Electrospun Chitosan/Polyethylene Oxide Nanofibrous Scaffolds with Potential Antibacterial Wound Dressing Applications

Electrospinning is a simple and versatile technique for the fabrication of nonwoven fibrous materials for biomedical applications. In the present study, chitosan (CS) and polyethylene oxide (PEO) nanofibrous scaffolds were successfully prepared using three different CS/PEO mass ratios and then evaluated for their physical, chemical, and biological characteristics. Scaffold morphologies were observed by scanning electron microscopy, which showed decreasing fiber diameters with increasing CS content. Higher CS concentrations also correlated with increased tensile strength and decreased elasticity of the scaffold. Degradation studies demonstrated that PEO was solubilized from the scaffold within the first six hours, followed by CS. This profile was unaffected by changes in the CS/PEO ratio or the pH of the media. Only the 2 : 1 CS/PEO scaffold demonstrated superior inhibition of both growth and attachment of Staphylococcus aureus. Finally, all scaffolds exhibited little impact on the proliferation of murine fibroblast monolayers. These data demonstrate that the 2 : 1 CS/PEO scaffold is a promising candidate for wound dressing applications due to its excellent antibacterial characteristics and biocompatibility

Development of Electrospun Chitosan-Polyethylene Oxide/Fibrinogen Biocomposite for Potential Wound Healing Applications

Abstract Normal wound healing is a highly complex process that requires the interplay of various growth factors and cell types. Despite advancements in biomaterials, only a few bioactive wound dressings reach the clinical setting. The purpose of this research was to explore the feasibility of electrospinning a novel nanofibrous chitosan (CS)-fibrinogen (Fb) scaffold capable of sustained release of platelet-derived growth factor (PDGF) for the promotion of fibroblast migration and wound healing. CS-Fb scaffolds were successfully electrospun using a dual-spinneret electrospinner and directly evaluated for their physical, chemical, and biological characteristics. CS-polyethylene/Fb scaffolds exhibited thinner fiber diameters than nanofibers electrospun from the individual components while demonstrating adequate mechanical properties and homogeneous polymer distribution. In addition, the scaffold demonstrated acceptable water transfer rates for wound healing applications. PDGF was successfully incorporated in the scaffold and maintained functional activity throughout the electrospinning process. Furthermore, released PDGF was effective at promoting fibroblast migration equivalent to a single 50 ng/mL dose of PDGF. The current study demonstrates that PDGF-loaded CS-Fb nanofibrous scaffolds possess characteristics that would be highly beneficial as novel bioactive dressings for enhancement of wound healing

Genetic Deletion of Mst1 Alters T Cell Function and Protects against Autoimmunity

<div><p>Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4⁺ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1^−/− B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1^−/− CD4⁺ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4⁺ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.</p></div

Mst1^−/− mice exhibit decreased incidence and severity of CIA.

<p>(<b>A</b>) Mice of indicated genotype (n = 11–13) were immunized with CII in CFA and observed for clinical signs of arthritis at the time points depicted on the X axis. Histological scores of synovial inflammation and cartilage/bone erosion were analyzed on day 45 after immunization. Data are expressed as mean (± SEM); * (p<0.05) indicates significant differences in comparison to WT littermates (Mann–Whitney U test). Similar data were obtained in two additional independent experiments. (<b>B</b>) Representative pictures of histological and radiographic signs of arthritis in the same mice as in (A), obtained on day 45 after immunization with CII (left and middle panels). Arrows of the H&E-stained sections of paw joints point to severe synovial inflammation and cartilage erosion in the Mst1^−/− animals. Representative µCT images of subchondral bone changes characteristic of arthritis were taken on day 45 after immunization (right panels).</p

Analysis of T cell apoptosis and functional immune responses in Mst1^−/− mice.

<p>(<b>A</b>) Increased apoptosis of activated Mst1^−/− splenic T cells in vitro. Early apoptotic T cells were quantitated by flow cytometry in after stimulation with the indicated mAbs for 48 hrs (n = 5 per genotype). (<b>B</b>) Analysis of apoptosis in unstimulated MST^−/− T cells. Early apoptotic T cells were quantitated by flow cytometry in CD44^low and CD44^high subsets of freshly isolated splenocytes (n = 5 per genotype). (<b>C</b>) The percentages of naïve (CD62L^highCD44^low), effector memory (CD62L^lowCD44^high), and CD279-, CD25-, and CD69-positive splenic CD4⁺ T cells were examined by flow cytometry (n = 5 per genotype). (<b>D</b>) Cytokine production by splenic CD62L^highCD44⁻ CD4⁺ T cells from WT and Mst1^−/− mice (n = 5 per genotype) was examined 48 hrs after stimulation with mAbs to CD3 and CD28 (both at 1 µg/ml). (<b>E</b>) Th1 and Th2 polarized cells were generated from naïve splenic CD62L^highCD44⁻ CD4⁺ T cells after in vitro culture in polarizing conditions for 5 days. The percentages of effector memory (CD62L^lowCD44^high) CD4⁺ T cells were examined by flow cytometry (n = 5 per genotype). The percentage of sub-G0/G1 apoptotic cells was determined by the BrdU/7-AAD Flow kit and flow cytometry (n = 5 per genotype) following restimulation in vitro with plate-bound CD3 mAb (5 µg/ml) for 48 hrs. (<b>F</b>) Mst1^−/− deficiency leads to decreased Ag-specific adaptive immune responses in vivo. Mst1^−/− and WT mice (n = 6 and 9, respectively) were immunized with OVA in CFA. On day 14, serum samples were analyzed for OVA-specific IgG1 and IgG2a concentrations. Values and statistical significance are expressed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098151#pone-0098151-g002" target="_blank">Fig. 2</a> and are representative of at least two independent experiments. Pre, preimmune serum.</p

Altered immune responses of activated Mst1^−/− T cells in vitro.

<p>(<b>A</b>) Th1 and Th2 cytokine production by splenic T cells from WT and Mst1^−/− mice was examined 48 hrs after stimulation with Con A (2.5 µg/ml) or mAbs to CD3 and CD28 (both at 1 µg/ml). (<b>B</b>) IgE concentration was measured by ELISA in serum samples of WT (n = 8) and Mst1^−/− mice (n = 10). (<b>C</b>) Deficient upregulation of lymphocyte activation markers on Mst1^−/− T cells. Splenic T cells were stimulated with plate-bound CD3 mAb (1 µg/ml) for 48 hrs, stained for CD69 and CD95 (Fas/APO-1), and analyzed by flow cytometry (n = 5 per genotype). (<b>D</b>) Suppression of Mst1^−/− T cell proliferative responses in allogeneic MLR in vitro. Splenic C57Bl/6-Albino/129SvEv (H-2b) Mst1^−/− and WT T cells (responder cells; 5 mice per genotype) were stimulated with the indicated numbers of MHC-mismatched irradiated stimulator cells obtained from Balb/c mice (H-2d). Proliferation of the responder cells was assessed by [³H]-thymidine incorporation after 90 hrs of culture. Values and statistical significance are expressed as in Fig. 2 and represent results of at least two independent experiments.</p

Mst1^−/− mice are resistant to EAE.

<p>(<b>A</b>) EAE was induced in mice of indicated genotype (n = 10) by immunization with MOGp35–55 in CFA. The mean EAE score ± SEM observed in each group is plotted against time after immunization (left panel). Representative H&E stained sections of the cervical spinal cord of WT and Mst1^−/− mice obtained on day 21 after immunization (middle panels). Higher (×40) magnification shows areas of prominent mononuclear multifocal inflammation, vacuolization and gliosis in the white matter of WT mice, which are nearly absent in Mst1^−/− mice. Aggregate histopathology scores were obtained on day 21 after immunization (right panel). Similar data were obtained in two additional independent experiments. * (p<0.05) and ** (p<0.01) indicate significant differences in comparison to WT animals (Mann–Whitney U test). (<b>B</b>) Infiltrating mononuclear cells were isolated from the spinal cord of mice with the indicated genotype (n = 5/group) on day 15 after immunization with MOGp35–55, and analyzed by flow cytometry for markers of T cell differentiation and activation. Numbers above the rectangular gates represent the percentages and absolute numbers (x10⁴/spinal cord) of infiltrating CD4⁺ and CD8+ T cells for each genotype. (<b>C</b>) Frequency of IL-2-, TNF-α-, IFN-γ-, IL-4-, IL-10, and IL-17A-producing CD4⁺ T cells infiltrating the spinal cords of indicated mice was measured by flow cytometry analysis of intracellular cytokine staining. (<b>D</b>) qPCR analysis of IL-2, TNF-α, IFN-γ, IL-4, and IL-10 mRNA in the spinal cords from mice of indicated genotype (n = 6–10) was performed on day 11 after immunization with MOGp35–55. Values are expressed relative to the expression of GAPDH. <i>Values and statistical significance are expressed as in Fig. 2 and represent at least two independent experiments.</i></p

Targeted disruption of the Mst1 gene locus.

<p>(<b>A</b>) Targeting strategy used to disrupt the Mst1 locus. Homologous recombination (represented by ×) between the targeting vector and the Mst1 gene results in the replacement of exons 3–4 with the selection cassette. (<b>B</b>) Southern hybridization indicating proper gene targeting in the embryonic stem cell clones. Clone 1F3 was selected for blastocyst injections to generate chimeric animals which were bred to C57BL/6 (albino) females, and the resulting heterozygous offspring were interbred to produce homozygous Mst1 deficient mice; Lex-2 represents untransfected embryonic stem cell DNA. (<b>C</b>) The effect of Mst1 disruption on peripheral lymphocyte subsets. Peripheral blood cell subsets were stained with a panel of lymphocyte lineage-specific mAbs and quantitated by FACS. Values are expressed as mean ± SEM; n = 9 per group. Student's <i>t</i> test was used for group comparison.</p