Antonia Icaro Cornaglia

Effect of Electrospun Fiber Diameter and Alignment on Macrophage Activation and Secretion of Proinflammatory Cytokines and Chemokines

Macrophage activation can be modulated by biomaterial topography according to the biological scale (micrometric and nanometric range). In this study, we investigated the effect of fiber diameter and fiber alignment of electrospun poly(L-lactic) (PLLA) scaffolds on macrophage RAW 264.7 activation and secretion of proinflammatory cytokines and chemokines at 24 h and 7 days. Macrophages were cultured on four different types of fibrous PLLA scaffold (aligned microfibers, aligned nanofibers, random microfibers, and random nanofibers) and on PLLA film (used as a reference). Substrate topography was found to influence the immune response activated by macrophages, especially in the early inflammation stage. Secretion of proinflammatory molecules by macrophage cells was chiefly dependent on fiber diameter. In particular, nanofibrous PLLA scaffolds minimized the inflammatory response when compared with films and microfibrous scaffolds. The histological evaluation demonstrated a higher number of foreign body giant cells on the PLLA film than on the micro- and nanofibrous scaffolds. In summary, our results indicate that the diameter of electrospun PLLA fibers, rather than fiber alignment, plays a relevant role in influencing in vitro macrophage activation and secretion of proinflammatory molecules.

Early effects of AZT on mitochondrial functions in the absence of mitochondrial DNA depletion in rat myotubes

Zidovudine (AZT) is a potent inhibitor of human immunodeficiency virus (HIV) replication. In humans, as well as in animal models, long-term treatment with AZT induces a severe myopathy characterised by structural and functional alterations of mitochondria associated with depletion of mitochondrial DNA (mtDNA). In the present work, we compared the effects induced by AZT on mitochondria upon short- or long-term treatments of cultured rat myotubes. Morphological alterations were investigated by electron microscopy, and mtDNA depletion and deletions were analysed by Southern blot. Mitochondrial membrane potential was determined after JC-1 staining by laser-scanning confocal microscopy in whole cells, and by flow cytometry in isolated muscle mitochondria. We found that the early effects of AZT on mitochondrial functions were a marked, yet reversible reduction in mitochondrial membrane potential, in the absence of any effect on mtDNA. The long-term treatment, in addition to mitochondrial membrane potential alterations, induced morphological changes in mitochondria, and a remarkable reduction in the amount of mtDNA, without any significant evidence of mtDNA deletions. In both treatments, a block of the spontaneous contraction of myotubes was observed. To study in more detail the early effects induced by AZT, the ability of the drug to interact with cardiolipin, an important component of internal mitochondrial membrane, was investigated by atomic force microscopy (AFM) in an artificial membrane model system. The results suggest that the primary effects of AZT may be related to a physical interference with the membrane structure leading to a consequent modification of its physical characteristics.

Mutation analysis of five new patients affected by prolidase deficiency: the lack of enzyme activity causes necrosis-like cell death in cultured fibroblasts.

Abstract. Prolidase, a ubiquitously distributed dipeptidase, is involved in the latter stage of degradation of endogenous and dietary proteins and is particularly important in collagen catabolism. It hydrolyzes dipeptides containing proline or hydroxyproline at the C-terminal position. Mutations in the gene encoding for prolidase cause prolidase deficiency (PD), an autosomal recessive disorder mainly characterized by skin lesions, mental retardation and recurrent infectious. In this work we reported the identification of the molecular defect in five PD patients. Direct sequencing of PCR amplified genomic DNA showed a homozygous G>A transversion in two siblings leading to a G448R substitution. A heterozygous IVS11+1G>C transition causing the skipping of exon 11 and a null allele were detected in a third proband. In two unrelated patients, a homozygous IVS7-1G>A transversion was identified and shown to cause multiple alternative spliced transcripts. All the mutations result in loss of prolidase activity. Long-term cultured fibroblasts from these PD patients were used to develop an in vitro model that allowed investigation of the affected cells. Light and electron microscopy revealed that PD cells were more round and branched out than controls with increased cytosolic vacuolization, interruptions of the plasma membrane, mitochondria swelling, mitochondrial matrix and cristae modifications. JC-1 labeling showed decreased mitochondrial membrane potential. A significant intracellular accumulation of the Gly-Pro dipeptide was detected by capillary electrophoresis analysis. Our results provide the first evidence that absence of prolidase activity causes the activation of a necrosis-like cellular death, which could be responsible for the typical skin lesions in PD.

Long-Term miR-669a Therapy Alleviates Chronic Dilated Cardiomyopathy in Dystrophic Mice

Background Dilated cardiomyopathy (DCM) is a leading cause of chronic morbidity and mortality in muscular dystrophy (MD) patients. Current pharmacological treatments are not yet able to counteract chronic myocardial wastage, thus novel therapies are being intensely explored. MicroRNAs have been implicated as fine regulators of cardiomyopathic progression. Previously, miR‐669a downregulation has been linked to the severe DCM progression displayed by Sgcb‐null dystrophic mice. However, the impact of long‐term overexpression of miR‐669a on muscle structure and functionality of the dystrophic heart is yet unknown. Methods and Results Here, we demonstrate that intraventricular delivery of adeno‐associated viral (AAV) vectors induces long‐term (18 months) miR‐669a overexpression and improves survival of Sgcb‐null mice. Treated hearts display significant decrease in hypertrophic remodeling, fibrosis, and cardiomyocyte apoptosis. Moreover, miR‐669a treatment increases sarcomere organization, reduces ventricular atrial natriuretic peptide (ANP) levels, and ameliorates gene/miRNA profile of DCM markers. Furthermore, long‐term miR‐669a overexpression significantly reduces adverse remodeling and enhances systolic fractional shortening of the left ventricle in treated dystrophic mice, without significant detrimental consequences on skeletal muscle wastage. Conclusions Our findings provide the first evidence of long‐term beneficial impact of AAV‐mediated miRNA therapy in a transgenic model of severe, chronic MD‐associated DCM.

Halloysite and chitosan oligosaccharide nanocomposite for wound healing

Halloysite is a natural nanotubular clay mineral (HNTs, Halloysite Nano Tubes) chemically identical to kaolinite and, due to its good biocompatibility, is an attractive nanomaterial for a vast range of biological applications. Chitosan oligosaccharides are homo- or heterooligomers of N-acetylglucosamine and D-glucosamine, that accelerate wound healing by enhancing the functions of inflammatory and repairing cells. The aim of the work was the development of a nanocomposite based on HNTs and chitosan oligosaccharides, to be used as pour powder to enhance healing in the treatment of chronic wounds. A 1:0.05 wt ratio HTNs/chitosan oligosaccharide nanocomposite was obtained by simply stirring the HTNs powder in a 1% w/w aqueous chitosan oligosaccharide solution and was formed by spontaneous ionic interaction resulting in 98.6% w/w HTNs and 1.4% w/w chitosan oligosaccharide composition. Advanced electron microscopy techniques were considered to confirm the structure of the hybrid nanotubes. Both HTNs and HTNs/chitosan oligosaccharide nanocomposite showed good in vitro biocompatibility with normal human dermal fibroblasts up to 300μg/ml concentration and enhanced in vitro fibroblast motility, promoting both proliferation and migration. The HTNs/chitosan oligosaccharide nanocomposite and the two components separately were tested for healing capacity in a murine (rat) model. HTNs/chitosan oligosaccharide allowed better skin reepithelization and reorganization than HNTs or chitosan oligosaccharide separately. The results suggest to develop the nanocomposite as a medical device for wound healing. STATEMENT OF SIGNIFICANCE The present work is focused on the development of halloysite and chitosan oligosaccharide nanocomposite for wound healing. It considers a therapeutic option for difficult to heal skin lesions and burns. The significance of the research considers two fundamental aspects: the first one is related to the development of a self-assembled nanocomposite, formed by spontaneous ionic interaction, while the second one is related to the possibility to find an effective treatment for cutaneous non healing lesions. The characterization of this hybrid system involves a multidisciplinary approach considering integrated techniques of solid state investigation and advanced electron microscopies, and in vitro/in vivo models to understand biocompatibility and proliferation properties (enhancement of in vitro fibroblast motility, proliferation and migration, and of in vivo burn healing), to understand safety and effectiveness of the developed nanocomposite.

Occurrence, distribution and possible role of the regulatory peptide endothelin in the nasal mucosa

Nasal blood flow is finely regulated by local release of neurotransmitters, neuropeptides and other bioactive molecules acting via paracrine mechanisms. We have investigated the occurrence and distribution in human nasal mucosa of endothelin, a potent vasoconstrictor peptide, by immunocytochemistry and the effect of systemic administration of endothelin-1 on vascular perfusion of rabbit nasal mucosa by laser Doppler flowmetry. Endothelin-like immunoreactivity was demonstrated within vascular endothelial cells in both developing and mature human mucosa. Nasal epithelial cells and some connective tissue cells, presumed to be macrophages, also displayed specific immunostaining. In rabbits injected with endothelin-1, a potent and prolonged nasal vasoconstriction was observed. It is suggested that endothelin released locally may participate in the regulation of nasal blood flow via paracrine mechanisms. Since endothelin has growth-promoting actions on several cell types, it is also tentatively proposed that this regulatory peptide may play a role during development of the nose.

Detection of Bromo-Deoxyuridine-and Proliferating Cell Nuclear Antigen-Immunoreactivities in Tooth Germ

The development of antibodies to cell cycle-related antigens provides the basis for immunochemical studies on cell kinetics. Bromo-deoxyuridine (BrdU) incorporated by S-phase traversing cells is an exogenous marker of replicating cells, whereas proliferating cell nuclear antigen (PCNA) is an endogenous marker of replicating cells. We have applied monoclonal antibodies to BrdU and PCNA to study cell kinetics in tooth germ by immunohistochemistry and flow cytometry. BrdU-antibody reacted only with S phase-traversing cells in pulse-labelling experiments, whereas PCNA-antibody reacted with G1, S and G2-M phases traversing cells. Although the number of PCNA-positive cells largely exceeded the number of BrdU-labelled cells, the pattern distribution of immunoreactive cells was similar using BrdU- and PCNA-antibodies as revealed by immunohistochemistry. The use of PCNA-antibody allowed the detection of proliferating cells also in human tooth germ. It is suggested that combined identification of BrdU and PCNA on one side and growth factors, oncoproteins or differentiation markers on the other side may constitute a useful approach to understand the mechanisms of cell differentiation in tooth germ.

Synthesis and characterization of strontium-substituted hydroxyapatite nanoparticles for bone regeneration.

The production of stable suspensions of strontium-substituted hydroxyapatite (Sr-HA) nanopowders, as Sr ions vector for bone tissue regeneration, was carried out in the present work. Sr-HA nanopowders were synthesized via aqueous precipitation methods using Sr2+ amount from 0 to 100mol% and were characterized by several complementary techniques such as solid-state Nuclear Magnetic Resonance spectroscopy, X-ray diffraction, Infrared spectroscopy, N2 physisorption and Transmission Electron Microscopy. The substitution of Ca2+ with Sr2+ in HA is always isomorphic with gradual evolution between the two limit compositions (containing 100% Ca and 100% Sr), this pointing out the homogeneity of the synthesized nanopowders and the complete solubility of strontium in HA lattice. Strontium addition is responsible for an increasing c/a ratio in the triclinic unit cell. A significant variation of the nanopowders shape and dimension is also observed, a preferential growth along the c-axis direction being evident at higher strontium loads. Modifications in the local chemical environment of phosphate and hydroxyl groups in the apatite lattice are also observed. Stable suspensions were produced by dispersing the synthesized nanopowders in bovine serum albumin. Characterization by Dynamic Light Scattering and ζ-potential determination allowed to show that Ca2+→Sr2+ substitution influences the hydrodynamic diameter, which is always twice the particles size determined by TEM, the nanoparticles being always negatively charged as a result from the albumin rearrangement upon the interaction with nanoparticles surface. The biocompatibility of the suspensions was studied in terms of cell viability, apoptosis, proliferation and morphology, using osteosarcoma cell line SAOS-2. The data pointed out an increased cell proliferation for HA nanoparticles containing larger Sr2+ load, the cells morphology remaining essentially unaffected.

Dysplastic Histogenesis of Cartilage Growth Plate by Alteration of Sulphation Pathway: A Transgenic Model

Mutations in the diastrophic dysplasia sulphate transporter (dtdst) gene causes different forms of chondrodysplasia in the human. The generation of a knock-in mouse strain with a mutation in dtdst gene provides the basis to study developmental dynamics in the epiphyseal growth plate and long bone growth after impairment of the sulphate pathway. Our microscopical and histochemical data demonstrate that dtdst gene impairment deeply affects tissue organization, matrix structure, and cell differentiation in the epiphyseal growth plate. In mutant animals, the height of the growth plate was significantly reduced, according to a concomitant decrease in cell density and proliferation. Although the pathway of chondrocyte differentiation seemed complete, alteration in cell morphology compared to normal counterparts was detected. In the extracellular matrix, it we observed a dramatic decrease in sulphated proteoglycans, alterations in the organization of type II and type X collagen fibers, and premature onset of mineralization. These data confirm the crucial role of sulphate pathway in proteoglycan biochemistry and suggest that a disarrangement of the extracellular matrix may be responsible for the development of dtdts cartilage dysplasia. Moreover, we corroborated the concept that proteoglycans not only are structural components of the cartilage architecture, but also play a dynamic role in the regulation of chondrocyte growth and differentiation.

Particulate systems based on pectin/chitosan association for the delivery of manuka honey components and platelet lysate in chronic skin ulcers

The aim of the present work was the development of a powder formulation for the delivery of manuka honey (MH) bioactive components and platelet lysate (PL) in chronic skin ulcers. In particular pectin (PEC)/chitosan (CS) particles were prepared by ionotropic gelation in the presence of calcium chloride and subsequently characterized for particle size, hydration properties and mechanical resistance. Different experimental conditions (calcium chloride and CS concentrations; rest time in the cationic solution) were considered in order to obtain particles characterized by optimal size, hydration properties and mechanical resistance. Two different fractions of MH were examined: one (Fr1), rich in methylglyoxal and the other (Fr2), rich in polyphenols. Particles were loaded with Fr1, fraction able to enhance in vitro proliferation of human fibroblasts, and with PL. The presence of CS in Fr1-loaded particles produced an improvement in cell proliferation. Moreover, PL loading into particles did not affect the biological activity of the hemoderivative. In vivo efficacy of PL- and Fr1-loaded particles was evaluated on a rat wound model. Both treatments markedly increased wound healing to the same extent.