoding the sarcomeric Z-disk protein myotilin cause three main myopathic phenotypes including proximal limb-girdle muscular dystrophy, spheroid body myopathy, and late-onset distal myopathy. We describe a family carrying a heterozygous MYOT deletion (Tyr4_His9del) that clinically was characterized by an early-adult onset distal muscle weakness and pathologically by a myofibrillar myopathy (MFM). Molecular modeling of the full-length myotilin protein revealed that the 4-YERPKH-9 amino acids are involved in local interactions within the N-terminal portion of myotilin. Injection of in vitro synthetized mutated human MYOT RNA or of plasmid carrying its cDNA sequence in zebrafish embryos led to muscle defects characterized by sarcomeric disorganization of muscle fibers and widening of the I-band, and severe motor impairments. We identify MYOT novel Tyr4_His9 deletion as the cause of an early-onset MFM with a distal myopathy phenotype and provide data supporting the importance of the amino acid sequence for the structural role of myotilin in the sarcomeric organization of myofibers.

Physical Training Chronically Stimulates the Motor Neuron Cell Nucleus in the Ts65Dn Mouse, a Model of Down Syndrome.

Down syndrome (DS) is a genetically-based disease based on the trisomy of chromosome 21 (Hsa21). DS is characterized by intellectual disability in association with several pathological traits among which early aging and altered motor coordination are prominent. Physical training or passive exercise were found to be useful in counteracting motor impairment in DS subjects. In this study we used the Ts65Dn mouse, a widely accepted animal model of DS, to investigate the ultrastructural architecture of the medullary motor neuron cell nucleus taken as marker of the cell functional state. Using transmission electron microscopy, ultrastructural morphometry, and immunocytochemistry we carried out a detailed investigation of possible trisomy-related alteration(s) of nuclear constituents, which are known to vary their amount and distribution as a function of nuclear activity, as well as the effect of adapted physical training upon them. Results demonstrated that trisomy per se affects nuclear constituents to a limited extent; however, adapted physical training is able to chronically stimulate pre-mRNA transcription and processing activity in motor neuron nuclei of trisomic mice, although to a lesser extent than in their euploid mates. These findings are a step towards understanding the mechanisms underlying the positive effect of physical activity in DS.

Physical training promotes remodeling of the skeletal muscle extracellular matrix: An ultrastructural study in a murine model of Down syndrome.

Down syndrome (DS) is a genetically based disease caused by triplication of chromosome 21. DS is characterized by multi-systemic premature aging associated with deficit in motor coordination, balance, and postural control. Using a morphological, morphometrical, and immunocytochemical ultrastructural approach, this study investigated in vastus lateralis muscle of Ts65Dn mouse, a murine model of DS, the effect of an adapted physical training on the extracellular matrix (ECM) characteristics and whether the forecasted exercise-induced ECM remodeling impacts on sarcomere organization. Morphometry demonstrated thicker basement membrane and larger collagen bundles with larger interfibrillar spacing as well as irregularly arrayed myofibrils and lower telethonin density on Z-lines in trisomic versus euploid sedentary mice. In agreement with the multi-systemic premature aging described in DS, these ECM alterations were similar to those previously observed in skeletal muscle of aged mice. Adapted physical training induced remodeling of ECM in both trisomic and euploid mice, that is, enlargement of the collagen bundles associated with hypertrophy of collagen fibrils and reduction of the interfibrillar spacing. A re-alignment of the myofibrils and a higher telethonin density on Z-line was found in trisomic mice. Altogether, our findings suggest that physical training is an effective tool in limiting/counteracting the trisomy-associated musculoskeletal structural anomalies. The current findings constitute a solid experimental background for further study investigating the possible positive effect of physical training on skeletal muscle performance. RESEARCH HIGHLIGHTS: Vastus lateralis muscle of trisomic mice shows aging-like alterations of extracellular matrix. Training promotes extracellular matrix remodeling. Training may be an effective tool to counteract trisomy-associated alterations of skeletal muscle.

Mitochondrial Features of Mouse Myoblasts Are Finely Tuned by Low Doses of Ozone: The Evidence In Vitro.

The mild oxidative stress induced by low doses of gaseous ozone (O3) activates the antioxidant cell response through the nuclear factor erythroid 2-related factor 2 (Nrf2), thus inducing beneficial effects without cell damage. Mitochondria are sensitive to mild oxidative stress and represent a susceptible O3 target. In this in vitro study, we investigated the mitochondrial response to low O3 doses in the immortalized, non-tumoral muscle C2C12 cells; a multimodal approach including fluorescence microscopy, transmission electron microscopy and biochemistry was used. Results demonstrated that mitochondrial features are finely tuned by low O3 doses. The O3 concentration of 10 µg maintained normal levels of mitochondria-associated Nrf2, promoted the mitochondrial increase of size and cristae extension, reduced cellular reactive oxygen species (ROS) and prevented cell death. Conversely, in 20 µg O3-treated cells, where the association of Nrf2 with the mitochondria drastically dropped, mitochondria underwent more significant swelling, and ROS and cell death increased. This study, therefore, adds original evidence for the involvement of Nrf2 in the dose-dependent response to low O3 concentrations not only as an Antioxidant Response Elements (ARE) gene activator but also as a regulatory/protective factor of mitochondrial function.

Endotracheal nebulization of gold nanoparticles for noninvasive pulmonary drug delivery.

Background & aims: Gold nanoparticles (AuNPs) are useful tools for noninvasive drug delivery. AuNP nebulization has shown poor deposition results, and AuNP tracking postadministration has involved methods inapplicable to clinical settings. The authors propose an intratracheal delivery method for minimal AuNP loss and computed tomography scans for noninvasive tracking. Materials & methods: Through high-frequency and directed nebulization postendotracheal intubation, the authors treated rats with AuNPs. Results & conclusion: The study showed a dose-dependent and bilateral distribution of AuNPs causing no short-term distress to the animal or risk of airway inflammation. The study demonstrated that AuNPs do not deposit in abdominal organs and show targeted delivery to human lung fibroblasts, offering a specific and noninvasive strategy for respiratory diseases requiring long-term therapies.

This study presents an alternative method for drug delivery involving gold nanoparticle aerosolization directly into the major airways. Direct nebulization prevents particle loss and avoids drug administration through the blood. The particles can be detected successfully via upper body scans, which are noninvasive and allow for on-demand monitoring. Nanoparticles are flexible tools that can be modified to target specific cells of interest and can be excreted upon completion of their function. These results could represent an alternative method of drug administration in patients needing repeated cytotoxic therapies with known off-target effects.

An Innovative Fluid Dynamic System to Model Inflammation in Human Skin Explants.

Skin is a major administration route for drugs, and all transdermal formulations must be tested for their capability to overcome the cutaneous barrier. Therefore, developing highly reliable skin models is crucial for preclinical studies. The current in vitro models are unable to replicate the living skin in all its complexity; thus, to date, excised human skin is considered the gold standard for in vitro permeation studies. However, skin explants have a limited life span. In an attempt to overcome this problem, we used an innovative bioreactor that allowed us to achieve good structural and functional preservation in vitro of explanted human skin for up to 72 h. This device was then used to set up an in vitro inflammatory model by applying two distinct agents mimicking either exogenous or endogenous stimuli: i.e., dithranol, inducing the contact dermatitis phenotype, and the substance P, mimicking neurogenic inflammation. Our in vitro system proved to reproduce inflammatory events observed in vivo, such as vasodilation, increased number of macrophages and mast cells, and increased cytokine secretion. This bioreactor-based system may therefore be suitably and reliably used to simulate in vitro human skin inflammation and may be foreseen as a promising tool to test the efficacy of drugs and cosmetics.

In memoriam of Prof. Stan Fakan.

In memoriam of Prof. Stan Fakan.

Nanoparticle-Based Techniques for Bladder Cancer Imaging: A Review.

Bladder cancer is very common in humans and is often characterized by recurrences, compromising the patient's quality of life with a substantial social and economic impact. Both the diagnosis and treatment of bladder cancer are problematic due to the exceptionally impermeable barrier formed by the urothelium lining the bladder; this hinders the penetration of molecules via intravesical instillation while making it difficult to precisely label the tumor tissue for surgical resection or pharmacologic treatment. Nanotechnology has been envisaged as an opportunity to improve both the diagnostic and therapeutic approaches for bladder cancer since the nanoconstructs can cross the urothelial barrier and may be functionalized for active targeting, loaded with therapeutic agents, and visualized by different imaging techniques. In this article, we offer a selection of recent experimental applications of nanoparticle-based imaging techniques, with the aim of providing an easy and rapid technical guide for the development of nanoconstructs to specifically detect bladder cancer cells. Most of these applications are based on the well-established fluorescence imaging and magnetic resonance imaging currently used in the medical field and gave positive results on bladder cancer models in vivo, thus opening promising perspectives for the translation of preclinical results to the clinical practice.

An ex vivo experimental system to track fluorescent nanoparticles inside skeletal muscle.

The development of novel nanoconstructs for biomedical applications requires the assessment of their biodistribution, metabolism and clearance in single cells, organs and entire organisms in a living environment. To reduce the number of in vivo experiments performed and to refine the methods used, in accordance with the 3Rs principle, this work proposes an ex vivo experimental system to monitor, using fluorescence microscopy, the distribution of nanoparticles in explanted murine skeletal muscle maintained in a bioreactor that can preserve the structural and functional features of the organ for long periods of time. Fluorescently-labelled liposomes and poly(lactide-co-glycolide) (PLGA)-based nanoparticles were injected into the intact soleus muscle (in the distal region close to the tendon) immediately after explants, and their distribution was analysed at increasing incubation times in cross cryosections from the proximal region of the belly. Both nanocarriers were clearly recognized in the muscle and were found to enter and migrate inside the myofibres, whereas their migration in the connective tissue seemed to be limited. In addition, some fluorescent signals were observed inside the macrophages, demonstrating the physiological clearance of the nanocarriers that did not enter the myofibres. Our ex vivo system therefore provides more information than previous in vitro experiments on cultured muscle cells, highlighting the need for the appropriate functionalization of nanocarriers if myofibre targeting is to be improved.

Repurposing pentamidine using hyaluronic acid-based nanocarriers for skeletal muscle treatment in myotonic dystrophy.

In a context of drug repurposing, pentamidine (PTM), an FDA-approved antiparasitic drug, has been proposed to reverse the splicing defects associated in myotonic dystrophy type 1 (DM1). However, clinical use of PTM is hinder by substantial toxicity, leading to find alternative delivery strategies. In this work we proposed hyaluronic acid-based nanoparticles as a novel encapsulation strategy to efficiently deliver PTM to skeletal muscles cells. In vitro studies on C2C12 myoblasts and myotubes showed an efficient nanoparticles' internalization with minimal toxicity. More interestingly, our findings evidenced for the first time the endosomal escape of hyaluronic acid-based nanocarriers. Ex vivo studies showed an efficient nanoparticles' internalization within skeletal muscle fibers. Finally, the therapeutic efficacy of PTM-loaded nanosystems to reduce the number of nuclear foci has been demonstrated in a novel DM1 in vitro model. So far, current data demonstrated the potency of hyaluronic acid-based nanosystems as efficient nanocarrier for delivering PTM into skeletal muscle and mitigate DM1 pathology.

Alcian Blue Staining to Visualize Intracellular Hyaluronic Acid-Based Nanoparticles.

Investigating at transmission electron microscopy the intracellular trafficking of hyaluronic acid-based nanoparticles remains a challenge due to their intrinsic weak electron density. Here we describe a simple protocol to stain hyaluronic acid that allows visualization of hyaluronic acid-based nanoparticles inside cells at both light and electron microscopy. By applying the critical-electrolyte-concentration Alcian blue method, these nanoparticles were observed as blue dots at bright-field microscopy or filled with fine electron dense precipitates at transmission electron microscopy.

Diaminobenzidine Photooxidation to Visualize Fluorescent Nanoparticles in Adhering Cultured Cells at Transmission Electron Microscopy.

Visualizing nanoparticles made of organic material (e.g., polysaccharides, proteins, non-osmiophilic lipids) inside cells and tissues at transmission electron microscopy is a difficult task due to the intrinsic weak electron density of these nanoconstructs, which makes them hardly distinguishable in the biological environment. We describe here a simple protocol to apply photooxidation to fluorescently labeled nanoparticles administered to cultured cells in vitro. The conversion of the fluorescent signal into a granular electron-dense reaction product through light irradiation in the presence of diaminobenzidine makes the nanoparticles clearly visible at the ultrastructural level. Our procedure proved to be reliable with various fluorophores and may be applied to any cell type.

Ex Vivo Evaluation of Ethosomes and Transethosomes Applied on Human Skin: A Comparative Study.

In this study, the transdermal fate of vesicular nanosystems was investigated. Particularly, ethosomes based on phosphatidylcholine 0.9% w/w and transethosomes based on phosphatidylcholine 0.9 or 2.7% w/w plus polysorbate 80 0.3% w/w as an edge activator were prepared and characterized. The vesicle mean size, morphology and deformability were influenced by both phosphatidylcholine and polysorbate 80. Indeed, the mean diameters of ethosome were around 200 nm, while transethosome's mean diameters were 146 or 350 nm in the case of phosphatidylcholine 0.9 or 2.7%, w/w, respectively. The highest deformability was achieved by transethosomes based on phosphatidylcholine 0.9%, w/w. The three types of vesicular nanosystems were applied on explanted human skin maintained in a bioreactor. Transmission electron microscopy demonstrated that all vesicles were able to enter the skin, keeping their structural integrity. Notably, the vesicle penetration capability was influenced by their physical-chemical features. Indeed, ethosomes reached keratinocytes and even the dermis, phosphatidylcholine 0.9% transethosomes were found in keratinocytes and phosphatidylcholine 2.7% transethosomes were found only in corneocytes of the outer layer. These findings open interesting perspectives for a differentiated application of these vesicles for transdermal drug delivery as a function of the cutaneous pathology to be addressed.

Assessing the interactions between nanoparticles and biological barriers in vitro: a new challenge for microscopy techniques in nanomedicine.

Nanoconstructs intended to be used as biomedical tool must be assessed for their capability to cross biological barriers. However, studying in vivo the permeability of biological barriers to nanoparticles is quite difficult due to the many structural and functional factors involved. Therefore, the in vitro modeling of biological barriers -2D cell monocultures, 2D/3D cell co-cultures, microfluidic devices- is gaining more and more relevance in nanomedical research. Microscopy techniques play a crucial role in these studies, as they allow both visualizing nanoparticles inside the biological barrier and evaluating their impact on the barrier components. This paper provides an overview of the various microscopical approaches used to investigate nanoparticle translocation through in vitro biological barrier models. The high number of scientific articles reported highlights the great contribution of the morphological and histochemical approach to the knowledge of the dynamic interactions between nanoconstructs and the living environment.