Biomineralized Poly(l‑lactic-<i>co</i>-glycolic acid)/Graphene Oxide/Tussah Silk Fibroin Nanofiber Scaffolds with Multiple Orthogonal Layers Enhance Osteoblastic Differentiation of Mesenchymal Stem Cells

Bone scaffolds with interconnected pores, good mechanical properties, excellent biocompatibility, and osteoinductivity are challenging to fabricate. In this study, we fabricated and characterized the morphology, hydrophilicity, protein adsorptivity, mechanical properties, and fibrous structure of nanofiber scaffolds with multiple, orthogonal layers of composite materials based on poly­(l-lactic-<i>co</i>-glycolic acid) (PLGA), graphene oxide (GO), tussah silk fibroin (TSF), and hydroxyapatite (HA). The data show that incorporation of 1 wt % GO into PLGA/TSF nanofibers significantly decreased the fiber diameter from 321 to 89 nm. On the other hand, incorporation of 10 wt % TSF accelerated the nucleation and growth of HA on composite PLGA/GO scaffolds exposed to simulated body fluid. Furthermore, the compressive modulus and stress of composite scaffolds with GO were 1.7-fold and 0.6-fold higher than those of similar scaffolds without GO. Interestingly, composite scaffolds with multiple orthogonal layers exhibited higher compressive modulus and stress compared to scaffolds with randomly oriented nanofibers. Biological assays indicated that mineralized scaffolds with multiple orthogonal layers significantly enhanced cell adhesion, proliferation, and differentiation of mesenchymal stem cells into osteoblasts. In summary, the data indicate that these scaffolds have excellent cytocompatibility and osteoinductivity and have potential as versatile substrates for bone tissue engineering

Nickel Ferrite Nanoparticles Anchored onto Silica Nanofibers for Designing Magnetic and Flexible Nanofibrous Membranes

Many applications proposed for magnetic silica nanofibers require their assembly into a cellular membrane structure. The feature to keep structure stable upon large deformation is crucial for a macroscopic porous material which functions reliably. However, it remains a key issue to realize robust flexibility in two-dimensional (2D) magnetic silica nanofibrous networks. Here, we report that the combination of electrospun silica nanofibers with zein dip-coating can lead to the formation of flexible, magnetic, and hierarchical porous silica nanofibrous membranes (SNM). The 290 nm diameter silica nanofibers act as templates for the uniform anchoring of nickel ferrite nanoparticles (size of 50 nm). Benefiting from the homogeneous and stable nanofiber–nanoparticle composite structure, the resulting magnetic SNM can maintain their structure integrity under repeated bending as high as 180° and can facilely recover. The unique hierarchical structure also provides this new class of silica membrane with integrated properties of ultralow density, high porosity, large surface area, good magnetic responsiveness, robust dye adsorption capacity, and effective emulsion separation performance. Significantly, the synthesis of such fascinating membranes may provide new insight for further application of silica in a self-supporting, structurally adaptive, and 2D membrane form

Pressure and Fluorescence Dual Signal Readout CuO-NiO/C Heterojunction Nanofibers-Based Nanoplatform for Imaging and Detection of Target Cancer Cells in Blood

The development of multifunctional nanoplatform, which integrates biosensing and imaging diagnosis, for accurate identification of cancer cells in blood or other bodily fluids is critical for early cancer diagnosis and treatment. Herein, by using a facile single spinneret electrospinning technique followed by thermal treatment, CuO-NiO/C heterojunction nanofiber was synthesized. The resultant CuO-NiO/C nanofibers exhibited excellent catalytic activity toward ammonia borane (AB) hydrolysis, producing a marked pressure increase in a closed reaction vessel. Moreover, the resultant nanofibers showed highly efficient catalytic activity for reduction of rhodamine 6G (Rh6G, fluorescent molecule), accompanied by the fluorescence quenching. On the basis of these findings, a novel sensing platform utilizing folic-acid (FA) conjugated CuO-NiO/C nanofibers as an artificial enzyme and recognition element for fluorescence imaging and pressure-based (pressure meter) detection of folate receptor (FR)-positive cancer cells was developed. The detection limit is as low as 50 cells/mL. What’s more, the clinical applicability of the sensor was also proven to be suitable for the sensing cancer cells in whole blood. The most important advantage of the synthesized nanofibers based-probe is the combination of pressure meter and fluorescence imaging to specifically see and sensitively count the cancer cells, paving a new way in early liver cancer diagnosis

2-AA enhances survival following BI.

<p>(A) Mice were injected with 2-AA (6.75 mg/kg mice) or PBS 6 h (n = 20), 2 d (n = 20), 4 d (n = 20), 8 d (n = 20), or 30 d (n = 20) prior to BI with PA14. The data shown are averages of two independent experiments. Significance of survival rate differences was determined using the Kaplan-Meier method, with a hazard ratio of 1.8932 (95% CI, 1.0664–6.0718). Infection (−) drastically reduced survival relative to (2-AA W/O BI) controls (p = 0.03). Delivery of 2-AA 4 d before BI (red) had a particularly powerful influence on survival versus mice not pretreated with 2-AA (p = 0.03). A less remarkable, but still significant, survival benefit was also observed in BI mice pre-exposed to 2-AA 6 h, 2 d, 8 d, or 30 d before BI (all p = 0.03 vs. non-infected 2-AA exposed controls). (B) Relative to the effects observed with 2-AA (n = 20), 4 d pretreatment with the 2-AA analogs 4-AA (n = 8; p = 0.03), 2-NA (n = 8; p = 0.03), or MA (n = 8; p = 0.03), or the 2-AA metabolite 3OH-2-AA (n = 8; p = 0.03) prior to PA14 infection had weak, though still statistically significant, positive effects on survival after infection. Significance of survival rate differences was calculated as in A. (C) Bacterial loads in the local muscle 7 d post-BI were significantly higher in mice pretreated with 2-AA 4 d before BI (n = 7) than in control mice subjected to BI without 2-AA pretreatment (n = 7; p<0.05, Kruskal-Wallis test). CFU data are presented on a log₁₀ scale. (D) CFU counts at the site of infection in mice 11 d postinfection. The 2-AA treated mice showed proliferation and higher counts than mice that were not treated with 2-AA. (n = 6; p<0.001, Kruskal-Wallis test). CFU data are presented on a log₁₀ scale.</p

Proposed model for 2-AA immunomodulatory mechanisms.

<p>In naïve cells (left), stimulation with 2-AA induces activation of NF-κB, which leads to the phosphorylation and degradation of I-κBα, releasing the NF-κB dimers p65 and p50. 2-AA also induces the p38 MAPK and JNK pathways to stimulate c-Jun and c-Fos. Activation of MAPK and NF-κB pathway upregulates pro-inflammatory genes. In contrast, in 2-AA pretreated cells (right) over-expression of ERK1/2 activates C/EBPβ, which binds directly to p65, resulting in c/EBPβ-p65 complex formation, and preventing 2-AA induced phosphorylation of p65 upon 2-AA stimulation. This interaction inhibits NF-κB mediated transactivation. The activation of JNK and p38 MAPK are repressed in 2-AA pretreated cells. All together, repression of the p38 MAPK, JNK, and NF-κB pathways abrogates the activation of pro-inflammatory mediators.</p

Transcription and NF-κB inhibitors can block the effects of 2-AA pretreatment.

<p>Western blots showing phosphorylation of JNK1/2 in 2-AA pretreated or untreated cells along with CAPE (1.5 µM) (A), MG-132 (1 µM) (B), and actinomycin D (1 µM) (C) following 0.2 mM or 0.4 mM 2-AA stimulation. Loading was normalized relative to mouse β-actin. One representative experiment (of three) is shown for JNK1/2.</p

Histopathology of lung tissues after 2-AA treatment.

<p>(A) Control healthy (non-infected) lung tissue 4 d after 2-AA treatment. (B) Inflammatory cell infiltration with large areas of consolidation in lung parenchyma 48 h after infection with PA14 (Black arrows indicate the infiltration and necrotic foci). (C) Lack of infiltration 48 h after PA14 infection in the lungs of mice pretreated with 2-AA 4 d prior to BI.</p

2-AA pretreatment modulates activation of the NF-κB pathway in mouse macrophages.

<p>(A) Schematic of 2-AA treatment. Macrophages were left untreated (No Pre) or pretreated with 0.8-mM 2-AA or 4-AA for 48 h (2-AA/4-AA Pre). The untreated and 2-AA pretreated cells were then stimulated with 0.2 mM, 0.4 mM, or 2.0 mM 2-AA (for experiment in B) or 4-AA (for experiment in C). (B) Pretreatment with 2-AA blocked NF-κB activation relative to cells not pretreated with 2-AA (0.8 mM). (C) NF-κB was activated by 2-AA analog 4-AA in 4-AA pretreated and not pretreated cells. Mean values calculated from 2–4 replicate experiments are depicted with SD error bars. (D and E) Following stimulation with 2-AA (0.4 mM), cellular extracts prepared from not pretreated and 2-AA pretreated macrophages. Western blots of I-κBα and I-κBβ degradation (D) and phosphorylation of NF-κB subunit p65 (E). Loading was normalized relative to mouse β-actin. (F and G) A TransAM NF-κB assay showed binding of NF-κB p65 and p50 with the NF-κB promoter in not pretreated and 2-AA pretreated cells following stimulation with 2-AA. Mean values calculated from three replicate experiments are depicted with SD error bars. (p<0.05, Student's t test).</p

Inhibition of p65 phosphorylation in 2-AA pretreated cells is accompanied by <i>de novo</i> formation of c/EBPβ-p65 complexes.

<p>(A) Western blots of cellular extracts incubated with c/EBPβ from macrophages that had been incubated for 48 h with 0.8 mM 2-AA (2-AA Pre) or plain medium (No Pre) and subsequently stimulated with 0.4 mM 2-AA for the indicated time periods. (B) Western blot showing inhibition of ERK1/2 and c/EBPβ in 2-AA pretreated cells in the presence of MEK1 inhibitor PD98059 (1 µM, 5 µM, or 10 µM). Loading was normalized relative to mouse β-actin. (C) In cells treated as above, c/EBPβ-p65 complex formation monitored by IP followed by immunoblotting with anti-c/EBPβ or anti-p65 antibodies.</p

2-AA pretreatment alters the expression of pro- and anti-inflammatory cytokines upon 2-AA stimulation in macrophages.

<p>Levels of TNF-α (A), IFN-γ (B), and TGF-β (C), following 6 h stimulation of 2-AA as measured by ELISA. The experiments were performed in triplicate and the results are expressed as means ± SD. (p<0.05, one-way ANOVA).</p