Effect of Magnesium on Dentinogenesis of Human Dental Pulp Cells.

Introducing therapeutic ions into pulp capping materials has been considered a new approach for enhancing regeneration of dental tissues. However, no studies have been reported on its dentinogenic effects on human dental pulp cells (HDPCs). This study was designed to investigate the effects of magnesium (Mg2+) on cell attachment efficiency, proliferation, differentiation, and mineralization of HDPCs. HDPCs were cultured with 0.5 mM, 1 mM, 2 mM, 4 mM, and 8 mM concentrations of supplemental Mg2+ and 0 mM (control). Cell attachment was measured at 4, 8, 12, 16, and 20 hours. Cell proliferation rate was evaluated at 3, 7, 10, 14, and 21 days. Crystal violet staining was used to determine cell attachment and proliferation rate. Alkaline phosphatase (ALP) activity was assessed using the fluorometric assay at 7, 10, and 14 days. Mineralization of cultures was measured by Alizarin red staining. Statistical analysis was done using multiway analysis of variance (multiway ANOVA) with Wilks' lambda test. Higher cell attachment was shown with 0.5 mM and 1 mM at 16 hours compared to control (P < 0.0001). Cells with 0.5 mM and 1 mM supplemental Mg2+ showed significantly higher proliferation rates than control at 7, 10, 14, and 21 days (P < 0.0001). However, cell proliferation rates decreased significantly with 4 mM and 8 mM supplemental Mg2+ at 14 and 21 days (P < 0.0001). Significantly higher levels of ALP activity and mineralization were observed in 0.5 mM, 1 mM, and 2 mM supplemental Mg2+ at 10 and 14 days (P < 0.0001). However, 8 mM supplemental Mg2+ showed lower ALP activity compared to control at 14 days (P < 0.0001), while 4 mM and 8 mM supplemental Mg2+showed less mineralization compared to control (P < 0.0001). The study indicated that the optimal (0.5-2 mM) supplemental Mg2+ concentrations significantly upregulated HDPCs by enhancing cell attachment, proliferation rate, ALP activity, and mineralization. Magnesium-containing biomaterials could be considered for a future novel dental pulp-capping additive in regenerative endodontics.

Effect of magnesium on the osteogenesis of normal human osteoblasts.

Biomaterials containing magnesium are used for implants and bone regeneration. However, mechanisms underlying the biologic effects of magnesium are still largely unknown and have not been examined on normal human osteoblasts. This study was designed to test the effect of supplemented Mg2+ concentrations between 0.5 mM and 16 mM on the osteogenic behaviors of normal human primary osteoblasts. Human primary osteoblasts were cultured in the groups with various concentrations of supplemented magnesium for various time intervals. Cell proliferation was measured using crystal violet staining. Degree of Alkaline Phosphatase (ALP) activity was measured by fluorometric assay. Expression of osteocalcin was measured by immunosorbent assay. Mineralization of cultures was determined by Alizarin Red S staining. Results showed that initial cell attachment efficiency was not affected by supplemented Mg2+ (P > 0.05). At 21 days, proliferation rates increased in groups containing 0.5 mM-4 mM supplemented Mg2+ and decreased in groups of supplemented 8 mM and 16 mM Mg2+. ALP activity and osteocalcin expression were upregulated in groups of supplemented Mg2+ between 0.5 mM-2.0 mM (P < 0.05), but downregulated in groups with supplemented Mg2+ concentrations of 4mM and above (P < 0.05). Cultures with 1 mM and 2 mM supplemented Mg2+ showed upregulated mineralization activity compared to the control (P < 0.05), but downregulated in groups with supplemented Mg2+ concentrations of 4 mM and above (P < 0.05). The present study based on an experimental design demonstrated the impact of 2 mM supplemented Mg2+ on induced-proliferation and differentiation of normal human osteoblasts.

Neurotoxicity of Silver Nanoparticles in Rat Brain After Intragastric Exposure.

It is known that the biological half-life of silver in the central nervous system is longer than in other organs. However, the potential toxicity of silver nanoparticles (NPs) on brain tissue and the underlying mechanism(s) of action are not well understood. In this study, neurotoxicity of silver NPs was examined in rat after intragastric administration. After a two-week exposure to low-dose (1 mg/kg, body weight) or high-dose (10 mg/kg) silver NPs, the pathological and ultrastructural changes in brain tissue were evaluated with H&E staining and transmission electron microscopy. The mRNA expression levels of key tight junction proteins of the blood-brain barrier (BBB) were analyzed by real-time RT-PCR, and several inflammatory factors were assessed in blood using ELISA assay. We observed neuron shrinkage, cytoplasmic or foot swelling of astrocytes, and extra-vascular lymphocytes in silver NP exposure groups. The cadherin 1 (2(-ΔΔCt): 1.45-fold/control) and Claudin-1 (2(-ΔΔCt): 2.77-fold/control) were slightly increase in mRNA expression levels, and IL-4 significantly increased after silver NP exposure. It was suggest that silver NP can induce neuronal degeneration and astrocyte swelling, even with a low-dose (1 mg/kg) oral exposure. One potential mechanism for the effects of silver NPs to the nervous cells is involved in inflammatory effects.

Toxic responses in rat embryonic cells to silver nanoparticles and released silver ions as analyzed via gene expression profiles and transmission electron microscopy.

After exposing rat embryonic cells to 20 µg/mL of silver nanoparticle (NP) suspension and their released ions for different time periods, silver nanoparticles were found in cellular nuclei, mitochondria, cytoplasm and lysosomes by transmission electron microscopy (TEM). We also observed mitochondrial destruction, distension of endoplasmic reticulum and apoptotic bodies. Global gene expression analysis showed a total of 279 genes that were up-regulated and 389 genes that were down-regulated in the silver-NP suspension exposure group, while 3 genes were up-regulated and 41 genes were down-regulated in the silver ion exposure group. Further, the GO pathway analysis suggested that these differentially expressed genes are involved in several biological processes, such as energy metabolism, oxygen transport, enzyme activities, molecular binding, etc. It is possible that inhibition of oxygen transport is mediated by the significant down-regulation of genes of the globin family, which might play an important role in silver ion-induced toxicity. KEGG pathway analysis showed that there were 23 signal pathways that were affected in the cells after exposure to silver-NP suspension, but not silver ion alone. The most significant change concerned inflammatory signal pathways, which were only found in silver-NP suspension exposed cells, indicating that inflammatory response might play an important role in the mechanism(s) of silver-NP-induced toxicity. The significant up-regulation of matrix metalloproteinases 3 and 9 suggests that silver NPs could induce extracellular matrix degradation via an inflammatory signaling pathway. The significant up-regulation of secretory leukocyte peptidase inhibitor and serine protease inhibitor 2c was considered to be an embryonic cellular defense mechanism in response to silver-NP-induced inflammation.

Genotoxicity and molecular response of silver nanoparticle (NP)-based hydrogel.

BACKGROUND: Since silver-nanoparticles (NPs) possess an antibacterial activity, they were commonly used in medical products and devices, food storage materials, cosmetics, various health care products, and industrial products. Various silver-NP based medical devices are available for clinical uses, such as silver-NP based dressing and silver-NP based hydrogel (silver-NP-hydrogel) for medical applications. Although the previous data have suggested silver-NPs induced toxicity in vivo and in vitro, there is lack information about the mechanisms of biological response and potential toxicity of silver-NP-hydrogel. METHODS: In this study, the genotoxicity of silver-NP-hydrogel was assayed using cytokinesis-block micronucleus (CBMN). The molecular response was studied using DNA microarray and GO pathway analysis. RESULTS AND DISCUSSION: The results of global gene expression analysis in HeLa cells showed that thousands of genes were up- or down-regulated at 48 h of silver-NP-hydrogel exposure. Further GO pathway analysis suggested that fourteen theoretical activating signaling pathways were attributed to up-regulated genes; and three signal pathways were attributed to down-regulated genes. It was discussed that the cells protect themselves against silver NP-mediated toxicity through up-regulating metallothionein genes and anti-oxidative stress genes. The changes in DNA damage, apoptosis and mitosis pathway were closely related to silver-NP-induced cytotoxicity and chromosome damage. The down-regulation of CDC14A via mitosis pathway might play a role in potential genotoxicity induced by silver-NPs. CONCLUSIONS: The silver-NP-hydrogel induced micronuclei formation in cellular level and broad spectrum molecular responses in gene expression level. The results of signal pathway analysis suggested that the balances between anti-ROS response and DNA damage, chromosome instability, mitosis inhibition might play important roles in silver-NP induced toxicity. The inflammatory factors were likely involved in silver-NP-hydrogel complex-induced toxic effects via JAK-STAT signal transduction pathway and immune response pathway. These biological responses eventually decide the future of the cells, survival or apoptosis.

Calcium upregulated survivin expression and associated osteogenesis of normal human osteoblasts.

Survivin is an antiapoptotic protein expressed in all phases of the normal cell cycle but is at its highest level during the G2/M interphase. This protein has been recently identified in normal human osteoblasts and has raised questions about the regulation of its expression. This study intends to verify if survivin expression could be manipulated by external factors such as calcium ions. Normal human alveolar bone explants recovered from six healthy donors were cultured to 2nd passage. Cells were cultured with essential medium as a control and with medium containing supplemental calcium ions at a concentration of 30 parts per million as a study group. Vitamin D(3) was added to all culture groups at the 5th and 18th days to promote differentiation. Differentiation markers were confirmed by performing mineralization, alkaline phosphatase (ALP), and osteocalcin assays at 7 and 21 days. Cell attachment was measured at 16 h and used as a reference for cell proliferation at 7 days and 21 days. Survivin levels were measured at 16 h, 7 and 21 days. Compared with the control group, the study group presented a significant increase of survivin expression at 16 h (p < 0.01), at 7 days (p < 0.01), and at 21 days (p < 0.05), a significant increase of cell proliferation, ALP activity and mineralization at 7 days (p < 0.05) and 21 days (p < 0.05), and a significant increase in osteocalcin expression only at 21 days (p < 0.01). This study demonstrated that survivin expression could be significantly upregulated by calcium-enhanced normal human osteoblast cultures, which might correlate to subsequent upregulation of cell proliferation and differentiation.

Effects of SiO2 nanoparticles on HFL-I activating ROS-mediated apoptosis via p53 pathway.

Silicon dioxide nanoparticles (SiO(2) NPs) have attracted increasing interest as nanovehicles for delivering drugs, genes and bio-active molecules into cells. However, it is still unknown whether SiO(2) NPs could cause side-effects to normal cells. In the present study, human lung fibroblasts (HFL-Is) were directly exposed to two different sizes of SiO(2) NPs. The effect of size and concentration on cell response was studied by analyzing the cell viability, the ratio of apoptosis and the pathway of cell injury. The results demonstrated that a size-associated and a dose-dependent toxicity of HFL-Is was induced by SiO(2) NPs. Meanwhile, the expression of reactive oxygen species in HFL-I was significantly increased. This activation effect was accompanied by upregulation of p53 expression, release of cytochrome C from chondriosomes, inhibition of Bcl2, and activation of Bax and caspase 9. These findings implied that SiO(2) NPs might induce apoptosis of HFL-Is by stimulating reactive oxygen species release and subsequently causing the activation of p53 pathway in vitro.

Silver nanoparticles crossing through and distribution in the blood-brain barrier in vitro.

Silver nanoparticles (SNPs) translocate to the brain through the blood stream after they are implanted in vivo. The aim of this study was to investigate the distribution of SNPs that crossed through the blood-brain barrier (BBB). An in vitro BBB model established by co-cultures of rat brain microvessel vascular endothelial cells (BMVECs) with astrocytes (ACs) was cultured with cell culture medium containing 100 microg/mL of either SNPs or silver microparticles (SMPs). After 4 hours of culture, the ultrastructure and its silver content of BBB was evaluated with transmission electronic microscopy (TEM) and inductively-coupled plasma mass spectrometry (ICP-MS) respectively. Results demonstrated that SNPs crossed the BBB and accumulated inside BMVECs, while the SMPs did not. The data indicated a special distribution of SNPs in the BBB and suggested that SNPs pass the BBB mainly by transcytosis of capillary endothelial cells. Further study would be necessary to evaluate the actual biological effects of SNPs on the brain.

Synthesis of a zwitterionic silane and its application in the surface modification of silicon-based material surfaces for improved hemocompatibility.

A phosphorylcholine-like silane coupling agent bearing zwitterionic molecular structure was synthesized and studied. The chemical structure of this silane coupling agent was characterized by FTIR, 1H NMR and 31P NMR. The zwitterionic structure was successfully constructed onto the surface of silicon as a self-assembled layer (SAL). Static water contact angle, and atomic force microscopy (AFM) were used to investigate the wettability and surface topography of the modified silicon surfaces. Static water contact angle results indicated that the hydrophilicity of the surfaces could be effectively improved by the modification with this zwitterionic silane coupling agent. The changes of the topography and water contact angle of the modified surfaces with different incubation periods in PBS solution were also measured to evaluate the stability of the SALs. Blood compatibility of the modified surfaces were evaluated by testing the full-blood activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT), as well as by observing the adhered blood platelets onto the surface. The modified surfaces showed prolonged clotting time and fewer adherent platelets, revealing that the blood compatibility was evidently improved by the modification using this zwitterionic silane.

The effect of a layer-by-layer chitosan-heparin coating on the endothelialization and coagulation properties of a coronary stent system.

A biomacromolecular layer-by-layer coating process of chitosan/heparin onto a coronary stent is designed for the acceleration of the re-endothelialization and healing process after coronary stent deployment. The results of in vitro culturing of porcine iliac artery endothelial cells as well as the measurements of the APTT, PT and TT supported the rationale that the combination of chitosan and heparin could bring both endothelial cell compatibility and haemocompatibility to the stent surface. A porcine coronary injury model and arteriovenous shunt model were used for the further evaluation of the application of this kind of surface-modified stainless steel stent in vivo. The final results proved that this facile coating approach could significantly promote re-endothelialization and was safer compared with bare metal stents for its much improved anticoagulation property.

Microbial profile on metallic and ceramic bracket materials.

The placement of orthodontic appliances creates a favorable environment for the accumulation of a microbiota and food residues, which, in time, may cause caries or exacerbate any pre-existing periodontal disease. The purpose of the present study was to compare the total bacterial counts present on metallic and ceramic orthodontic brackets in order to clarify which bracket type has a higher plaque retaining capacity and to determine the levels of Streptococcus mutans and Lactobacillus spp on both types of brackets. Thirty-two metallic brackets and 24 ceramic brackets were collected from orthodontic patients at the day of debonding. Two brackets were collected from each patient; one from a maxillary central incisor and another from a maxillary second premolar. Sixteen patients who used metallic brackets and 12 patients who used ceramic brackets were sampled. Bacterial populations were studied using "checkerboard" DNA-DNA hybridization, which uses DNA probes to identify species in complex microbial samples. The significance of differences between groups was determined using the Mann-Whitney U-test. Results showed no significant differences between metallic and ceramic brackets with respect to the caries-inducing S mutans and L acidophilus spp counts. Mean counts of 8 of 35 additional species differed significantly between metallic and ceramic brackets with no obvious pattern favoring one bracket type over the other. This study showed higher mean counts of Treponema denticola, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum ss vincentii, Streptococcus anginosus, and Eubacterium nodatum on metallic brackets while higher counts of Eikenella corrodens, Campylobacter showae, and Selenomonas noxia were found on ceramic brackets.