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13 p.-7 fig.-1 tab., Inducible T-cell costimulator (ICOS) upon binding to its ligand (ICOSL) mediates adaptive immunity and antitumor response. Thus, antitumor therapies targeting the ICOS/ICOSL pathway hold great promise for cancer treatment. In this regard, ICOSL triggering by a soluble recombinant form of ICOS (ICOS-Fc) hampered adhesiveness and migration of dendritic, endothelial, and tumor cells in vitro. Furthermore, in vivo treatment with ICOS-Fc previously showed the capability to inhibit lung metastatization of ICOSL+ B16-F10 melanoma cells when injected intravenously in mice, but it failed to block the growth of established subcutaneous B16-F10 murine tumors. Thus, we asked whether passive targeting of solid tumors with ICOS-Fc-loaded biocompatible and biodegradable nanoparticles (NPs) could instead prove effectiveness in reducing tumor growth. Here, ICOS-Fc was loaded in two types of polymer nanoparticles, i.e. cross-linked β-cyclodextrin nanosponges (CDNS) and poly(lactic-co-glycolic acid) (PLGA) NPs and in vitro characterized. In vivo experiments showed that treatment of C57BL6/J mice with ICOS-Fc loaded into the two nanoformulations inhibits the growth of established subcutaneous B16-F10 tumors. This anticancer activity appears to involve both anti-angiogenic and immunoregulatory effects, as shown by decreased tumor vascularization and downmodulation of IL-10 and Foxp3, two markers of regulatory T cells (Tregs). Overall, the substantial in vivo anticancer activity of ICOS-Fc-loaded CDNS and PLGA NPs against different components of the tumor microenvironment makes these nanoformulations attractive candidates for future combination cancer therapy., This work was supported by the Associazione Italiana Ricerca sul Cancro (IG 20714, AIRC, Milano), Fondazione Amici di Jean (Torino), and Fondazione Cariplo (2017–0535).

Aims Monocytes/macrophages are essential in innate immune response against pathogens also because their ability to release extracellular traps named METs (monocytes/macrophages extracellular traps). These structures are composed of DNA fibers decorated with nuclear and cytoplasmic proteins and their production process is called METosis. In this study attention has been focused on the ability of differently charged molecular systems (polyhedral oligomeric silsesquioxanes, POSS positively or negatively charged) to induce METosis. Main methods METs formation was induced by lipopolysaccharide (250 μg/ml, positive control) and POSS positive and negative (0.05–1 mg/ml) treatment. METs were visualized and quantified by confocal microscopy using Sytox green staining. Oxidative stress, autophagy, as well as endocytosis involvement in the POSS induced METosis was evaluated. Key findings Results obtained indicate a POSS positive or negative dose dependent ability in inducing MET release independently to their charge and that this phenomenon is a consequence of POSS +/− internalization. Moreover, studies using many reactive oxidative species (ROS) blockers and autophagy inhibitor showed a strong reduction in POSS induced METosis indicating their involvement. Significance POSS +/− induce extracellular traps production in human monocytes/macrophages by oxidative and autophagic pathway.

Photodinamic therapy (PDT) has gained an increasing interest as a new tool to treat skin cancers such as melanoma. This clinical approach take advantage from the combination of a photosensitizer and a specific light wavelength able to induce singlet oxygen production. Mesoporous silica nanoparticles (MSNs) have been widely investigated as drug nanocarriers as their structure and morphology could be customized to produce suitable nanoplatforms enabling high cargo capacity. In the present study MSNs were successfully conjugated with the second generation photosensitizer verteporfin and the resulting nanoplatform (Ver-MSNs) was tested in an in vitro PDT model as a potential tool for melanoma treatment. Ver-MSNs based PDT did not affect cell proliferation of neither a normal human keratinocyte cell line (HaCaT) or a low mestastatic melanoma cell line (A375P). On the other hand Ver-MSNs based PDT deeply affect the highly invasive SK-MEL-28 melanoma cell line behavior, as testified by the strong reduction in cell proliferation along with the dramatic change in cellular morphology, through a nanoparticle internalization dependent mechanism. In fact, experiments performed in the presence of endocytosis inhibitors (chlorpromazine and amiloride) resulted in an attenuation of Ver-MSNs based PDT induced cell death, along with a recover in cellular morphology. MSN doped with verteporfin could thus represent a promising and useful tool for PDT treatment of highly invasive melanoma.

Melanoma is one of the most lethal tumors among the skin cancers, arising from complex genetic mutations in melanocyte. Melanoma microenvironment is very heterogeneous, showing complex vascular networks and immunogenicity, as well as induced acquired resistance to treatments by upregulation of multidrug resistance (MDR) mechanisms. Different studies have showed that Photodynamic Therapy (PDT) could be considered a new potential approach for melanoma treatment. PDT combines a light with a specific wavelength and a photosensitizer: when these two elements interact reactive oxygen species (ROS) are generated leading to tumor cell destruction. In this study verteporfin (Ver), a second-generation photosensitizer, has been conjugated with mesoporous silica nanoparticles (MSNs): the resulting Ver-MSNs are an efficient nanoplatforms used to enhance cargo capacity and cellular uptake. Our in vitro and in vivo studies investigated whether Ver-MSNs were able to reduce or inhibit melanoma growth. In vitro experiments performed using B16F10 mouse melanoma cells showed that Ver-MSNs stimulated by red light (693 nm) significantly decreased in vitro cells proliferation in a range of concentration between 0.1 μg/ml to 10 μg/ml. When Ver-MSNs (5 μg/ml in glycerol) were topically administrated to melanoma tumor mass developed in mice and stimulated by red light for four times in 16 days, they were able to reduce the tumor mass of 50.2 ± 6,6% compared to the untreated (only glycerol) mice. In the light of this information, PDT performed using Ver-MSNs could be considered a new promising and potential approach to treat melanoma.

The deposition of organic functionalities on biomaterials to immobilize biomolecules is a research area of great interest in the medical field. The surface functionalization of a 3D porous scaffolds of PDLLA with carboxyl (-COOH) and amino (-NH2) groups by cold plasma treatment at atmospheric pressure is described in this paper. Two methods of continuous and pulsed plasma deposition were compared to assess the degree of functionalization of the internal porous 3D scaffold. In particular, the pulsed plasma treatment was found to functionalize uniformly not only the sample surface but also inside the open cavities thanks to its permeability and diffusion in the porous 3D scaffold. The species developed in the plasma were studied by optical emission spectroscopy (OES) technique, while the functionalization of the sponges was evaluated by the Diffuse Reflectance Fourier-Transform Infrared Spectroscopy (DR-FTIR) technique using also the adsorption of ammonia (NH3) and deuterated water (D2O) probe molecules. The functional groups were deposited only on the front of the sponge, then the structural characterization of both front and back of the sponge has demonstrated the uniform functionalization of the entire scaffold.

A highly efficient nanoplatform for photodynamic therapy was obtained by covalent conjugation of verteporfin with mesoporous silica nanoparticles. Verteporfin has an intense absorption band in the red region of the Vis spectrum ensuring a deeper tissue penetration; this feature makes verteporfin-based formulations particularly attractive for in vivo PDT applications. The verteporfin loading, dispersion and efficiency in the photoproduction and delivery of singlet oxygen (1O2) were carefully evaluated. In vitro tests have evidenced that the optimized nanoplatform is able to largely reduce HeLa cell proliferation after red-light irradiation.

Case-control study.To assess serum myostatin levels, bone mineral density (BMD), appendicular skeletal muscle mass (ASMM) and serum sclerostin levels in chronic spinal cord injured (SCI) patients and healthy controls.SCI centre in Italy.Blood samples, whole-body bioelectrical impedance analysis and BMD measurement with the ultrasound technique at the calcaneus level were taken from patients suffering from chronic SCI (both motor complete and incomplete) and healthy control subjects.A total of 28 SCI patients and 15 healthy controls were enrolled. Serum myostatin levels were statistically higher (P0.01) in SCI patients compared with healthy controls. Similar results were found comparing both the motor complete and the motor incomplete SCI subgroups to healthy controls. Serum sclerostin was significantly higher in patients with SCI compared with healthy controls (P0.01). BMD, stiffness and mean T-score values in SCI patients were significantly lower than those in healthy controls. Serum myostatin concentrations in the motor complete SCI subgroups correlated only with serum sclerostin levels (r(2)=0.42; P=0.001) and ASMM (r(2)=0.70; P=0.002) but not in healthy controls.Serum myostatin and serum sclerostin are significantly higher in chronic SCI patients compared with healthy controls. They are potential biomarkers of muscle and bone modifications after SCI. This is the first study reporting an increase in serum myostatin in patients suffering from chronic SCI and a correlation with ASMM.

Photobiomodulation is a widely used tool in regenerative medicine thanks to its ability to modulate a plethora of physiological responses. Wound re-epithelialization is strictly regulated by locally produced chemical mediators, such as nitric oxide (NO), a highly reactive free radical generated by the nitric oxide synthase (NOS) enzymatic family. In this study, it has been hypothesized that a 980-nm low-level laser stimulation could increase NO production in human keratinocytes and that such event might be directly related to the re-epithelialization process. Human keratinocytes were irradiated with increasing energy outputs (10–75 J) in the absence or presence of L-NAME, a NOS inhibitor. Laser stimulation induced an increase in NO production, resulting in an energy-dependent increase in both keratinocytes proliferation and re-epithelialization ability. The direct link between increased NO production and the observed physiological responses was confirmed by their inhibition in L-NAME pre-treated samples. Since NO production increase is a quick event, it is conceivable that it is due to an increase in existing NOS activity rather than to a de novo protein synthesis. For this reason, it could be hypothesized that photobiomodulation-derived NO positive effects on keratinocytes behavior might rely on a near infrared mediated increase in NOS conformational stability and cofactors as well as substrate binding ability, finally resulting in an increased enzymatic activity.

Organically modified mesoporous silica nano- particles (MSNs) containing rose bengal (RB), a xanthene dye, were successfully synthesized. RB-modified MSNs have shown a relevant photostability and a high efficiency in the photoproduction and delivery of singlet oxygen ( 1 O2), which is particularly promising for photodynamic therapy (PDT) applications. In vitro tests have evidenced that RB-MSNs are able to reduce cell proliferation in one of the most aggressive skin cancer types (SK-MEL-28) after green-light irradiation.

Low-level laser therapy (LLLT) is widely used in regenerative medicine and in dental therapy by virtue of its beneficial effects in a plethora of pathological conditions. In this study, the effect of a 980 nm diode laser on pre-osteoblasts proliferation has been evaluated, along with reactive oxygen species (ROS) production. We hypothesized that ROS were a key factor in LLLT-induced pre-osteoblasts proliferation, as it is known that ROS can induce the activation of many biological pathways, leading to cell proliferation, differentiation or apoptosis. Murine pre-osteoblasts MC3T3 cells were irradiated with different energy outputs (1-50 J) in the absence or presence of the antioxidant N-Acetyl-L-cysteine (NAC). Laser treatment, in the absence of NAC, was able to induce a fluence-dependent statistically significant increase in ROS generation, while the presence of NAC strongly inhibited it. Cell proliferation, measured after laser stimulation, was significantly increased both at low and higher energy, with a peak at 10 J in the absence of the antioxidant. On the contrary, in the presence of NAC, laser irradiation was not able to induce any cell proliferation, suggesting a crucial role of ROS in this laser-induced cell effect. These results suggest that LLLT may be a useful tool for bone regeneration therapy and an effective range of fluences to be used is indicated.

Background/Aims: The 1α,25-dihydroxycholecalciferol (Vit. D) induces eNOS dependent nitric oxide (NO) production in human umbilical vein endothelial cells (HUVEC). To our knowledge, there are no reports directly relating Vit. D induced NO production to proliferation and/or migration in endothelial cells (EC). The aim of this study was to evaluate whether Vit. D addition to porcine EC could affect their proliferation and/or migration in a three-dimensional matrix via NO production. Materials and Methods: Porcine aortic endothelial cells (PAE) were used to evaluate Vit. D effects on cell proliferation and migration in a three-dimensional matrix. Results: Vit. D induced NO production in PAE cells. Moreover, it induced a significant increase in cellular proliferation and migration in a three-dimensional matrix. These effects were NO dependent, as inhibiting eNOS activity by L-NAME PAE migration was abrogated. This effect was strictly related to MMP-2 expression and apparently dependent on Vit. D and NO production. Conclusions: Vit. D can promote both endothelial cells proliferation and migration in a three-dimensional matrix via NO-dependent mechanisms. These findings cast new light on the role of Vit. D in the angiogenic process, suggesting new applications for Vit. D in such fields as tissue repair and wound healing.

Aim: To evaluate, in an in vitro wound healing model, the effect of zoledronate on keratinocyte cellular behavior. -Materials and Methods: Human spontaneously immortalized keratinocytes (HaCaT) were treated with low zoledronate concentrations (10 nmol/l to 10 µmol/l) and its effect on cell proliferation was evaluated by means of a fluorescent assay (Tox-8), along with the analysis of cytokeratin 5 and filaggrin gene expression. Moreover, zoledronate effects on cell migration were evaluated by in vitro wound healing, and matrix metalloproteinase (MMP) activity was investigated by gelatin zymography. Results: At the tested concentrations, zoledronate stimulated keratinocyte proliferation, upregulating cytokeratin 5 while downregulating filaggrin expression and wound healing ability, without any significant effect on MMP-9 activity. The lack of zoledronate effects on MMP-9 activity indicates that, in our experimental model, wound closure is mainly due to an increase in cell proliferation rather than an increase in cell migration. Moreover, the observed increase in cell proliferation could be ascribed to a zoledronate-mediated reduction of farnesyl pyrophosphate endogenous levels. Conclusions: These results could foster new clinical applications for this ‘old' drug in the field of epithelial regeneration.

Biocompatible and resorbable polylactic acid (PLA) derived polymers are used for clinical applications. Antioxidant vitamin E (Vit.E) and vitamin E acetate (Vit.E Ac), a Vit.E commercial analog, were added to PLA during films preparation. Wettability, protein adsorption analysis, platelet adhesion and whole blood coagulation experiments highlighted that Vit.E incorporation improved PLA hemocompatibility. NMR analysis showed that Vit.E and Vit.E Ac blending differentially altered PLA polymer organization with Vit.E leading to the formation of microcavities, as observed by SEM analysis, while reducing surface hydrophobicity. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013

Novel hydrogel composites based on natural gelatin matrix and containing different loading of a water soluble octa-am- monium POSS (POSS-NH3 þ ), (from 0.5 to 2 wt%) have been prepared in this work and their biocompatibility and mechanical behavior have been tested. The presence of 1 wt% of POSS-NH3 þ molecules in the hydrogel matrix increased the amount of human plasma proteins adsorption and human keratinocyte adhesion at 24 hours. In addition, the presence of increasing amounts of POSS in the hydrogel matrix allowed to a significantly increase of the matrix swelling degree at acid and neutral pH. Finally, a significant modulation of the mechanical characteristics (Young modulus, strain at break) of the hydrogel matrix induced by the presence of dif- ferent amounts of POSS-NH3 þ was observed. The novel ability of POSS-NH3 þ to modulate both biological and mechanical proper- ties of a natural hydrogel could be useful for the production of new composites of interest for soft tissue engineering. V C 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 129: 699-706, 2013

Our skin is in close contact with clothes most of the time thus risking potentially noxious chemicals contact. One of the potentially harmful manufacturing by-products that can be released by textiles when sweating is formaldehyde, used as an anti-crease treatment. As it is known to be carcinogenic to humans and a potent skin sensitizer, the aim of this study was to investigate its effects on both normal human keratinocytes (HaCaT cells) and on a highly invasive malignant melanoma cell line (SK-MEL-28) in order to contribute to the definition of safety cut-off to be applied to the production processes. Formaldehyde concentrations below the commonly accepted limits (10-50μM) were obtained by diluting formaldehyde in simulated sweat (UNI EN ISO 105-E04). The effects on cell proliferation were evaluated by cell counting, while ERK pathway activation was evaluated by western blot. Low concentrations of formaldehyde (10μM) in both acidic and alkaline simulated sweat were able to increase malignant melanoma cell proliferation, while not affecting normal keratinocytes. Melanoma proliferation increase was greater in acidic (pH=5.5) than in alkaline (pH=8) conditions. Moreover, formaldehyde stimulation was able to induce ERK pathway activation. The data obtained suggest the need for an even increasing attention to the potentially harmful effects of textile manufacturing by-products.

This in vitro experimental study has been designed to assess the effects of photodynamic antimicrobial chemotherapy (PACT) on human keratinocytes proliferation. Human keratinocytes (HaCaT) monolayers (∼0.5 cm(2)) have been irradiated with 635 nm red laser light with a fluence of 82.5 or 112.5 J/cm(2) in the absence or presence of toluidine (TB). Cell proliferation, monolayer area coverage, cytokeratin 5 (K5) and filaggrin (Fil) expression, and metalloproteinase (MMP)-2 and MMP-9 activity were measured after 72 h from laser irradiation. HaCaT proliferation was reduced by TB staining. Cell exposure to both low- and high-fluence laser irradiation in both presence and absence of TB staining reduced their proliferation and monolayer area extension. Moreover both laser treatments were able to reduce K5 and Fil expression and MMP-9 production in keratinocytes not treated with TB. These data indicate that PACT could exert toxic effects on normal proliferating keratinocytes present around parodontal pockets. The observed reduced cell proliferation along with a reduced production of enzymes involved in wound healing could alter the clinical outcome of the patients treated with PACT.

Polylactic acid (PLA)-derived polymers are widely used in many biotechnological fields, thanks to their biocompatibility and resorbability through natural pathways. Moreover, PLA is one of the few polymers in which both the properties and the stereochemical structure can be easily modified, making possible a specific ‘tailoring’ of the final polymer according to the desired use. In this study, we obtained, by salt leaching technique, P(d,l)LA sponges in which various concentrations of α-tocopherol (Vit. E 10–40%, w/w) were incorporated. Vit.E is a natural biological antioxidant, also known to have anti-inflammatory activity, which has been extensively used to improve biostability and biocompatibility of different biomaterials. To assess whether Vit.E could modify the main physical–chemical properties of P(d,l)LA sponges, their morphology, water uptake and hydrolytic degradation kinetics, along with protein loading and releasing attitudes, were investigated. Our results highlighted that incorporation of Vit.E into P(d,l)LA sponges modified the sponge morphology, decreased P(d,l)LA water uptake and degradation, and modified protein releasing kinetics. These Vit.E-related effects could make P(d,l)LA more suitable as drug delivery system and tissue engineering scaffold.

J Oral Pathol Med (2011) 40: 33–36 Oral epithelial keratinocytes express nicotinic cholinergic receptors which activation modulates keratinocytes differentiation and migration through different metabolic pathways. Matrix metalloproteinases (MMPs) are Zn-dependent enzyme involved in cell migration. Among them, gelatinase B (MMP-9) and epilysin (MMP-28) are two MMPs expressed by human keratinocytes during both wound healing and proliferation. Their expression has been investigated in a reconstituted human oral epithelium (HOE) exposed to nicotine (Nic, 1–50 μM) for 72 h both in the absence and presence of the nicotinic antagonist mecamylamine (Mec), H7, a PKC inhibitor and PD98059, a MAPK inhibitor (PD). At the end of treatment, MMP-28 expression has been analyzed in epithelium sections using an anti-MMP-28 antibody, whereas MMP-9 presence and activity has been measured in cell-conditioned medium analyzed by gelatine zymography. The expression of MMP-9 was reduced by Nic in a dose-dependent fashion and this effect was antagonized by Mec, H7 and PD. On the other hand, Nic increased the expression of MMP-28, and this effect was blocked both by H7 and PD, whereas Mec even enforced it. Nic effects on MMP-9 and MMP-28 expression by oral keratinocytes were not previously reported and these data suggest MMPs expression mediated by PKC and MAPK as a possible target for Nic toxicity in oral epithelium.

Lasers devices are widely used in various medical fields (eg, surgery, dermatology, dentistry, rehabilitative medicine, etc.) for different applications, ranging from surgical ablation of tissues to biostimulation and pain relief. Laser is an electromagnetic radiation, which effects on biological tissues strongly depends on a number of physical parameters. Laser wavelength, energy output, irradiation time and modality, temperature and tissue penetration properties have to be set up according to the clinical target tissue and the desired effect. A less than optimal operational settings, in fact, could result in a null or even lethal effect. According to the first law of photobiology, light absorption requires the presence of a specific photoacceptor that after excitation could induce the activation of downstream signaling pathways. Low-level lasers operating in the red/near infrared portion of the light spectra are generally used for biostimulation purposes, a particular therapeutic application based on the radiant energy ability to induce nonthermal responses in living cells. Biostimulation process generally promotes cell survival and proliferation. Emerging evidences support a low-level laser stimulation mediated increase in "good" reactive oxygen species, able to activate redox sensitive signal transduction pathways such as Nrf-2, NF-kB, ERK which act as key redox checkpoints.

Poly(D,L-lactic acid) (P(D,L)LA) is a biocompatible and biodegradable polymer whose use is limited to orthopaedic applications. In fact, the mechanical properties of P(D,L)LA are not usually utilized for cardiovascular applications, as the polymer has been proven to activate both granulocyte- and platelet-causing inflammation. In order to improve P(D,L)LA haemocompatibility vitamin E (alpha-tocoferol, 10-30% (w/w)), a natural biological anti-oxidant and anti-inflammatory agent, was added during the solvent casting of P(D,L)LA film. The P(D,L)LA films obtained were then analysed using FT-IR analysis to assess vitamin E presence; polymer surface wettability and human plasma protein adsorption were measured by sessile drop test, spectrophotometric protein quantification and Western blot, respectively, and polymer haemocompatibility was assessed measuring platelet and granulocyte adhesion and whole blood coagulation. Vitamin E presence caused an increase in polymer surface wettability and human plasma protein adsorption. The combination of both effects may account for the decrease in platelet and granulocyte adhesion and for the doubling of whole blood clotting time measured onto vitamin-E-enriched P(D,L)LA compared to control P(D,L)LA. Our results indicate that vitamin E addition improves P(D,L)LA haemocompatibility, making this polymer suitable for cardiovascular application.

Ultra-high molecular weight polyethylene (UHMWPE) is a semicrystalline biomaterial widely used in the components for articular prosthesis for its excellent mechanical qualities. Two major problems limit the UHMWPE prosthesis life-wearing and delamination, both phenomena being mainly due to chemical oxidation of polymer. Wearing causes the release of generated particulate that triggers a macrophage reaction leading to chronic inflammation and osteolysis, while delamination, due to the mechanical stress, macroscopically alters the surfaces. The most diffused method to reduce wearing is UHMWPE molecular cross-linking by high-energy irradiation followed by melting that also reduces polymer fatigue strength. For this reason, the use of vitamin E (alpha-tocopherol), as an anti-oxidative and biocompatible additive for normal and cross-linked UHMWPE, has been suggested as an alternative method to improve polymer wearing resistance without altering its mechanical strength. This paper describes briefly the rationale of vitamin E as UHMWPE additive and its possible use as an emerging perspective in the orthopaedic field.

Vitamin E (Vit.E, α −tocoferol) is a natural agent with anti-oxidative and anti-inflammatory properties and it has been suggested that it could act as a stimulating factor for osteoblast proliferation and maturation. We produced poly( d,l )-lactic acid films enriched with Vit.E (1, 5 and 10% w/w) and investigated their surface properties using the FTIR analysis, sessile measure of wettability and serum protein adsorption, and evaluated attachment and spreading of MC-3T3 E1 murine osteoblast cells. FTIR analysis showed the presence of Vit.E on the polymer surface and Vit.E increased the polymer wettability in a concentration-dependent manner. The serum total protein adsorption increased significantly onto the 10% Vit.E P( d,l )-LA and the main protein adsorbed was albumin. The presence of albumin, considered as an anti-adhesive protein, on the surface of Vit.E enriched P( d,l )-LA films (especially 5 and 10% Vit.E) could explain, at least in part, the behaviour of MC-3T3 osteoblast cells seeded onto the polymers. Cell adhesion and spreading were strongly decreased by Vit.E (5 and 10%) in spite of the increased wettability. This reaction could be cell type-specific, independent by the surface wettability and linked to cell-specific characteristics (membrane phospholipid composition, integrins expression). Moreover a direct effect of Vit.E on cell adhesion and spreading cannot be completely excluded.

Epilysin, designated matrix metalloproteinase (MMP)-28, is the newest member of this family of proteases expressed by keratinocytes in response to an injury. MMP-28's physiological role and specific substrates are unknown, but its expression pattern suggests that it may serve a role in both tissue homeostasis and wound healing. The aim of this preliminary study was to observe the presence of MMP-28 protein in normotrophic and hypertrophic scars and to evaluate the effect of in vitro mechanical compression on its expression. Biopsies from normotrophic and hypertrophic scars resulting from burns were divided into two samples, one to be used as control (uncompressed) and the other to be compressed in an oxygenated organ chamber for 24 hours in the presence of a serum-free medium, using an electromechanical load transducer (stable pressure = 35 mmHg). Analysis of MMP-28 protein secretion, assessed by Western blot and beta-casein zymography in scar conditioned media, revealed that normotrophic scar did not release MMP-28 in any condition while hypertrophic scar released active MMP-28 both in control conditions and after compression. MMP-28 immunohistochemistry revealed a light protein presence in normotrophic scar keratinocytes and a strong MMP-28 positivity in hypertrophic scar keratinocytes in control conditions, while compression increased MMP-28 staining in normotrophic scar and induced a significant reduction of the protein presence in hypertrophic scar keratinocytes. As it has been suggested that MMP-28 may restructure the skin basal membrane (Saarialho-Kere et al., 2002), our data indicate that mechanical compression directly acts to modulate the remodeling phase of wound healing, altering release and activity of MMP-28 in hypertrophic scars.

Vitamin E (Vit.E, -tocopherol) is a natural biological antioxidant and antinflammatory agent, which protects cells from the effects of free radicals and inhibits inflammation. For such properties Vit.E has been used to improve the biocompatibility of materials such as cellulose membrane for hemodialysis. In this study granulocytes adhesion and activation have been studied after contact with normal cell culture grade polystyrene (PS) and Vit.E-coated polystyrene (Vit.E 0.1 and 0.3% (v/v)) using optical microscopy, flow cytometry and substrate zymography. Vit.E increased the number of adherent granulocytes both at 0.1% (11470 ± 1064 cells/cm 2 , P< 0.01) and 0.3% (13706 ± 818 cells/cm 2 , P< 0.001) concentration compared to normal PS (5529 ± 692 cells/cm 2 ). The morphology of granulocytes adherent to Vit.E–PS appeared lightly altered and no differences have been observed in their respiratory burst compared to control granulocyte, while matrix metalloproteinase 9 or gelatinase B (MMP-9) release and activation were increased compared to the normal PS samples. Our data indicate that Vit.E-coated surface induced an increase in granulocytes adhesion and MMP-9 release in the absence of the typical oxidative stress, hallmark of granulocytes activation. A possible explanation of the phenomenon is that Vit.E modifies the surface protein adsorption thus increasing cell adhesion and in turn MMP-9 releasing. © 2004 Elsevier B.V. All rights reserved.

Ultra-high molecular weight polyethylene (UHMWPE), a biopolymer widely used in orthopaedic implants, is oxidized during gamma-ray sterilization; such surface oxidation is considered as major responsible for inflammation and prosthesis failure. As granulocytes are involved in first contact inflammation, we have measured their oxidative burst by flow cytometry using dihydrorhodamine 123 (DRH) to evaluate their activation following contact with normal and oxidized UHMWPE. Peripheral blood cells (obtained by lysed blood) were loaded with DRH, seeded onto polystyrene, normal and heat-oxidized UHMWPE disks for 30min and then collected for analysis. Granulocytes were individuated using FSC and SSC signals and their cell associated green fluorescence was analyzed. Both normal and oxidized UHMWPE stimulated granulocytes activation as showed by the mean fluorescence emitted (109.3+/-3.8 and 150.1+/-9.2, respectively) compared to control samples (81.6+/-0.3). Moreover oxidized UHMWPE activated a significantly higher percentage of granulocytes (73.35+/-5.2%) compared to not-oxidized UHMWPE (21.5+/-3.8%). UHMWPE surface oxidation responsible for increased granulocyte activation seems to play a role in tissue response to implants.

Ultra-high molecular weight polyethylene (UHMWPE) sterilization with gamma rays induced high oxidation levels both on the surface and in the bulk that alter its structure and mechanical properties. The oxidation process of gamma-radiated UHMWPE induces a reduction of molecular weight and, consequently, a less abrasive resistance that has been related, among others, to the failure of UHMWPE in vivo. To explain the role of cells in such events, human osteoblast-like cells were seeded onto UHMWPE and oxidized UHMWPE discs. Cellular viability and morphology were evaluated along with matrix metalloproteinases (MMPs) production and activity. Oxidized UHMWPE did not induce any significant cytotoxic effects as observed by lactate dehydrogenase activity compared to the nonoxidized form; no changes in the cell morphology after 4 and 8 days proliferation were observed. In growth medium metalloproteinase 2 (gelatinase-A, MMP-2) was produced and released by osteoblast-like cells. We observed that cells grown onto oxidized UHMWPE discs decreased the release and activity of MMP-2 after 4 and 8 days culture compared to cells grown on control and non-oxidized UHMWPE discs; metalloproteinase 9 (gelatinase-B, MMP-9) release was not significantly influenced. The absence of cytotoxic and morphological effects in the presence of a down-regulation of MMP-2 release and activity suggest that oxidized polyethylene surfaces may modulate matrix remodeling and, consequently, bone formation.

Ultra high molecular weight polyethylene (UHMWPE) used in orthopedic prosthesis is often sterilized with γ-rays and the subsequent oxidation was suggested to favor the in vivo wear. UHMWPE debries produced by wearing trigger an inflammatory response that can led to the implant failure. To explore direct effects of UHMWPE oxidation on immunocompetent cells and their possible role in the prosthesis failure, peripheral blood cells (PBCs) have been grown for 24 and 48 h onto plastic (Ct), UHMWPE (PE) and heat oxidized UHMWPE (PEOx) discs. PBCs necrosis and apoptosis were assessed in flow cytometry using propidium iodide staining. After 24 h, no statistically significant differences were observed in the amount of apoptotic and necrotic cells between Ct, PE and PEOx samples while, after 48 h, both necrotic and apoptotic cells were strongly increased in PEOx samples where also the granulocytes population appeared strongly reduced (6.3±1.1%) compared to PE (10.5±1.5%) and Ct (15.1±0.9%) samples. We conclude that surface oxidation may interfere with prosthetic failure and/or integration via granulocytes modulation.

Objectives Textiles used to make clothing can represent a source, often ignored, of chemicals potentially noxious to both skin and the whole organism. Among the most frequently produced potentially noxious chemical manufacturing by-products are formaldehyde (FA), nickel (Ni) and hexavalent chromium (Cr); they are of potential clinical interest as all are known to be carcinogenic to humans and to be potent skin sensitizers. The aim of this study was to investigate, in vitro, effects of these potentially dangerous compounds on two different melanoma cell lines. In particular, attention was focused on A375P, a poorly metastatic and low invasive cell line and SK-MEL-28, a highly metastatic cell line. Materials and methods Effects of these compounds was evaluated on A375P and SK-MEL-28 cells. FA (1–5 × 10−5 m), NiSO4 (10−6–10−3 m), K2Cr2O7 (10−7–10−6 m) effects on cell proliferation were evaluated by cell counting, while ERK pathway involvement was evaluated by Western blot analysis. Results Low concentrations of the chemicals, covering a range that corresponds to commonly accepted limits in textile production, induced a significant increase in cell proliferation concomitant with transient activation of phosphorylated ERK expression. Conclusions Data obtained suggest that increasing attention must be focused on these by-products' potentially harmful effects in chemical manufacturing of clothes and accessories, that remain for long periods of time, in contact with human skin.

Titanium has long been used to produce dental implants. Problems related to its manufacturing, casting, welding, and ceramic application for dental prostheses still limit its use, which highlights the need for technologic improvements. The aim of this in vitro study was to evaluate the biologic performance of titanium dental implants coated with zirconium nitride in a murine preosteoblast cellular model.The purpose of this study was to evaluate the chemical and morphologic characteristics of titanium implants coated with zirconium nitride by means of physical vapor deposition.Chemical and morphologic characterizations were performed by scanning electron microscopy and energy dispersive x-ray spectroscopy, and the bioactivity of the implants was evaluated by cell-counting experiments.Scanning electron microscopy and energy dispersive x-ray spectroscopy analysis found that physical vapor deposition was effective in covering titanium surfaces with zirconium nitride. Murine MC-3T3 preosteoblasts were seeded onto titanium-coated and zirconium nitride-coated screws to evaluate their adhesion and proliferation. These experiments found a significantly higher number of cells adhering and spreading onto zirconium nitride-coated surfaces (P.05) after 24 hours; after 7 days, both titanium and zirconium nitride surfaces were completely covered with MC-3T3 cells.Analysis of these data indicates that the proposed zirconium nitride coating of titanium implants could make the surface of the titanium more bioactive than uncoated titanium surfaces.

The behaviour of plasma membrane was studied in UV-treated cells to investigate its involvement in apoptosis. It was studied in HL60 cells, in which DNA oligonucleosomic cleavage occurs, and in Molt-4 cells, which are characterised by a different fragmentation pattern. During the early stages of apoptosis, a membrane lipid rearrangement occurs, which involves phosphatidylserine translocation from the inner to the outer leaflet. This molecular alteration was investigated by annexin V-FITC binding, analysed by flow cytometry and confocal microscopy. It was correlated with transmission electron microscopy, subdiploid peak appearance and DNA fragmentation. Our data indicate that the plasma membrane represents an early apoptotic target, even if its alterations are not detectable by ultrastructural analysis, which indicates its good preservation until late apoptotic stages. In addition, the study of apoptotic cells with absent or inactivated endonuclease demonstrates the independence of this membrane mechanism from nuclear activity.

Mechanical properties of poly(D,L)lactic acid films enriched with Vitamin E and Vitamin E Acetate (5-40% w=w) were investigated. The addition of both formulations resulted in increased polymer Young's modulus and tensile strength. Human foreskin fibroblasts and murine pre-osteoblasts were used to assess the biocompatibility of polymers. Pre-osteoblasts adhesion and prolifera- tion were strongly decreased by Vitamin E, whereas Vitamin E Acetate did not alter cell proliferation. Collagen deposition was lower onto Vitamin E blended polymers than onto native and Vitamin E Acetate blended ones. Fibroblasts adhesion and proliferation were increased by both Vitamin E and Vitamin E Acetate addition. V C 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 39970.

Amino-bisphosphonates (N-BPs) have a wide range of clinical applications to treat bone diseases. Their activity lowered farnesyl pyrophosphate (FPP) endogenous levels by inhibiting FPP synthase. In epithelial cells it has been demonstrated that FPP reduces both cell proliferation and migration activting glucocorticoid receptor. In this study two N-BPs (zoledronate and neridronate) used at low concentrations (100 nM to 10 μM) are able to stimulate human keratinocytes proliferation reducing glucocorticoid receptor activation.

Purpose The distribution and relationship of hydroxyapatite debris, nanometric organic and metal wear particles and metal ions on periimplant interface membranes following aseptic and septic arthroplastic loosening were investigated. Methods Scanning electron microscopy and X-ray spectroscopic analysis were used to analyze debris and ion distribution. Results Hydroxyapatite debris appeared with different morphology in a particular distribution among several membranes. These differences may reflect the occurrence of different friction forces taking place between prosthesis and bone interface in the several types of prostheses studied. Metal wear particles were detected in greater numbers in membranes from noncemented prostheses compared with those from cemented ones. In contrast, more organic particles were present in membrane from cemented prosthesis. No differences were observed between aseptic and septic membranes. Conclusion Our findings support the need to evaluate the occurrence of friction forces that periprosthetic bone debris production may induce to exacerbate cellular reactivity. Furthermore, cellular engulfment of debris and the high level of different ions released indicate the occurrence of a toxic environment that may induce failure of any reparative pathways.

Nanoparticles (NPs) entering the human body are immediately confronted with the innate part of human immune system. In particular, monocyte and neutrophil granulocytes readily clear particles by phagocytosis, even if in the case of NPs the uptake mechanism may be classified as macropinocytosis. Among engineered nanoparticles, in the last years, siliceous materials have emerged as promising materials for several applications ranging from catalysis to biomedical. The polyhedral oligomeric silsesquioxanes (POSS) are nanodimensional, easily synthesizable molecular compounds and POSS-based systems are promising carriers for biological molecules. In this work, the ability of human granulocytes to uptake positively and negatively charged POSS was measured using a simple flow cytometry analysis based on cell size modifications. The data obtained showed that after a 30 min exposure only positive NPs were uptaken by human granulocyte using both macropinocytosis and clathrin-mediated mechanisms as demonstrated by uptake inhibition mediated by amiloride and chlorpromazine.

An atmospheric pressure plasma deposition for P(d,L)LA (PLA) film was used to modify polymer surface properties using 1,2-diaminopropane and acrylic acid as precursors. These two different plasma coatings result in a high density of amino groups (PLA-NH2) and carboxylic groups (PLACOOH) onto PLA surface as demonstrated by Fourier transform infra-red (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). Plasma coatings modified PLA surface wettability and proteins adsorption from fetal bovine serum (FBS), influencing cell adhesion and proliferation of 3T3 mouse fibroblast, MC-3T3 E1mouse pre-osteoblast, and HaCaT cells (human keratinocytes). In particular both coatings increased pre-osteoblast and keratinocyte adhesion while no effect was observed on fibroblast. Moreover, cell proliferation assessed after 48h by Tox-8 assay was significantly higher for osteoblast cells and keratinocyte seeded onto both PLA-NH2 and PLACOOH compared to cells seeded onto normal PLA. On the basis of the obtained data, the atmospheric pressure plasma deposition described might represent an innovative and useful tool for bone and skin tissue engineering.

Various lines of evidence suggest that muscarinic receptor-stimulated phosphoinositide hydrolysis during postnatal development in the rat brain may play a relevant role in glial cell proliferation and neuronal differentiation. We have previously shown that administration of ethanol to developing rats during the brain growth spurt causes microencephaly and selectively decreases muscarinic receptor-stimulated phosphoinositide hydrolysis. In the present study we have investigated the sensitivity of the phosphoinositide system coupled to muscarinic receptors to ethanol inhibition during distinct stages of the brain growth spurt. Different groups of rats were treated for 3 days with ethanol (4 g/kg per day) on postnatal days 2-4 (initial), 6-8 or 10-12 (peak), 13-15 (final stage of the brain growth spurt). The results show that the period of maximal sensitivity to ethanol of muscarinic receptor-stimulated phosphoinositide hydrolysis coincides with the peak of the brain growth spurt and with the period of maximal efficacy of muscarinic receptor agonists to induce inositol phosphates accumulation. Interestingly, only when muscarinic receptor-stimulated phosphoinositide hydrolysis was inhibited, a significant reduction of brain weight was observed. The close parallel between inhibition of this second messenger response and reduction of brain weight suggests that the phosphoinositide system coupled to muscarinic receptors may represent a target for the neurotoxic effects of ethanol during this stage of brain development.

Amino-bisphosphonates (N-BPs) are widely used to treat a great variety of clinical conditions involving altered calcium metabolism, as well as to prevent bone metastases. The use of N-BPs, however, display well-known side effects, including cellular toxicity, mainly at soft tissue and mucosal level, that arise from N-BPs ability to induce cell apoptosis when administered at clinically relevant concentrations. The aim of this study was to evaluate, in an in vitro wound healing model, the effect of N-BPs low concentration (10 nM-10 µM) stimulation on keratinocyte cellular behaviour. Human keratinocytes were treated with neridronate and zoledronate, two N-BPs with different chemical structure and clinical potency, but sharing a common pharmacological target: farnesyl pyrophosphate (FPP) synthase. Surprisingly, at the tested concentrations, both drugs stimulated keratinocytes proliferation, upregulating cytokeratin 5 while downregulating filaggrin expression, and wound healing ability, without any significant effect on matrix metalloproteinase (MMP)-9 activity. The lack of N-BPs effect on MMP-9 activity indicates that wound closure, in our experimental model, is mainly due to an increase in cell proliferation rather than to an increase in cell migration. Therefore, it can be hypothesised that the observed wound healing results could be ascribed to an N-BPs mediated reduction of FPP endogenous levels, thus suggesting new possible clinical applications for these compounds.

Ionic hydrogels are biocompatible candidates for skin tissue engineering. Two hydrogels synthesized by crosslinking gelatin with polylysine (positively charged HG1) or polyglutamic acid (negatively charged HG2) were tested using spontaneously immortalized human keratinocytes (HaCaT). HaCaT cells displayed higher adhesion and proliferation onto HG2, forming a continuous and stratified epithelium after 7 days. Moreover HaCaT cells grown onto HG2 showed a decreased Epilysin and Filaggrin expression, while transglutaminase-1 expression was increased. Those data indicate that human keratinocyte can form a stratified epithelium onto HG2 that could therefore be an useful tool for skin tissue engineering.

Cells within human skin are exposed to mechanical stretching that is considered a trigger stimulus for keratinocyte proliferation, while its effect on keratinocyte migration has been poorly investigate. In order to explore the effect of stretching on keratinocyte migration spontaneously immortalized human keratinocyte (HaCaT) monolayers seeded onto collagen I-coated silicon sheets were stimulated three times for 1 hour every 24 hours (total time = 72 hours) by mechanical stretching increasing substrate deformations (10%) applied both as static (0 Hz) and cyclic (0.17 Hz) uniaxial stretching. At the end of stimulations monolayer areas measured in both static and cyclic samples appeared reduced and strongly oriented in a direction perpendicular to the stress direction compared to unstimulated ones. Moreover during the mechanical stimulation period HaCaT monolayers strongly increased the release in the medium of matrix metalloproteinase 9 (MMP-9), a proteolytic enzyme necessary for keratinocyte migration.

Matrix metalloproteinases (MMPs) are zinc-dependent enzymes involved in the remodelling of connective tissues during the development and wound healing. Moreover, two MMPs, Gelatinase A (MMP-2) and Gelatinase B (MMP-9), are also present in body fluids such as blood and urine and, therefore, they can be in contact with implanted biomaterials and can be adsorbed onto their surface. In order to test this hypothesis disks of different polymers (polystyrene (PS), polyvinyl chloride (PVC), poly(D,L-lactide) (PLA), polymethyl methacrylate (PMMA) and poly(2-hydroxyethyl methacrylate) (PHEMA)) have been exposed to human plasma and adsorbed proteins have been eluted and analyzed. Using Western blot and substrate zymography analysis, we observed that both MMP-2 and MMP-9 adsorbed onto the surfaces of all the polymers, especially hydrophilic ones (PMMA and PHEMA) and PLA, in both the active and inactive forms. Furthermore, we observed that adhesion of human granulocyte neutophils to PMMA, the polymer that adsorbed the higher quantity of MMP-2 and MMP-9 compared to the others, was reduced by more that 50% by the presence of a gelatinase inhibitor. This data suggest a surprising role of these absorbed enzymes in the adhesion of neutrophil onto some polymeric biomaterials surface and, therefore, in the setting of inflammation.

The production of artificial epidermis using reabsorbable polymeric matrices is one of possible goals; one of most used strategies in this field is the polymer substrate functionalitation using specific growth factors, in order to accelerate and improve keratinocyte adhesion and proliferation. In this study films of poly(D,L)lactide (P(D,L)LA), have been functionalized with various concentrations of galactose (GAL, 1-5-10%, w/v) conjugated with poly-L-lysine (PLL) using 1-etil-3-(3-diaminopropil) carbodiimide (EDC) as a coupling agent. GAL is a disaccharide present in the extracellular matrix (ECM) and it is bind by Galectines, a family of cell receptors whose activation regulate the cell-matrix interaction and cell growth and apoptosis. One of these receptors, Galectin-7 (Gal-7), is selectively expressed by human keratinocytes. Spontaneously immortalized human keratinocytes (HaCaT) that express high level of Gal-7 were allowed to adhere for 4 h in serum free condition on control P(D,L)LA (PLA), and on PLA-GAL and cell proliferation; the production of matrix metalloproteinases (MMP-2, MMP-9, and MMP-28), involved in cellular migration and tissue homeostasis have been analyzed after 24 h. The presence of GAL onto the polymer surface increased both cell adhesion, spreading and proliferation along with MMP-9 and MMP-28 production, suggesting that polymer functionalization using GAL could be an useful tool for the production of an artificial epidermis.

In-stent restenosis is a process that occurs in 10-50% of cases currently treated with stent and it is caused by an abnormal smooth muscle cell (SMC) proliferation and migration in the vascular lumen. One of the most promising strategy to reduce restenosis is stent coating with biodegradable polymers to deliver in situ anti-proliferative drugs. Poly(D,L)lactic acid (P(D,L)LA), one of the most interesting candidate for stent coating, has been observed to induce inflammation and neointimal proliferation. In our laboratory, we developed P(D,L)LA enriched with Vitamin E (Vit.E), an anti-oxidative and anti-inflammatory agent that reduces also SMC proliferation. In order to evaluate the in vitro cellular behaviour of neointima cells onto Vitamin E-enriched P(D,L)LA, cell adhesion and proliferation along with the expression of two SMC migration markers (MMP-9 and hyaluronic acid receptor CD44) were measured in rat vascular SMC A10 cells seeded onto control P(D,L)LA (PLA) and P(D,L)LA films containing 10-30% (w/w) Vit.E (PLA10-30). Cell adhesion, proliferation and MMP-9 production were unaffected by the Vit.E presence in the PLA films after 24 h, while proliferation was slowed or blocked after 48 and 72 h onto PLA10, 20 and 30. MMP-9 production was almost blocked and CD44 density decreased significantly after 72 h for cells grew onto PLA30 compare to cells seeded onto PLA. These data indicate that Vit.E-enriched P(D,L)LA could be an interesting polymer for stent coating.

Major trauma such as severe bums and extensive surgery could result in accelerated macrophage differentiation and hyperactivation causing an excessive release of proinflammatory cytokines and prostaglandin E2 (PGE2) with consequent severe impairment of immunologic reactivity. HL-60 cells stimulated with phorbol 12-myristate 13-acetate (PMA) have been used as a model to asses the PGE2 role in the macrophage differentiation observed after major trauma. Cell adhesion, matrix metalloproteinase-9 (MMP-9) and tumor necrosis factor-alpha (TNF-alpha) production were measured after 24 h of PMA treatment in the presence of PGE2 (1 nM - 1 microM). PGE2 increased both the PMA-induced cell adhesion and MMP-9 production via EP2/EP4 receptors while it had no effect on the induced TNF-alpha release. The cAMP/PKA pathway, usually linked to EP2/EP4 activation, was not involved in the phenomenon, suggesting that an alternative signalling pathway could be linked to a PKC-activated enzyme. In fact PGE2 activity was partially inhibited by Wortmannin, a phosphoinositide-3 kinase (PI-3K) inhibitor indicating that PGE2 act as a co-factor able to increase macrophage differentiation in vitro via a PI-3K dependent pathway that could be also involved in the immunosuppression observed in the aftermath of trauma.