纳米银粒子改性种植体的研究进展

关节置换术是治疗终末期关节疾病和老年股骨颈骨折最常用的有效手段,在临床研究中,无菌性松动和假体周围感染是造成假体植入失败的主要原因。而良好的骨整合性是使假体保持长期稳定的关键,因此如何提高骨整合能力是骨组织工程研究关注的热点。纳米银粒子(Silver nanoparticles)的抗菌能力备受推崇,它可以有效减少假体周围感染;同时有研究指出它还具备一定的促成骨能力,这将对无菌性松动有改善作用。以上优点使纳米银改性种植体受到广泛关注,本文就纳米银改性种植体的应用作一综述。     

纳米银溶胶的体外细胞毒性评价及其机制研究

目的 对纳米银溶胶的体外细胞毒性进行评价,初步探讨纳米银对细胞的毒性作用机制.方法利用化学还原法制备纳米银溶胶,通过紫外-可见分光光度计和透射电镜对其物理特性进行检测;以小鼠纤维肉瘤细胞( L929)为研究对象,通过细胞形态观察、LIVE/DEAD染色分析和MTT检测来评价纳米银对细胞的毒性作用;在电子显微镜下观察纳米...     

Inhibitory effects of silver nanoparticles against adenovirus type 3 in vitro

Adenoviruses are associated with respiratory, ocular, or gastrointestinal disease. With various species and high morbidity, adenoviruses are increasingly recognized as significant viral pathogen among pediatric and immunocompromised patients. However, there is almost no specific drug for treatment. Silver nanoparticles are demonstrated to be virucidal against influenza A (H1N1) virus, human immunodeficiency virus and Hepatitis B virus. Currently, there is no data regarding whether the silver nanoparticles inhibit the adenovirus or not. The aim of this study is to investigate the effect of silver nanoparticles on adenovirus type 3 (Ad3). The results revealed that HeLa cells infected with silver nanoparticles treated Ad3 did not show obvious CPE. The viability of HeLa cells infected with silver nanoparticles treated Ad3 was significantly higher than that of cells infected with untreated Ad3. There was a significant difference of fluorescence intensity between the cells infected with silver nanoparticles treated and untreated Ad3. The transmission electron microscopy (TEM) showed that silver nanoparticles could directly damage the structure of Ad3 particle. The PCR amplification products of DNA isolated from silver nanoparticles treated Ad3 was decreased in a dose-dependent manner. The decreased DNA loads were also confirmed by real-time PCR experiment. The present study indicates silver nanoparticles exhibit remarkably inhibitory effects on Ad3 in vitro, which suggests silver nanoparticles could be a potential antiviral agent for inhibiting Ad3 infection.     

Impact of exposure time,particle size and uptake pathway on silver nanoparticle effects on circulating immune cells in mytilus galloprovincialis

Nanomaterials have increasingly emerged as potential pollutants to aquatic organisms. Nanomaterials are known to be taken up by hemocytes of marine invertebrates including Mytilus galloprovincialis. Indeed, assessments of hemocyte-related parameters are a valuable tool in the determination of potentials for nanoparticle (NP) toxicity. The present study assessed the effects from two size types of silver nanoparticles (AgNP: <50?nm and <100?nm) on the frequency of hemocytes subpopulations as immunomodulation biomarkers exposed in a mollusk host. Studies were performed using exposures prior to and after inhibition of potential NP uptake pathways (i.e. clathrin- and caveolae-mediated endocytosis) and over different durations of exposure (3, 6 and 12?h). Differential hemocyte counts (DHC) revealed significant variations in frequency of different immune cells in mussels exposed for 3?hr to either AgNP size. However, as exposure duration progressed cell levels were subsequently differentially altered depending on particle size (i.e. no significant effects after 3?h with larger AgNP). AgNP effects were also delayed/varied after blockade of either clathrin- or caveolae-mediated endocytosis. The results also noted significant negative correlations between changes in levels hyalinocytes and acidophils or in levels basophils and acidophils as a result of AgNP exposure. From these results, we concluded AgNP effects on mussels were size and duration of exposure dependent. This study highlighted how not only was NP size important, but that differing internalization mechanisms could be key factors impacting on the potential for NP in the environment to induce immunomodulation in a model/test sentinel host like M. galloprovincialis.     

Biocompatibility of silver nanoparticles and silver ions in primary human mesenchymal stem cells and osteoblasts

The prevention of implant-related infections is an important issue in medical research. The aim is to exploit the strong antimicrobial effect of silver nanoparticles (AgNP) to develop new antibacterial coatings for implants. However, there is still a serious lack of information on the influence of AgNP on bone metabolism. In the present study we have evaluated the influence of AgNP on cell stress, viability, proliferation and differentiation of primary human mesenchymal stem cells (MSC) and osteoblasts (OB). Finally, cellular uptake of the AgNP was examined. After 21 days impairment of cell viability of MSC and OB occurred at a concentration of 10 μg/g of AgNP. Cytotoxicity and inhibition of proliferation was highly time and dose dependent. No influence on cell differentiation, but an increase in cell stress, was observed. Uptake of AgNP into MSC and OB could be confirmed. In summary, these results demonstrate AgNP-mediated cytotoxicity at higher concentrations. Therefore, a therapeutical window for the application of AgNP in medical products might exist. However, the antibacterial benefits and potential health risks of AgNP need to be weighed in further studies.     

Phagocytosis and endocytosis of silver nanoparticles induce interleukin-8 production in human macrophages

Phagocytosis or endocytosis by macrophages is critical to the uptake of fine particles, including nanoparticles, in order to initiate toxic effects in cells. Here, our data enhance the understanding of the process of internalization of silver nanoparticles by macrophages. When macrophages were pre-treated with inhibitors to phagocytosis, caveolin-mediated endocytosis, or clathrin-mediated endocytosis, prior to exposure to silver nanoparticles, Interleukin-8 (IL-8) production was inhibited. Although cell death was not reduced, the inflammatory response by macrophages was compromised by phagocytosis and endocytosis inhibitors.     

Monoisotopic silver nanoparticles-based mass spectrometry imaging of human bladder cancer tissue: Biomarker discovery

PurposeBladder cancer (BC) is the 10th most common form of cancer worldwide and the 2nd most common cancer of the urinary tract after prostate cancer, taking into account both incidence and prevalence.Materials/methodsTissues from patients with BC and also tissue extracts were analyzed by laser desorption/ionization mass spectrometry imaging (LDI-MSI) with monoisotopic silver-109 nanoparticles-enhanced target (¹⁰⁹AgNPET).ResultsUnivariate and multivariate statistical analyses revealed 10 metabolites that differentiated between tumor and normal tissues from six patients with diagnosed BC. Selected metabolites are discussed in detail in relation to their mass spectrometry (MS) imaging results. The pathway analysis enabled us to link these compounds with 17 metabolic pathways.ConclusionsAccording to receiver operating characteristic (ROC) analysis of biomarkers, 10 known metabolites were identified as the new potential biomarkers with areas under the curve (AUC) higher than >0.99. In both univariate and multivariate analysis, it was predicted that these compounds could serve as useful discriminators of cancerous versus normal tissue in patients diagnosed with BC.     

The influence of lysosomal stability of silver nanomaterials on their toxicity to human cells

How silver nanomaterials (Ag NMs) could induce toxicity has been debated heatedly by many researchers. We utilized Ag nanoclusters (Ag NCs) with the same size and ligand protection but different core surface speciation. Ag⁺-rich NCs (Ag⁺-R NCs) and their counterpart, the reduced Ag⁰-rich NCs (Ag⁰-R NCs) are synthesized to represent possible dichotomous stages in silver nanomaterial degradation process. Here we show Ag⁰-R NCs induce higher cellular toxicity when compared to Ag⁺-R NCs. This cellular toxicity is brought about via the modulation of reactive oxygen species (ROS) in cells as a result of the more rapid release of Ag species from Ag⁰-R NCs and subsequent oxidation into Ag⁺ in the lysosomal compartment. The weaker Ag⁰-R bond greatly potentiated the release of Ag species in the acidic and enzymatic processes within the lysosomes. Since lysosomes are absent in bacteria, increasing silver nanomaterials stability may lower toxicity in mammalian cells whilst not reducing their efficacy to fight bacteria; this redesign can result in a safer silver nanomaterial.     

Rapid determination of plasmonic nanoparticle agglomeration status in blood

Plasmonic nanomaterials as drug delivery or bio-imaging agents are typically introduced to biological systems through intravenous administration. However, the potential for agglomeration of nanoparticles in biological systems could dramatically affect their pharmacokinetic profile and toxic potential. Development of rapid screening methods to evaluate agglomeration is urgently needed to monitor the physical nature of nanoparticles as they are introduced into blood. Here, we establish novel methods using darkfield microscopy with hyperspectral detection (hsDFM), single particle inductively-coupled plasma mass spectrometry (spICP-MS), and confocal Raman microscopy (cRM) to discriminate gold nanoparticles (AuNPs) and their agglomerates in blood. Rich information about nanoparticle agglomeration in situ is provided by hsDFM monitoring of the plasmon resonance of primary nanoparticles and their agglomerates in whole blood; cRM is an effective complement to hsDFM to detect AuNP agglomerates in minimally manipulated samples. The AuNPs and the particle agglomerates were further distinguished in blood for the first time by quantification of particle mass using spICP-MS with excellent sensitivity and specificity. Furthermore, the agglomeration status of synthesized and commercial NPs incubated in blood was successfully assessed using the developed methods. Together, these complementary methods enable rapid determination of the agglomeration status of plasmonic nanomaterials in biological systems, specifically blood.     

A modified protocol for the cytochalasin B in vitro micronucleus assay using whole human blood or separated lymphocyte cultures

A modified protocol is described for the in vitro analysis ofmicronuclei in whole blood or separated lymphocyte cultures.The induction of binucleate cells by various concentrationsof cytochalasin B (3, 4.5 or 6 µg/ml) was examined attwo harvest times (68 or 72 h). An optimal yield was obtainedby adding cytochalasin B at a dose of 6 µg/ml to cultures44 h after initiation with harvest 24 h (whole blood) or 28h (separated lymphocytes) later. Cytocentrifuge preparationsof lymphocytes (separated from whole blood using commercialpreparations of Ficoll either at the commencement of the assayor upon harvest) were stained with Acridine Orange. Using thismethod, cytokinesis-blocked lymphocytes remain intact and micronucleiare readily identified. The method is suitable for both wholeblood and separated lymphocyte, thus cultures, thus allowingdirect comparisons of sensitivity to genotoxic agents.     

Hemocompatibility of human whole blood on polymers with a phospholipid polar group and its mechanism.

The hemocompatibility of a polymer containing a phospholipid polar group, poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate(BMA)), with human whole blood was evaluated. When human whole blood without an anticoagulant was contacted with polymers, the blood cell adhesion and aggregation on the polymer without the MPC moiety was extensive, and considerable fibrin deposition was observed. This phenomenon was suppressed with an increase in the polymer MPC composition. Thus, the MPC moiety in the copolymer plays an important role in the nonthrombogenic behavior of the copolymer. These results were also confirmed by the whole blood coagulation time on the polymer surface which was determined by Lee-White method. The adsorption of phospholipids and proteins from human plasma on poly(MPC-co-BMA) was investigated to clarify the mechanism of the nonthrombogenicity observed with the polymer. The amount of phospholipids was increased; whereas, adsorbed proteins were decreased with an increase in the MPC composition. From these results, we concluded that the phospholipids adsorbed on poly(MPC-co-BMA) play the most important role in the nonthrombogenicity of the MPC copolymer.     

Effect of low fibrinogen concentrations on the rheology of human blood in vitro.

The influence of low concentrations of fibrinogen on the rheology of normal human blood was investigated with an instrument that permitted simultaneous determination of viscosity and the state of red blood cell aggregation and deformation. Fibrinogen, in concentrations of 9-82 mg/100 ml, decreased blood viscosity at all shear rates below the value obtained with red blood cells suspended in serum. At concentrations above 116 mg/100 ml viscosity was increased. Aggregate formation increased progressively as the fibrinogen concentration increased, necessitating higher dispersing shear rates. The deformation and alignment of the red cells, occurring at a shear rate of 230 s-1, was facilitated by low concentrations. The effect of fibrinogen on low-shear viscosity is explained by the formation of different kinds of aggregates. At low concentrations, the aggregates consist of only few cells forming spherelike particles displaying hemodynamic properties better than those of the single discoid cells. At normal or high concentrations big rodlike aggregates occur and increase resistance to flow.     

Antibacterial effect of silver nanoparticles on Staphylococcus aureus

Antibacterial activity of silver nanoparticles (AgNPs) was investigated using Staphylococcus aureus PTCC1431 as a model of Gram-positive bacteria. The mechanism of antibacterial activity of AgNPs was then studied by analyzing the growth, morphology, and molecular variations in the cell wall. Experimental data showed that AgNPs at a concentration of 4 μg/ml completely inhibited bacterial growth. Transmission electron microscopy results confirmed cell wall damage produced by AgNPs as well as accumulation of AgNPs in the bacterial membrane. Meanwhile, the AgNP-treated bacteria were monitored by circular dichroism to reveal peptidoglycan variations. Some degree of variation in the α-helix position of the peptide chain was observed. Moreover, increasing the AgNP concentration to 8 μg/ml resulted in release of muramic acid (MA) into the medium, which could be attributed to cell wall distraction. A gas chromatography-tandem mass spectrometry analysis and release of MA, as a bacterial indicator, showed that glycan strands may also be decomposed as a result of AgNP treatment.     

Development of a new dynamic method for quantitative evaluation of in vitro hemocompatibility of biomedical materials

In this study a new dynamic method is introduced allowing the estimation of blood cell adhesion on flat test surfaces by measuring the cell loss in the bulk phase of surface contacting test blood under defined rheological conditions. This was achieved by constructing a novel test chamber permitting the contact of small amounts of blood with a large geometrical test surface. The construction consists of a spiral-shaped flow channel of 0.3 cm width, 0.02 cm height and 78 cm length covered with the biomaterials to be tested from both sides. Laminarity of blood flow in the conduit was confirmed theoretically by the calculation of an equivalent to the Reynolds number for curved systems the so-called Dean number. Furthermore, flow laminarity was proved experimentally finding that the flow rate of blood with different hematocrit values was proportional to the hydrostatic pressure applied. The applicability of the novel 'spiral method' for the estimation of hemocompatibility was demonstrated by evaluation of platelet adhesion onto different polymers in comparison to siliconized and fibrinogen coated glass as reference surfaces. Additionally, it was possible under distinct conditions to determine the adhesion of leucocytes and the detachment of platelet aggregates. Therefore, it was concluded that the spiral method can be used for the assessment of the hemocompatibility of flat biomedical polymers. As main advantages of the new method can be considered the high time efficiency and accuracy without labelling or optical detection of adherent cells.     

Antibiofilm activity of a monolayer of silver nanoparticles anchored to an amino-silanized glass surface

Biofilm production is the crucial pathogenic mechanism of the implant-associated infection and a primary target for new anti-infective strategies. Silver nanoparticles (AgNPs) are attracting interest for their multifaceted potential biomedical applications. As endowed with highest surface/mass ratio and potent antibacterial activity, they can profitably be applied as monolayers at biomaterial surfaces. Desirably, in order to minimize the risks of toxic effects from freely circulating detached nanoparticles, AgNPs should firmly be anchored to the modified biomaterial surfaces. Here we focus on a newly designed glass surface modified with AgNPs and on its antibiofilm properties. Link of a self-assembled monolayer of AgNPs to glass was obtained through preliminary amino-silanization of the glass followed by immersion in an AgNPs colloidal suspension. Static contact angle measure, AFM, TEM, UV-Vis spectroscopy, ICP atomic emission spectroscopy were used for characterization. Antibiofilm activity against the biofilm-producer Staphylococcus epidermidis RP62A was assayed by both CFU method and CLSM. Performances of AgNPs-glasses were: i) excellent stability in aqueous medium; ii) prolonged release and high local concentration of Ag⁺ without any detaching of AgNPs; iii) strong antibiofilm activity against S. epidermidis RP62A. This AgNPs surface-modification can be applied to a large variety of biomaterials by simply depositing glass-like SiO₂ films on their surfaces.     

Photochemical effect of silver nanoparticles on flesh fly larval biological system

With the progress of nanoscience and its applications, silver nanoparticles (AgNPs) have become one of the most interesting nanoparticles owing to their use in different fields. However, the excessive use of AgNPs and its products may cause toxicity in both the environment and in human health. The main goal of this research is to study the toxic and photochemical effects of AgNPs against Sarcophaga argyrostoma larvae through ultrastructure, morphological change, and DNA damage. Treating midgut epithelium with AgNPs led to many alterations in dark conditions, disintegrated epithelium, swollen cells, and shrunken nucleus. Organelles appeared in a loose manner and mitochondria were without cristae, endoplasmic reticulum had dark spots, and peritrophic membrane was loose in appearance. Fatty tissues were vacuolized and muscle fibers lacked normal striations and had many gaps and lysosomal bodies. In the light conditions, the epithelium appeared with detached cells and many vacuoles, organelles were ruptured with many gaps in between, and secretory vesicles were scattered. Peritrophic membrane disappeared. Muscles collapsed and vacuolized loosed fatty tissues were detected. On the other hand, control larvae epithelium appeared regularly distinct, with organelles intact and muscles had clear normal striations. Data showed that AgNPs caused ultrastructural and morphological changes of the external cuticle of the 4th instar larvae along with a significant effect on DNA damage that occurred after the larval treatment, reflecting the toxicity of AgNPs.     

Immunomodulatory effect of gelatin-coated silver nanoparticles in mice: Ultrastructural evaluation

Silver nanoparticles (SNP) are used in many pharmaceutical, cosmetic, and industrial products already available in the market. Although they are considered relatively safe, many toxic and pathological alterations in different organs including immune organs were reported after SNP administration. In this study, 10-week-old male mice (n = 20) were divided into two groups. Ten mice received greenly synthesized gelatin-coated silver nanoparticles in a dose of 10 mg/kg body weight for five consecutive days while the other 10 received 0.5 ml of distilled water daily for 5 days and kept as control. At the sixth day, all mice were sacrificed; blood and tissue samples were collected and prepared for pathological analysis. Liver and kidney lesions were in the form of degenerative and inflammatory changes. Interestingly, the immune organs were drastically affected by SNP treatment. Severe hyperplasia of the Peyer’s patches was noticed in the intestines of intoxicated animals both in gross and microscopic examination. Spleen was enlarged and showed large number of megakaryocytes. The particles were encountered in membrane-bound phagosomes inside macrophages in different organs like lungs and spleen. Blood picture complied to morphological findings with an increase in monocytes and eosinophils accompanied by drop in the platelets count in the intoxicated animals.     

不同浓度重组人促红细胞生成素对神经干细胞体外培养增殖的影响

背景：重组人促红细胞生成素是一种糖蛋白,近年的研究表明其对神经细胞的许多功能活动均具有调节作用。 目的：观察不同浓度重组人促红细胞生成素对神经干细胞体外培养增殖的影响。 方法：提取新生SD大鼠神经干细胞,用含不同浓度(5,50,500 U/mL)重组人促红细胞生成素和20 μg/L碱性成纤维细胞生长因子的无血清培养基进行培养,以不含重组人促红细胞生成素无血清培养基为对照组。细胞培养7 d后计算神经干细胞克隆形成率,培养10 d后计数NSE和GFAP免疫阳性细胞数。 结果与结论：添加重组人促红细胞生成素组细胞增殖较快,最终神经球的数量多于对照组,以50 U/mL重组人促红细胞生成素组作用显著;50 U/mL重组人促红细胞生成素组的生长速度显著快于对照组。50 U/mL重组人促红细胞生成素组中NSE和GFAP免疫阳性细胞明显多于对照组(P < 0.01)。结果表明重组人促红细胞生成素对神经干细胞体外培养增殖有促进作用,尤以适中浓度(50 U/mL) 作用更加明显。