Effects of four types of hydroxyapatite nanoparticles with different nanocrystal morphologies and sizes on apoptosis in rat osteoblasts

The oxidative stress-induced osteoblast apoptosis plays an important role in the pathological process of osteoporosis, but the roles of autophagy in oxidative stress and apoptosis of osteoblasts remain unclear. This study aimed to observe the role of autophagy in oxidative stress injury of osteoblasts and the relationship between autophagy and apoptosis. Mc3T3-E1 cells were stimulated with different concentrations (0.1, 0.5, and 1 mM) of hydrogen peroxide. The cell viability was detected via cell counting kit 8 (CCK8) at different time points (0, 2, 6, 8, and 12 h), the apoptosis was detected via Western blotting and flow cytometry, and the autophagy was detected via macrophage-derived chemokine (MDC) and transmission electron microscope. The changes in expression of autophagy-associated protein, Beclin1, and LC3II/I ratio, were detected via Western blotting. Moreover, the intracellular reactive oxygen species (ROS) level and extracellular superoxide dismutase (SOD) level were observed using the autophagy regulators, rapamycin (Rap) and 3-methyladenine (3-MA), so as to clarify the interaction between autophagy and cellular oxidation. Hydrogen peroxide-induced apoptosis and autophagy of osteoblasts were in dose- and time-dependent manners; the hydrogen peroxide inhibitors could inhibit the autophagy level, and autophagy inhibitor (3-MA) could significantly enhance the hydrogen peroxide-induced ROS level and apoptosis rate in cells. Besides, Western blotting confirmed that the cleaved caspase-3 and cleaved poly adenosine diphosphate ribose polymerase (PARP) proteins were increased. The autophagy inducer (Rap) partially inhibited the hydrogen peroxide-induced oxidative stress and apoptosis. Autophagy inhibits the oxidative stress-mediated apoptosis of osteoblasts, which is a potential target for the osteoporosis treatment.

BackgroundOsteoblasts play important roles in the process of osteogenesis and prevention of osteonecrosis. Dexamethasone (Dex), a type of glucocorticoids (GCs), induces apoptosis of osteoblasts and leads to the occurrence of non-traumatic osteonecrosis. This study aimed to explore the effects of phosphatidylinositol 3-kinase/Protein kinase 3 (PI3K/AKT) and glycogen synthase kinase 3β (GSK3β) on Dex-induced osteoblasts apoptosis. MethodsViabilities, proliferation, and apoptosis of primary osteoblasts and pre-osteoblast MC3T3-E1 cells after Dex treatment were detected using cell counting kit-8 (CCK-8) assay, 5-bromo-2′-deoxyuridine (BrdU) incorporation assay, FITC-Annexin V/PI staining and western blotting, respectively. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) staining was performed to measure the intracellular reactive oxygen species (ROS) levels after Dex treatment. N-acetyl-l-cysteine (NAC) was used as ROS scavenger in this research. The expressions of PI3K/AKT and GSK3β in osteoblasts and MC3T3-E1 cells after Dex treatment were analyzed using western blotting and qRT-PCR, respectively. Then the effects of GSK3β knockdown on Dex-induced apoptosis of osteoblasts were explored. Alkaline phosphatase (ALP) activity assay was used to detect the role of Dex in regulating ALP activity. ResultsDex remarkably inhibited proliferation and induced apoptosis of osteoblasts and MC3T3-E1 cells. Dex potentially attenuated the osteoblast differentiation. The intracellular ROS levels were significantly increased after Dex treatment. Dex suppressed the activation of PI3K/AKT pathway in osteoblasts and MC3T3-E1 cells by down-regulating the expressions of p-PI3K and p-AKT. The expressions of GSK3β in osteoblasts and MC3T3-E1 cells were obviously up-regulated after Dex treatment. Knockdown of GSK3β alleviated Dex-induced osteoblast and MC3T3-E1 cell apoptosis by decreasing the expressions of Bax, cleaved-caspase 3, cleaved-caspase 9 and increasing the expression of Bcl-2. ConclusionOur research verified that Dex induced osteoblasts apoptosis by ROS-PI3K/AKT/GSK3β signaling pathway.

Molecular mechanism of nitric oxide-induced osteoblast apoptosis

CaMKⅡ mediates cadmium induced apoptosis in rat primary osteoblasts through MAPK activation and endoplasmic reticulum stress

Hydroxyapatite nanoparticles (HA-NPs) have recently been proposed as a novel phosphorus (P) fertiliser that not only provide increased efficiency through controlled release but also minimise adverse environmental impacts. In the present study, three types of HA-NPs were synthesised with differences in surface charge. The P release dynamics of the three HA-NPs was investigated by incubating the compounds in two P-deficient soils (an Ultisol and a Vertisol) in the laboratory for up to 240 d. Their fertilising effect on sunflower (Helianthus annuus) was assessed by a glasshouse pot experiment using the two soils, their mobility in both soils was examined through a column leaching experiment with sand as a control. The surface charge of the three HA-NPs was +21 (±4.3), 0 (±3.5) and −12 (±2.7) mV in a neutral medium (pH 7) [hereafter defined as HA-NPs(+), HA-NPs(0), and HA-NPs(‒)], with an average particle size of 25.7 nm. In the Ultisol (pH 4.7), the addition of HA- NPs resulted in a smaller initial increase in P availability than triple superphosphate (TSP) due to their lower solubility. However, P availability in the TSP treatment decreased over time due to adsorption of P to the soil constituents, with a concomitant increase in the NaOH-extractable P fraction. In contrast, the release of P from the HA-NPs remained relatively constant over the entire incubation period. After 45 d, the soils amended with negatively charged HA-NPs had higher levels of available P when estimated using diffusive gradients in thin films (DGT-P) compared to both the neutral and the positively charged HA-NPs. In the Vertisol (pH 8.2), although the addition of TSP increased P availability markedly, amendment with the three HA-NPs did not increase P availability due to the low solubility of hydroxyapatite at this high pH. Indeed, for the Vertisol, most of the P added as HA-NPs remained in the Ca-P fraction. For the glasshouse experiment, in the Ultisol (pH 4.7), addition of TSP or any of the three HA-NP compounds increased plant biomass by 6.4-11.6 times, with the HA-NPs(‒) being significantly more effective than the other three P compounds (including TSP), increasing plant fresh biomass 16.5-fold for the shoots and 8-fold for the roots. The rock phosphate (RP) increased plant biomass the least (only by 1.5-fold). In the Vertisol, none of the three HA-NPs compounds or the RP significantly increased plant growth, whereas TSP increased plant fresh biomass by 28 % after 35 d. Through the column experiment, it was also found that although overall mobility of P in both soils is low, the application of HA-NPs(‒) in the Ultisol slightly increased P mobility, moving 5 % of the P to 100 mm depth of the column. The results show that for the acidic Ultisol, HA-NPs with altered surface charge is a competitive form of P fertiliser. However, HA-NPs were not effective in an alkaline soil due to their lower solubility. This information is important in the development of HA-NPs as a novel fertiliser, and it is important to consider the development of HA-NPs with altered surface properties (i.e. particle size, surface charge) to match the soil properties.

Correlation of particle properties with cytotoxicity and cellular uptake of hydroxyapatite nanoparticles in human gastric cancer cells

Long-term excessive fluoride intake is linked to skeletal disease. Skeletal health is influenced by the balance between bone formation and resorption of which osteoblast function is critical. The objectives of this study were to determine the effect of fluoride treatment on osteoblast proliferation, apoptosis and caspase-3 and caspase-9 mRNA expression in vitro. Neonatal rat osteoblasts were cultured in the presence of varying concentrations (0.5-30 mg/l) of sodium fluoride and effects of treatments were determined. Treatment with sodium fluoride inhibited osteoblast proliferation in a dose-dependent fashion and effects were maximal after 120 h incubation. A significant increase in osteoblast apoptosis was observed (after 24 and 72-h treatment) in response to the lowest dose of sodium fluoride (0.5 mg/l) and osteoblast apoptosis was further increased in response to higher doses. Increased-osteoblast caspase-3 and caspase-9 mRNA was also observed in response to sodium fluoride treatment (5 mg/l) for 72 h. Results indicate that negative effects of excess fluoride on skeletal health may be mediated in part by inhibition of osteoblast survival.

Tailoring hydroxyapatite nanoparticles to increase their efficiency as phosphorus fertilisers in soils

Iron overload induces apoptosis of osteoblast cells via eliciting ER stress-mediated mitochondrial dysfunction and p-eIF2α/ATF4/CHOP pathway in vitro

Activation of the transcription factor hypoxia inducible factor‑1α (HIF-1α) is considered critical for the stimulation of osteogenic markers including runt‑related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteocalcin, which are closely associated with forkhead boxclassO1 (Foxo1) levels in osteoblasts. The present study explored the associations between HIF‑1α and Foxo1 in the regulation of cell viability, proliferation and apoptosis of osteoblasts. Osteoblasts obtained from children's iliac cancellous bone were used in the present study, which were confirmed by immunofluorescence staining for the osteoblast marker osteocalcin. The results revealed that the levels of reactive oxygen species and apoptosis were markedly increased in cells with knockdown of HIF‑1α. By contrast, these were reduced in response to overexpressed HIF‑1α. In addition, HIF‑1α overexpression significantly stimulated cell viability, which was suppressed by silencing HIF‑1α. HIF‑1α overexpression also significantly increased the transcriptional and translational levels of Foxo1. Conversely, silencing HIF‑1α markedly suppressed the expression levels of Foxo1. Furthermore, silencing HIF‑1α reduced the expression of osteogenic markers, including Runx2, ALP and osteocalcin. Runx2 and ALP expression induced by HIF1α were markedly reversed by Foxo1 small interfering (si)RNA, whereas osteocalcin was not significantly affected by Foxo1 siRNA. Therefore, the cooperation of and interactions between HIF‑1α and Foxo1 may be involved in the regulation of osteoblast markers, and serve a pivotal role in the proliferation and apoptosis of osteoblast. The HIF1α‑induced expression of Runx2 and ALP may be completely dependent on the expression levels of Foxo1, and in turn, osteocalcin may be partially dependent on Foxo1 expression.

Exposure to cadmium (Cd) induces apoptosis in osteoblasts (OBs); however, little information is available regarding the specific mechanisms of Cd-induced primary rat OB apoptosis. In this study, Cd reduced cell viability, damaged cell membranes and induced apoptosis in OBs. We observed decreased mitochondrial transmembrane potentials, ultrastructure collapse, enhanced caspase-3 activity, and increased concentrations of cleaved PARP, cleaved caspase-9 and cleaved caspase-3 following Cd treatment. Cd also increased the phosphorylation of p38-mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases (ERK)1/2 and c-jun N-terminal kinase (JNK) in OBs. Pretreatment with the caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, ERK1/2 inhibitor (U0126), p38 inhibitor (SB203580) and JNK inhibitor (SP600125) abrogated Cd-induced cell apoptosis. Furthermore, Cd-treated OBs exhibited signs of oxidative stress protection, including increased antioxidant enzymes superoxide dismutase and glutathione reductase levels and decreased formation of reactive oxygen species. Taken together, the results of our study clarified that Cd has direct cytotoxic effects on OBs, which are mediated by caspase- and MAPK pathways in Cd-induced apoptosis of OBs.

Cadmium (Cd) is a common environmental pollutant that can damage many organs and the fetus. We previously reported that Cd induced apoptosis in primary rat osteoblasts (OBs). OB apoptosis induced by Cd will eventually lead to osteoporosis. In this study, a novel pharmacotherapeutic approach was investigated involving the regulation of autophagy to prevent Cd osteoporosis. The results showed that Cd treatment induced apoptosis in OBs, as demonstrated by the ratio of Bax/Bcl-2, activation of poly (ADP-ribose) polymerase (PARP) and nuclear condensation. In addition, cells treated with Cd were observed to undergo autophagic cell death by monitoring the induction of the beclin 1, autophagy gene 5 (Atg5) and the expression of microtubule-associated protein 1 light chain 3 (LC3). The results indicated that promotion of apoptotic cell death by Cd is accompanied by induction of autophagy in OBs. Interestingly, Cd-mediated apoptotic cell death was suppressed by pretreatment with the autophagy activator rapamycin (RAP) and potentiated by the autophagy inhibitor chloroquine (CQ) or small interfering RNA against beclin 1. These findings suggest that the autophagic response plays a protective role that impedes eventual cell death. Activation of autophagy could therefore be an adjunctive strategy for treatment of Cd-induced osteoporosis.

It is widely accepted that physiological mechanical stimulation suppresses apoptosis and induces synthesis of extracellular matrix by osteoblasts; however, the effect of stress overloading on osteoblasts has not been fully illustrated. In the present study, we investigated the effect of cyclic compressive stress on rat osteoblasts apoptosis, using a novel liquid drop method to generate mechanical stress on osteoblast monolayers. After treatment with different levels of mechanical stress, apoptosis of osteoblasts and activations of mitogen-activated protein kinases (MAPKs) and PI3-kinase (PI3K)/Akt signaling pathways were investigated. Osteoblasts apoptosis was observed after treated with specific inhibitors prior to mechanical stimulation. Protein levels of Bax/Bcl-2/caspase-3 signaling were determined using western blot with or without inhibitors of PI3K/Akt and phosphorylation of c-jun N-terminal kinase (JNK) MAPK. Results showed that mechanical stimulation led to osteoblasts apoptosis in a dose-dependent manner and a remarkable activation of MAPKs and PI3K/Akt signaling pathways. Activation of PI3K/Akt protected against apoptosis, whereas JNK MAPK increased apoptosis via regulation of Bax/Bcl-2/caspase-3 activation. In summary, the PI3K/Akt and JNK MAPK signaling pathways played opposing roles in osteoblasts apoptosis, resulting in inhibition of apoptosis upon small-magnitude stress and increased apoptosis upon large-magnitude stress.

Caffeine consumption is a risk factor for osteoporosis, but the precise regulatory mechanisms are currently unknown. Here, we show that cell viability decreases in osteoblasts treated with caffeine in a dose-dependent manner. This cell death is attributed primarily to apoptosis and to a smaller extent, necrosis. Moreover, caffeine directly stimulates intracellular oxidative stress. Our data support caffeine-induced apoptosis in osteoblasts via a mitochondria-dependent pathway. The apoptotic biochemical changes were effectively prevented upon pretreatment with ROS scavengers, indicating that ROS plays a critical role as an upstream controller in the caffeine-induced apoptotic cascade. Additionally, p21-activated protein kinase 2 (PAK2) and c-Jun N-terminal kinase (JNK) were activated in caffeine-treated osteoblasts. Experiments further found that PAK2 activity is required for caffeine-induced JNK activation and apoptosis. Importantly, our data also show that caffeine triggers cell death via inactivation of the survival signal, including the ERK- and Akt-mediated anti-apoptotic pathways. Finally, exposure of rats to dietary water containing approximately 10-20 microM caffeine led to bone mineral density loss. These results demonstrate for the first time that caffeine triggers apoptosis in osteoblasts via activation of mitochondria-dependent cell death signaling and inactivation of the survival signal, and causes bone mineral density loss in vivo.

Hydroxyapatite (HAp) is the most important constituent of biological tissues such as bone and teeth and exhibits several characteristic features. HAp nanoparticles (NPs) are good host materials and can be functionalized with various kinds of dopants and substrates. By endowing HAp NPs with desired properties in order to render them suitable for biomedical applications including cellular imaging, non-invasive and quantitative visualisation of molecular process occurring at cellular and subcellular levels becomes possible. Depending on their functional properties, HAp based nanoprobes can be divided into three classes, i.e., luminescent HAp NPs (for both downconversion and upconversion luminescence), magnetic HAp NPs, and luminomagnetic HAp NPs. Luminomagnetic HAp NPs are particularly attractive in terms of bimodal imaging and even multimodal imaging by virtue of their luminescence and magnetism. Functionalised HAp NPs are potential candidates for targeted drug delivery applications. This review (with 166 references) spotlights the cellular imaging applications of three types of HAp NPs. Specific sections cover aspects of molecular imaging and the various imaging modes, a comparison of the common types of nanoprobes for bioimaging, synthetic methods for making the various kinds of HAp NPs, followed by overviews on fluorescent NPs for bioimaging (such as quantum dots, gold nanoclusters, lanthanide-doped or fluorophore-doped NPs), magnetic HAp NPs for use in magnetic resonance imaging (MRI), luminomagnetic HAp NPs for bimodal imaging, and sections on drug delivery as well as cellular imaging applications of HAp based nanoprobes (including targeted imaging).