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Objective Interleukin-17 (IL-17) and interferon-gamma (IFN-γ) are all pro-inflammatory cytokines produced by specific subsets of T-cells and are also considered crucial regulators in bone remodeling, but their effects on osteogenesis have not been carefully studied. So, this study aimed to investigate the effects of secreting cytokines IL-17 and IFN-γ on the osteogenesis of primary osteoblasts and to clarify the potential roles of the related Janus activated kinase 2 (JAK2) and downstream signal transducer and activator of transcription 3 (STAT3) signaling pathway in bone remodeling. Methods The proliferation of osteoblasts was evaluated by MTT assay. Osteogenic activity was tested by alkaline phosphatase (ALP) activity assay and alizarin red staining. The mRNA levels of ALP, osteocalcin, osteoprotegerin (OPG), Runt-related transcription factor 2 (Runx2) and receptor activator of nuclear factor-kappa B ligand (RANKL) were also measured by real-time quantitative PCR. The JAK2-STAT3 pathway was evaluated by Western blot. Results Osteoblasts showed no obvious proliferation when treated with IL-17 and/or IFN-γ, but higher ALP activities were observed in primary osteoblasts treated with IL-17 or IL-17 + IFN-γ in induction medium. We also found that IL-17 could promote the gene expression of Alp , Runx2 , Osteocalcin , Opg , and Rankl , while IFN-γ might attenuate this effect. Nevertheless, IL-17 and IFN-γ exhibited an inhibitory effect on the calcification of p rimary osteoblasts. We also found that IL-17 could directly facilitate RANKL expressions by JAK2-STAT3 pathway. Conclusion The positive effects of IL-17 and IFN-γ on the early-stage differentiation and the negative effects on the calcification of murine calvarial osteoblasts contribute to our understanding of the role and interaction of inflammatory factors in the bone remodeling and as fundamental mechanisms involved in the destruction of alveolar bone.

We report on the electrodeposition of a 3-aminopropyltriethoxysilane-chitosan (APTES–CS) hybrid gel film for in situ immobilization of glucose oxidase (GOx) on an Au or platinized Au (Pt nano /Au) electrode for biosensing of glucose. Controllable electroreduction of p -benzoquinone is used to lift the electrode-surface pH for the GOx–APTES–CS codeposition, which was monitored by an electrochemical quartz crystal microbalance. The fabrication procedures of the biosensor and the parameters influencing the biosensing performance were optimized. The prepared porous GOx–APTES–CS/Pt nano /Au and GOx–APTES–CS/Au electrodes can be used to detect the enzymatically generated H 2 O 2 at 0.5 and 0.7 V vs SCE, respectively. The enzyme electrodes exhibited linear responses to glucose concentration from 0.2 μM to 8.2 mM ( R = 0.998, at Pt nano /Au substrate) and from 0.2 μM to 5.5 mM ( R = 0.998, at Au substrate), with current sensitivities of 69.5 (Pt nano /Au) and 65 (Au) μA mM −1 cm −2 , respectively, and a detection limit of 0.2 μM ( S / N = 3) was achieved for each electrode. The response time was less than 5 (Pt nano /Au) or 8 (Au) s. It is striking that the enzyme electrodes remained their initial response sensitivity after storage for 5 (Au) and >6 (Pt nano /Au) months in 0.10 M PBS (pH 7.0) at 4 °C.

Electrodeposition has been so widely used to immobilize biomacromolecules, and it is always an important topic to increase the load and activity of the immobilized biomacromolecules. We report here on a new, simple and rather universal method for the highly efficient immobilization of enzymes by aqueous electrodeposition of enzyme-tethered chitosan (CS) for sensitive amperometric biosensing. Glucose oxidase (GOx) is chosen here to examine the proposed protocol in detail. GOx was crosslinked to CS with low-concentration glutaraldehyde (GA, 0.080 wt%), and the electroreduction of added H(2)O(2) increased the electrode-surface pH and triggered the electrodeposition of a GOx-GA-CS composite film. The GOx-GA-CS electrodeposition was monitored by an electrochemical quartz crystal microbalance and is theoretically discussed based on an electrogenerated base-to-acid titration model. The prepared first-generation enzyme electrode (CS-GA-GOx/Pt(nano)/Au) exhibits a current sensitivity as high as 102 microA mM(-1) cm(-2) at 0.70 V vs SCE, being 13 times that of the CS-GOx/Pt(nano)/Au prepared similarly but without the GOx-CS precrosslinking. UV-vis spectrophotometric determination of the GOx remains in the supernatant liquids after pH-induced CS precipitation suggested a high enzyme load in the GOx-GA-CS film, and amperometric measurements suggested a negligible decrease in the enzymatic activity of GOx after its reaction with the low-concentration GA. Also, the proposed protocol works well for the precrosslinking manner of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysulfosuccinimide activation, the water-electroreduction-triggered CS electrodeposition, the second-generation biosensing mode, a 5.0-microm-radius Pt ultramicroelectrode, and immobilization of alkaline phosphatase for phenyl phosphate biosensing. The proposed protocol of pretethering the target biomacromolecules to the electrodeposition precusor for immobilization of the biomacromolecule at high load/activity is recommended for wide applications.

We report on the aqueous preparation of novel polymeric bionanocomposites (PBNCs) with polydopamine (PDA) as an efficient matrix to support antibody and uniformly dispersed abundant Pt nanoparticles (PtNPs) for high performance sandwich-type amperometric immunoassay. We prepare three kinds of PBNCs (PBNCs 1 , 2 , and 3 ) via chemical polymerization synthesis ( 1 ) and further adsorption ( 2 ) or glutaldehyde (GA) covalent crosslinking ( 3 ) of antibody. Transmission electron microscopy, atomic force microscopy, and quartz crystal microbalance are used to characterize the PBNCs. The results show that PDA is an excellent matrix to support antibody and PtNPs due to its high biocompatibility, adsorbability, and processibility, and the thus-prepared PBNCs are of high immuno-recognition efficiency and high catalytic activity toward H 2 O 2 reduction. Then, we examine the applicability of the prepared PBNCs for sandwich-type amperometric immunosensing using a model immuno-pair of human immunoglobulin G (hIgG) and anti-hIgG, and obtain the signal by detecting the electro-reduction of H 2 O 2 catalyzed by the PtNPs label. The constructed immunosensors using PBNCs 1 , 2 and 3 exhibit detection limits of 0.068, 0.037, and 0.018 ng mL −1 , respectively, being competitive with or better than the reported analogues. Our immunosensors also present good reproducibility, stability, regeneration ability, specificity, and satisfactory feasibility for target assay in clinical human serum samples. The suggested protocol for the preparation of PBNCs with high signal-transduction ability is expected to find wide bioassay applications.