Subcutaneous Application of a Gelatin/Hyaluronic Acid Hydrogel Induces the Production of Skin Extracellular Matrix.

The development of injectable hydrogels with natural biopolymers such as gelatin (Ge) and hyaluronic acid (Ha) is widely performed due to their biocompatibility and biodegradability. The combination of both polymers crosslinked with N-Ethyl-N'-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) can be used as an innovative dermal filler that stimulates fibroblast activity and increases skin elasticity and tightness. Thus, crosslinked Ge/Ha hydrogels with different concentrations of EDC were administered subcutaneously to test their efficacy in young and old rats. At higher EDC concentrations, the viscosity decreases while the particle size of the hydrogels increases. At all concentrations of EDC, amino and carboxyl groups are present. The histological analysis shows an acute inflammatory response, which disappears seven days after application. At one and three months post-treatment, no remains of the hydrogels are found, and the number of fibroblasts increases in all groups in comparison with the control. In addition, the elastic modulus of the skin increases after three months of treatment. Because EDC-crosslinked Ge/Ha hydrogels are biocompatible and induce increased skin tension, fibroblast proliferation, and de novo extracellular matrix production, we propose their use as a treatment to attenuate wrinkles and expression lines.

Gelatin/Hyaluronic Acid Scaffold Coupled to CpG and MAGE-A5 as a Treatment against Murine Melanoma.

The half-time of cells and molecules used in immunotherapy is limited. Scaffolds-based immunotherapy against cancer may increase the half-life of the molecules and also support the migration and activation of leukocytes in situ. For this purpose, the use of gelatin (Ge)/hyaluronic acid (HA) scaffolds coupled to CpG and the tumor antigen MAGE-A5 is proposed. Ge and HA are components of the extracellular matrix that stimulate cell adhesion and activation of leucocytes; CpG can promote dendritic cell maturation, and MAGE-A5 a specific antitumor response. C57BL/6 mice were treated with Ge/HA/scaffolds coupled to MAGE-A5 and/or CpG and then challenged with the B16-F10 melanoma cell line. Survival, tumor growth rate and the immune response induced by the scaffolds were analyzed. Ge/HA/CpG and Ge/HA/MAGE-A5 mediated dendritic cell maturation and macrophage activation, increased survival, and decreased the tumor growth rate and a tumor parenchyma with abundant cell death areas and abundant tumor cells with melanin granules. Only the scaffolds coupled to MAGE-A5 induced the activation of CD8 T cells. In conclusion, Ge/HA scaffolds coupled to CpG or MAGE-A5, but not the mixture, can induce a successful immune response capable of promoting tumor cell clearance and increased survival.

Immunomodulatory Properties of Masticadienonic Acid and 3α-Hydroxy Masticadienoic Acid in Dendritic Cells.

Dendritic cells are antigen-presenting cells, which identify and process pathogens to subsequently activate specific T lymphocytes. To regulate the immune responses, DCs have to mature by the recognition of TLR ligands, TNFα or IFNγ. These ligands have been used as adjuvants to activate DCs in situ or in vitro, with toxic effects. It has been shown that some molecules affect the immune system, e.g., Masticadienonic acid (MDA) and 3α-hydroxy masticadienoic acid (3α-OH MDA) triterpenes naturally occurring in several medicinal plants, since they activate the nitric oxide synthase in macrophages and induce T lymphocyte proliferation. The DCs maturation induced by MDA or 3a-OH MDA was determined by incubating these cells with MDA or 3α-OH MDA, and their phenotype was afterwards analyzed. The results showed that only 3α-OH MDA was able to induce DCs maturation. When mice with melanoma were inoculated with DCs/3α-OH MDA, a decreased tumor growth rate was observed along with an extended cell death area within tumors compared to mice treated with DCs incubated with MDA. In conclusion, it is proposed that 3α-OH MDA may be an immunostimulant molecule. Conversely, it is proposed that MDA may be a molecule with anti-inflammatory properties.

Epicutaneous Administration of 17ß-Estradiol Induces Langerhans Cells Depletion.

BACKGROUND: Langerhans cells (LC) number and function in mouse vaginal mucosa are affected by 17ß-estradiol (E2) application; nonetheless, its effect on epidermal LC has not been studied. The purpose of this study was to evaluate the effect of topical administration of E2 on the number, phenotype, and migratory ability of LC in mouse skin. METHODS: Ears of adult CD1 male mice were topically treated once with several doses. Immunohistochemical staining for CD207 and TUNEL staining were performed. LC migration to lymph nodes and the effect on the expression of costimulatory molecules on cultured dendritic cells (DC) were also evaluated. RESULTS: E2 decreased the number of CD207+ LC in a dose-dependent manner. One hour after treatment, 1 and 10 µg/mL E2 significantly reduced the LC number by 21% and 26%, respectively, after two hours, the reduction was 23% and 41%, respectively. After 48 hours, LC recovered, and after 96 hours of treatment, the CD207+/MHCII+ DC numbers were increased in regional lymph nodes. However, CD86 and CD40 molecules were expressed at lower levels than in positive control. The TUNEL assay did not show apoptotic cells. Furthermore, in cultured DC, E2 promoted a decrease in CD40 and CD86 expression and an increase in CD273, CD274, MHCII, and CCR7. CONCLUSIONS: The topical administration of E2 induced a transitory local diminution of LC population and a tolerogenic phenotype. This decrease in epidermal LC suggests that E2 may affect skin immune responses, inducing an inhibitory response, which should be considered when prescribing topical E2 medications.

Three-Dimensional Porous Scaffolds Derived from Bovine Cancellous Bone Matrix Promote Osteoinduction, Osteoconduction, and Osteogenesis.

The use of three-dimensional porous scaffolds derived from decellularized extracellular matrix (ECM) is increasing for functional repair and regeneration of injured bone tissue. Because these scaffolds retain their native structures and bioactive molecules, in addition to showing low immunogenicity and good biodegradability, they can promote tissue repair and regeneration. Nonetheless, imitating these features in synthetic materials represents a challenging task. Furthermore, due to the complexity of bone tissue, different processes are necessary to maintain these characteristics. We present a novel approach using decellularized ECM material derived from bovine cancellous bone by demineralization, decellularization, and hydrolysis of collagen to obtain a three-dimensional porous scaffold. This study demonstrates that the three-dimensional porous scaffold obtained from bovine bone retained its osteoconductive and osteoinductive properties and presented osteogenic potential when seeded with human Wharton's jelly mesenchymal stromal cells (hWJ-MSCs). Based on its characteristics, the scaffold described in this work potentially represents a therapeutic strategy for bone repair.

Cellulose-Chitosan-Nanohydroxyapatite Hybrid Composites by One-Pot Synthesis for Biomedical Applications.

The development of organic-inorganic hybrid materials deserves special interest for bone tissue engineering applications, where materials must have properties that induce the survival and activation of cells derived from the mesenchyme. In this work, four bio-nanocomposites based on cellulose and variable content of chitosan, from 15 to 50 w% based on cellulose, with nanohydroxyapatite and ß-Glycerophosphate as cross-linking agent were synthesized by simplified and low-energy-demanding solvent exchange method to determine the best ratio of chitosan to cellulose matrix. This study analyzes the metabolic activity and survival of human dermal fibroblast cells cultivated in four bio-nanocomposites based on cellulose and the variable content of chitosan. The biocompatibility was tested by the in vitro cytotoxicity assays Live/Dead and PrestoBlue. In addition, the composites were characterized by FTIR, XRD and SEM. The results have shown that the vibration bands of ß-Glycerophosphate have prevailed over the other components bands, while new diffraction planes have emerged from the interaction between the cross-linking agent and the biopolymers. The bio-nanocomposite micrographs have shown no surface porosity as purposely designed. On the other hand, cell death and detachment were observed when the composites of 1 and 0.1 w/v% were used. However, the composite containing 10 w% chitosan, against the sum of cellulose and ß-Glycerophosphate, has shown less cell death and detachment when used at 0.01 w/v%, making it suitable for more in vitro studies in bone tissue engineering, as a promising economical biomaterial.

Differential adhesion and fibrinolytic activity of mesenchymal stem cells from human bone marrow, placenta, and Wharton's jelly cultured in a fibrin hydrogel.

Mesenchymal stem cells isolated from different tissues should share associated markers and the capability to differentiate to mesodermal lineages. However, their behavior varies in specific microenvironments. Herein, adhesion and fibrinolytic activity of mesenchymal stem cells from placenta, bone marrow, and Wharton's jelly were evaluated in fibrin hydrogels prepared with nonpurified blood plasma and compared with two-dimensional cultures. Despite the source, mesenchymal stem cells adhered through focal adhesions positive for vinculin and integrin αV in two dimensions, while focal adhesions could not be detected in fibrin hydrogels. Moreover, some cells could not spread and stay rounded. The proportions of elongated and round phenotypes varied, with placenta mesenchymal stem cells having the lowest percentage of elongated cells (~10%). Mesenchymal stem cells degraded fibrin at distinct rates, and placenta mesenchymal stem cells had the strongest fibrinolytic activity, which was achieved principally through the plasminogen-plasmin axis. These findings might have clinical implications in tissue engineering and wound healing therapy.

Toxicity of silver nanoparticles in mouse bone marrow-derived dendritic cells: Implications for phenotype.

Silver nanoparticles (AgNP) are one of the most studied nanoparticles due to their anti-bacterial, -fungal, -viral, -parasitic, and -inflammatory properties. This raises the need to evaluate the toxicity and biological effects of AgNP in the immune system in order to develop new safer biomedical products. In this study, an AgNP formulation currently approved for veterinary applications was applied to mouse bone marrow-derived dendritic cells (BMDC), considered important antigen-presenting cells of the immune system, to evaluate cytotoxicity, genotoxicity, and any significant influence on expression of cellular markers associated with BMDC phenotype and maturation status. The results showed that after 12 h of AgNP exposure, a significant decrease in BMDC viability occurred at the highest concentration tested (1.0 µg AgNP/ml) and at lower doses, the cells maintained membrane integrity and metabolic activity. DNA damage was not significant with any AgNP level aside from the 1.0 µg AgNP/ml level. Regarding phenotype, no differences in expression of CD40 (co-stimulatory molecule highly present in mature BMDC) or in CD273 (a marker for inhibitory T-cell response) were observed. The current results showed that the toxicity of this AgNP formulation was dose-related. The findings also suggest BMDC could maintain structural conservation of co-stimulatory/co-inhibitory surface molecules after 12 h of exposure to this AgNP. This work represents the first step in identifying the toxic effects of this AgNP formulation on dendritic cells.

Influence of the PLGA/gelatin ratio on the physical, chemical and biological properties of electrospun scaffolds for wound dressings.

Chronic wounds are a global health problem, and their treatments are difficult and long lasting. The development of medical devices through tissue engineering has been conducted to heal this type of wound. In this study, it was demonstrated that the combination of natural and synthetic polymers, such as poly (D-L lactide-co-glycolide) (PLGA) and gelatin (Ge), were useful for constructing scaffolds for wound healing. The aim of this study was to evaluate the influence of different PLGA/gelatin ratios (9:1, 7:3 and 5:5 (v/v)) on the physical, chemical and biological properties of electrospun scaffolds for wound dressings. These PLGA/Ge scaffolds had randomly oriented fibers with smooth surfaces and exhibited distances between fibers of less than 10 µm. The 7:3 and 5:5 PLGA/Ge scaffolds showed higher swelling, hydrophilicity and degradation rates than pure PLGA and 9:1 (v/v) PLGA/Ge scaffolds. Young's moduli of the scaffolds were 72 ± 10, 48 ± 6, 58 ± 6 and 6 ± 1 MPa for the pure PLGA scaffold and the 9:1, 7:3 and 5:5 (v/v) PLGA/Ge scaffolds, respectively. Mesenchymal stem cells (MSCs) seeded on all the PLGA/Ge scaffolds were viable, and the cells were attached to the fibers at the different analyzed timepoints. The most significant proliferation rate was observed for cells on the 7:3 PLGA/Ge scaffolds. Biocompatibility analysis showed that all the scaffolds produced inflammation at the first week postimplantation; however, the 7:3 and 5:5 (v/v) PLGA/Ge scaffolds were degraded completely, and there was no inflammatory reaction observed at the fourth week after implantation. In contrast, the 9:1 PLGA/Ge scaffolds persisted in the tissue for more than four weeks; however, at the eighth week, no traces of the scaffolds were found. In conclusion, the scaffolds with the 7:3 PLGA/Ge ratio showed suitable physical, chemical and biological properties for applications in chronic wound treatments.

The mitochondrial alternative oxidase Aox1 is needed to cope with respiratory stress but dispensable for pathogenic development in Ustilago maydis.

The mitochondrial alternative oxidase is an important enzyme that allows respiratory activity and the functioning of the Krebs cycle upon disturbance of the respiration chain. It works as a security valve in transferring excessive electrons to oxygen, thereby preventing potential damage by the generation of harmful radicals. A clear biological function, besides the stress response, has so far convincingly only been shown for plants that use the alternative oxidase to generate heat to distribute volatiles. In fungi it was described that the alternative oxidase is needed for pathogenicity. Here, we investigate expression and function of the alternative oxidase at different stages of the life cycle of the corn pathogen Ustilago maydis (Aox1). Interestingly, expression of Aox1 is specifically induced during the stationary phase suggesting a role at high cell density when nutrients become limiting. Studying deletion strains as well as overexpressing strains revealed that Aox1 is dispensable for normal growth, for cell morphology, for response to temperature stress as well as for filamentous growth and plant pathogenicity. However, during conditions eliciting respiratory stress yeast-like growth as well as hyphal growth is strongly affected. We conclude that Aox1 is dispensable for the normal biology of the fungus but specifically needed to cope with respiratory stress.

Activation of Janus kinase/signal transducers and activators of transcription pathway involved in megakaryocyte proliferation induced by vanadium resembles some aspects of essential thrombocythemia.

Vanadium (V) is an air pollutant released into the atmosphere by burning fossil fuels. Also, it has been recently evaluated for their carcinogenic potential to establish permissible limits of exposure at workplaces. We previously reported an increase in the number and size of platelets and their precursor cells and megakaryocytes in bone marrow and spleen. The aim of this study was to identify the involvement of Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway and thrombopoietin (TPO) receptor, and myeloproliferative leukemia virus oncogene (Mpl), in megakaryocyte proliferation induced by this compound. Mice were exposed twice a week to vanadium pentoxide inhalation (0.02 M) and were killed at 4th, 6th, and 8th week of exposure. Phosphorylated JAK2 (JAK2 ph), STAT3 (STAT3 ph), STAT5, and Mpl were identified in mice spleen megakaryocytes by cytofluorometry and immunohistochemistry. An increase in JAK2 ph and STAT3 ph, but a decrease in Mpl at 8-week exposure was identified in our findings. Taking together, we propose that the morphological findings, JAK/STAT activation, and decreased Mpl receptor induced by V leads to a condition comparable to essential thrombocythemia, so the effect on megakaryocytes caused by different mechanisms is similar. We also suggest that the decrease in Mpl is a negative feedback mechanism after the JAK/STAT activation. Since megakaryocytes are platelet precursors, their alteration affects platelet morphology and function, which might have implications in hemostasis as demonstrated previously, so it is important to continue evaluating the effects of toxics and pollutants on megakaryocytes and platelets.

GK-1 improves the immune response induced by bone marrow dendritic cells loaded with MAGE-AX in mice with melanoma.

The aim of dendritic cell (DC) vaccination in cancer is to induce tumor-specific effector T cells that may reduce and control tumor mass. Immunostimulants that could drive a desired immune response are necessary to be found in order to generate a long lasting tumor immune response. GK-1 peptide, derived from Taenia crassiceps, induces not only increase in TNFα, IFNγ, and MCP-1 production in cocultures of DCs and T lymphocytes but also immunological protection against influenza virus. Moreover, the aim of this investigation is the use of GK-1 as a bone marrow DCs (BMDCs) immunostimulant targeted with MAGE antigen; thus, BMDC may be used as immunotherapy against murine melanoma. GK-1 induced in BMDCs a meaningful increment of CD86 and IL-12. In addition, the use of BMDCs TNFα/GK-1/MAGE-AX induced the highest survival and the smallest tumors in mice. Besides, the treatment helped to increase CD8 lymphocytes levels and to produce IFNγ in lymph nodes. Moreover, the histopathological analysis showed that BMDCs treated with GK-1/TNFα and loaded with MAGE-AX induced the apparition of more apoptotic and necrotic areas in tumors than in mice without treatment. These results highlight the properties of GK-1 as an immunostimulant of DCs and suggest as a potential candidate the use of this immunotherapy against cancer disease.

The effect of vanadium on platelet function.

Vanadium pentoxide (V(2)O(5)) inhalation effect on platelet function in mice was explored, as well as the in vitro effect on human platelets. Mouse blood samples were collected and processed for aggregometry and flow cytometry to assess the presence of P-selectin and monocyte-platelet conjugates. Simultaneously, human platelets were processed for aggregometry(.) The mouse results showed platelet aggregation inhibition in platelet-rich-plasma (PRP) at four-week exposure time, and normality returned at eight weeks of exposure, remaining unchanged after the exposure was discontinued after four weeks. This platelet aggregation inhibition effect was reinforced with the in vitro assay. In addition, P-selectin preserved their values during the exposure, until the exposure was discontinued during four weeks, when this activation marker increased. We conclude that vanadium affects platelet function, but further studies are required to evaluate its effect on other components of the hemostatic system.

Ultrastructural megakaryocyte modifications after vanadium inhalation in spleen and bone marrow.

Previous reports from our laboratory informed in mice an increase in platelets in blood, and megakaryocytes in spleen and bone marrow after vanadium inhalation. This element has become important in recent years because of its increased presence as an air pollutant. With this precedent, we evaluate the ultrastructural modifications in MKs from the spleen and bone marrow in our mouse experimental model. Mice inhaled 0.02 M V(2)O(5) 1 h twice a week for 12 weeks. Tissues were processed for transmission electron microscopy. Results indicate an increase in the size and cytoplasmic granular content, as well as nuclear changes in MKs of exposed mice, changes which correlate with the time of exposure. Modifications in MKs described here suggest that inhaled vanadium induce megakaryocytic maturation, a raise in its granules content and demarcation membrane systems, which may lead to a rise in circulating platelet production and an increased risk for thromboembolic events.

Spleen and bone marrow megakaryocytes as targets for inhaled vanadium.

An increased incidence in ischemic and thromboembolic events in the population of cities with rising air suspended particle pollution has suggested the interaction of some of the components of these particles in the coagulation system. A previous report from our laboratory identified thrombocytosis as a consequence of the subacute and chronic inhalation of vanadium. With this preceding information we decided to evaluate the effects of this element in the spleen and bone marrow in a mouse experimental model. CD-1 male mice inhaled V2O5 0.02 M for one hour twice a week for twelve weeks. The spleen and bone marrow were processed for light microscopy. The increase in quantity and size of megakaryocytes (MKs) in the exposed group in both organs was striking. Also, modifications in the cytoplasm, granule content and nuclear ultrastructure were evident. Our results indicate the influence of vanadium on megakaryopoyesis, an effect which could be the onset of the thrombocytosis previously reported by our group. The modifications in MKs described here suggest that inhaled vanadium could induce megakaryocytic proliferation, which may result in increased production of platelets and increased risk for thromboembolic events.

Ultrastructural findings in murine seminiferous tubules as a consequence of subchronic vanadium pentoxide inhalation.

Vanadium (V) is a transition metal emitted to the atmosphere during the combustion of fossil fuels. Its current status as an atmospheric pollutant increases the need for information about the effects that this element might have on the reproductive health of exposed populations. The present study investigated changes in testicular ultrastructure following inhalation exposure of male mice to V (as vanadium pentoxide). Tissue V level was constant during the 12-week time period. We observed necrosis of spermatogonium, spermatocytes and Sertoli cells, as well as pseudo-nuclear inclusion and disruption of cellular junctions. Our findings stressed the importance of the hemato-testicular barrier in supporting the function of Sertoli cells and suggest as a possible target of V, tight junction proteins. Further analysis is needed in order to identify the role that reactive oxidative species (ROS) might have on these cellular junctions, and if a specific protein is the target of its toxic effects. The relevance of this report concerns the impact that metal air pollution could have on male fertility in dense cities with vehicular traffic problems.

Thrombocytosis induced in mice after subacute and subchronic V2O5 inhalation.

Reports about vanadium (V) inhalation toxicity on the hematopoietic system, specifically about coagulation are limited. Therefore, we decided to evaluate the effects of V with a complete blood count and morphologic analysis of platelets on blood smears. CD-1 male mice inhaled V2O5 0.02 M 1 h twice weekly over 12 weeks. Blood samples were obtained by direct heart puncture; Wright stained smears were used for platelet quantification. An increase in platelet count from the third week of exposure was observed, as well as the presence of megaplatelets. Our results demonstrate, for the first time, that V induces thrombocytosis and it might correlate with some thromboembolic diseases. Further analysis is needed to evaluate the functionality of these platelets as well as the cause of its increase.

Inhaled vanadium pentoxide decrease gamma-tubulin of mouse testes at different exposure times.

Vanadium is an important environmental and industrial pollutant whose concentrations have increased in the last decades. Due to its status as reproductive toxicant and a microtubule damaging agent, the present study investigated by immunohistochemistry the effect of the inhalation of vanadium pentoxide on gamma-tubulin within somatic and testicular germ cells. Male mice inhaled vanadium pentoxide (V2O5) (0.02 M) 1 h/twice a week for 12 weeks. Our results demonstrated that vanadium accumulates in the testes starting with the initial inhalation (24 h), and this pattern remained until the last week of treatment. In general, vanadium was capable of significantly decreasing the percentage of gamma-tubulin in all analyzed testicular cells (Sertoli, Leydig and germ cells) starting with the first week of treatment. For all cell types studied, regression analysis revealed a negative and significant relationship between the percentage of immunopositive cells to gamma-tubulin and exposure time, showing a time dependent response in all cases. Our findings suggest that alterations on this protein might imply changes in microtubule-involved function such as cell division, which in the testes might lead to damage in the spermatogenesis, leading probably to infertility.