Intracellular Alkaline Phosphatase Research Articles

An acid-responsive DNA hydrogel-mediated cascaded enzymatic nucleic acid amplification system for the sensitive imaging of alkaline phosphatase in living cells.

Alkaline phosphatase (ALP) is a class of hydrolase that catalyzes the dephosphorylation of phosphorylated species in biological tissues, playing an important role in many physiological and pathological processes. Sensitive imaging of ALP activity in living cells is contributory to the research on these processes. Herein, we propose an acid-responsive DNA hydrogel to deliver a cascaded enzymatic nucleic acid amplification system into cells for the sensitive imaging of intracellular ALP activity. The DNA hydrogel is formed by two kinds of Y-shaped DNA monomers and acid-responsive cytosine-rich linkers. The amplification system contained Bst DNA polymerase (Bst DP), Nt.BbvCI endonuclease, a Recognition Probe (RP, containing a DNAzyme sequence, a Nt.BbvCI recognition sequence, and a phosphate group at the 3'-end), and a Signal Probe (SP, containing a cleavage site for DNAzyme, Cy3 and BHQ2 at the two ends). The amplification system was trapped into the DNA hydrogel and taken up by cells, and the cytosine-rich linkers folded into a quadruplex i-motif in the acidic lysosomes, leading to the collapse of the hydrogel and releasing the amplification system. The phosphate groups on RPs were recognized and removed by the target ALP, triggering a polymerization-nicking cycle to produce large numbers of DNAzyme sequences, which then cleaved multiple SPs, restoring Cy3 fluorescence to indicate the ALP activity. This strategy achieved sensitive imaging of ALP in living HeLa, MCF-7, and NCM460 cells, and realized the sensitive detection of ALP in vitro with a detection limit of 2.0 × 10-5 U mL-1, providing a potential tool for the research of ALP-related physiological and pathological processes.

Isolation, purification, and antiosteoporosis activity of donkey bone collagen from discarded bone and its antioxidant peptides

Oxidative stress plays a crucial role in the development of osteoporosis. In this study, it was observed that donkey bone collagen (DC) at a concentration of 500 μg/mL scavenged 17.89 % of 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radicals, indicating its antioxidant properties. Additionally, when an oxidative damage osteoblast model was created using H2O2, 100 μg/mL DC demonstrated the ability to enhance cell survival by 27.31 %. Furthermore, 50 μg/mL DC increased the intracellular differentiation marker alkaline phosphatase (ALP) level by 62.65 %. Additionally, the study revealed that DC significantly increased the expression of osteoporosis-related factors in serum and effectively restored the abnormal structure of spongy bone in mice osteoporosis model. Peptides (GGWFL, ANLGPA, and GWFK) isolated from DC through gastrointestinal digestion and subsequent enzymatic purification in vitro demonstrated the ability to safeguard osteoblasts from H2O2-induced damage by reducing intracellular reactive oxygen species (ROS). This protection resulted in enhanced cell survival and promoted osteoblast differentiation. This investigation underscores that DC can shield oxidative damage osteoblast model from oxidative stress, ameliorate osteoporosis, and enhance bone density in mice osteoporosis model. These findings suggest various DC applications in the food and medicine industries.

Antimicrobial Activity and Mechanisms of Walnut Green Husk Extract.

Walnut green husks (WGHs), by-products of walnut production, are believed to possess antimicrobial properties, making them a potential alternative to antibiotics. In this study, the antibacterial activities of three extracts, derived from WGH, against Staphylococcus aureus, Bacillus subtilis, and Escherichia coli were investigated, and the antibacterial mechanisms of an anhydrous ethanol extract of WGH (WGHa) were examined. The results showed that WGHa exhibited inhibitory effects on all tested bacteria. The ultrahigh-performance liquid chromatography-tandem mass spectrometry analysis revealed that the major active compounds present in WGHa were terpenoids, phenols, and flavonoids. Treatment with WGHa resulted in the leakage of intracellular ions and alkaline phosphatase; a reduction in intracellular ATP content, ATPase activity, and nucleic acid content; as well as cellular metabolic viability. The transmission electron microscopy images showed varying degrees of cell deformation and membrane damage following WGHa treatment. The transcriptome sequencing and differentially expressed gene enrichment analyses revealed an up-regulation in pathways associated with RNA degradation, translation, protein export, and oxidative phosphorylation. Conversely, pathways involved in cell movement and localization, as well as cell wall organization and carbohydrate transport, were found to be down-regulated. These findings suggest that WGHa alters cell membrane permeability and causes damage to the cell wall. Additionally, WGHa interferes with cellular energy metabolism, compromises RNA integrity, and induces DNA replication stress, consequently inhibiting the normal growth and proliferation of bacteria. These findings unveiled the antimicrobial mechanisms of WGHa, highlighting its potential application as an antibiotic alternative.

Synergistic effect of FGF-2 and TGF-β1 on the mineralization of human umbilical cord perivascular cells

ObjectiveHuman umbilical cord perivascular cells (HUCPVCs) are derived from the human umbilical cord perivascular tissue and are expected to replace mesenchymal stromal cells in the future. We investigated the synergistic effects of fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 1 (TGF-β1) on HUCPVC mineralization. DesignWe prepared HUCPVCs with (FGF(+)HUCPVCs) or without FGF-2 (FGF(-)HUCPVCs) in the presence of activated vitamin D3, a bone morphogenic protein inhibitor, and TGF-β1. We examined the cell proliferative capacity, expression of various hard tissue-forming cell gene markers, and mineralization induction ability and identified the crystalline phases of the mineralized nodules. ResultsFGF(+)HUCPVCs exhibited higher intracellular alkaline phosphatase (ALP) gene expression and ALP activity, and their cell proliferation rate was higher than that of FGF(-)HUCPVCs. The expression levels of osteoblast marker genes increased in FGF(+)HUCPVCs, whereas those of elastic fiber and muscle cell markers increased in FGF(-)HUCPVCs. The expression of genes related to matrix vesicle-mediated mineralization was increased in FGF(+)HUCPVCs. While FGF(-)HUCPVCs displayed myofibroblast-like properties and could not induce mineralization, FGF(+)HUCPVCs demonstrated the ability to produce mineralized nodules. The resulting mineralized nodules consisted of hydroxyapatite as the major phase and minor amounts of octacalcium phosphate. The mineralized nodules exhibited the morphological characteristics of bone hydroxyapatite, composed of fibrous hydroxyapatite nanorods and polycrystalline sheets. ConclusionWe found that FGF-2 synergizes with TGF-β1 and is a key factor in the differentiation of HUCPVCs into osteoblast-like cells. Thus, HUCPVCs can potentially serve as a new stem cell source for future bone regeneration and dental treatments.

Potential Use of Nucleic Acids as a Preceramic Polymer to Synthesize Nanodiamond-Embedded Phosphate Glass for Hard Tissue Engineering.

In recent years, nucleic acid has emerged as a versatile molecule that has been strategically used in material synthesis and biomedical applications. Keeping in mind the presence of the phosphate group, a glass former in the nucleic acids, we synthesized a transparent glass-like material by the thermal treatment of nucleic acids (DNA and RNA) at 900 °C at atmospheric pressure. Characterization of this material by transmission electron microscopy, X-ray photoelectron spectroscopy, and confocal fluorescence microscopy suggested the presence of in situ-formed nanodiamonds within the phosphate glass matrix. The molecular structure of glass investigated by X-ray photoelectron and infrared spectroscopy indicated a nearly equal proportion of metaphosphates and smaller phosphate units (pyro- and ortho-phosphate) that form the phosphate glass matrix. Thereafter, in vitro biological experiments showed that the nucleic acid-derived glass was non-toxic and cytocompatible, enhanced extracellular matrix secretion, and increased intracellular alkaline phosphatase activity, with potential application in hard tissue engineering. Our work offers insights into nanodiamond synthesis at atmospheric pressure and proves that nucleic acids could be used as a precursor to making an innovative glass-ceramic biomaterial.

Effect of insulin on bone formation ability of rat alveolar bone marrow mesenchymal stem cells.

The alveolar bone marrow mesenchymal stem cells (ABM-MSCs) play an important role in oral bone healing and regeneration. Insulin is considered to improve impaired oral bones due to local factors, systemic factors and pathological conditions. However, the effect of insulin on bone formation ability of ABM-MSCs still needs to be elucidated. The aim of this study was to determine the responsiveness of rat ABM-MSCs to insulin and to explore the underlying mechanism. We found that insulin promoted ABM-MSCs proliferation in a concentration-dependent manner, in which 10-6 M insulin exerted the most significant effect. 10-6 M insulin significantly promoted the type I collagen (COL-1) synthesis, alkaline phosphatase (ALP) activity, osteocalcin (OCN) expression and mineralized matrix formation in ABM-MSCs, significantly enhanced the gene and protein expressions of intracellular COL-1, ALP and OCN. Acute insulin stimulation significantly promoted insulin receptor (IR) phosphorylation, insulin receptor substrate-1 (IRS-1) protein expression and mammalian target of rapamycin (mTOR) phosphorylation, but chronic insulin stimulation decreased these values, while inhibitor NT219 could attenuate these responses. When seeded on β-tricalcium phosphate (β-TCP), ABM-MSCs adhered and grew well, during the 28-day culture period, ABM-MSCs+β-TCP+10-6 M Insulin group showed significantly higher extracellular type I collagen amino-terminus prolongation peptide (TPINP) content, ALP activity, OCN secretion, Ca and P concentration. When implanted subcutaneously in severe combined immunodeficient mice for 1 month, the ABM-MSCs+β-TCP+10-6 M Insulin group obtained the most bone formation and blood vessels. These results showed that insulin promoted the proliferation and osteogenic differentiation of ABM-MSCs in vitro, and enhance osteogenesis and angiogenesis of ABM-MSCs in vivo. Inhibition studies demonstrated that the insulin-induced osteogenic differentiation of ABM-MSCs was dependent of insulin/mTOR signaling. It suggests that insulin has a direct anabolic effect on ABM-MSCs.

Panax quinquefolius saponin inhibits vascular smooth muscle cell calcification via activation of nuclear factor-erythroid 2-related factor 2

BackgroundPanax quinquefolius saponin (PQS) is the main active component of Panax quinquefolius. Emerging evidence suggests that PQS exerts beneficial effects against cardiovascular diseases. However, the role and mechanism of PQS in vascular calcification are not unclear. The present study investigated the effects of PQS on the calcification of vascular smooth muscle cell (VSMCs).MethodsThe present study used calcification medium containing 3 mM inorganic phosphate (Pi) to induce rat VSMCs calcification. We investigated the effects of PQS on VSMCs calcification using alizarin red staining and alkaline phosphatase (ALP) activity assays. The intracellular reactive oxygen species (ROS) levels and the transcriptional activity of nuclear factor-erythroid 2-related factor 2 (Nrf2) were determined. The mRNA and protein expression levels of Nrf2, the antioxidant gene heme oxygenase-1 (HO-1), osteogenic markers, including runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 (BMP2), and Kelch-like ECH-associated protein 1 (Keap1) were also measured.ResultsTreatment with Pi significantly increased intracellular calcium deposition and ALP activity, which were suppressed by PQS in a concentration-dependent manner. During VSMCs calcification, PQS inhibited the mRNA and protein expression of Runx2 and BMP2. PQS treatment reduced intracellular ROS production and significantly upregulated Nrf2 transcriptional activity and the expression of Nrf2 and its target antioxidant gene HO-1. PQS suppressed the Pi-induced protein expression of Keap1, which is an endogenous inhibitor of Nrf2. Keap1 siRNA treatment induced Nrf2 expression and downregulated Runx2 expression in the presence of Pi and PQS.ConclusionTaken together, these findings suggest that PQS could effectively inhibit VSMCs calcification by ameliorating oxidative stress and regulating osteogenic genes via the promotion of Nrf2 expression.

The Synergistic Antimicrobial Effect and Mechanism of Nisin and Oxacillin against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for skin and soft tissue infections with multi-resistance to many antibiotics. It is thus imperative to explore alternative antimicrobial treatments to ensure future treatment options. Nisin (NIS), an antibacterial peptide produced by Lactococcus lactis, was selected to combine with Oxacillin (OX), to evaluate the antimicrobial effect and potential mechanism against MRSA. The synergistic antimicrobial effect of OX and NIS was verified by Minimal Inhibitory Concentration (MIC) assays, checkerboard analysis, time-kill curve, biofilm producing ability, and mice skin infection model in vivo. For the potential synergistic antimicrobial mechanism, the microstructure and integrity change of MRSA cells were determined by Scanning and Transmission Electron Microscope (SEM and TEM), intracellular alkaline phosphatase activity and propidium iodide staining were assayed; And transcription of mecA, main gene of MRSA resistant to OX, were detected by qRT-PCR. The results showed NIS could restore the sensitivity of MRSA to OX and inhibit biofilm production; OX + NIS can make MRSA cell deform; NIS may recover OX sensitivity by inhibiting the transcription of mecA. In vivo, mice skin infection models indicate that OX + NIS can substantially alleviate MRSA infections. As a safe commercially available biological compound, NIS and the combination of antibiotics are worth developing as new anti-MRSA biomaterials.

Rational synthesis of carbon dots with phosphate ester group for direct mapping of endogenous alkaline phosphatase and polarity monitoring in living cells

Carbon dots (CDs) have been extensively employed in biomolecule imaging. However, the imaging of biological enzymes with CDs has not been reported, which greatly limits their application in biological imaging. Herein, for the first time, a new type of fluorescent CDs is elaborately designed to realize the direct mapping of alkaline phosphatase (ALP) in cells. The obtained phosphorus and nitrogen co-doped CDs (P, N-CDs) generate specific structures including xanthene oxide and phosphate ester, thereby enabling P, N-CDs to be exclusively cleaved by ALP without auxiliary media. The fluorescence intensity of P, N-CDs can be specifically turned on in the presence of ALP, making them powerful probes for sensitive sensing of ALP activity with a detection limit of 1.27 U·L−1. Meanwhile, P, N-CDs possessing electron deficiency structure fulfill sensitive responding to polarity variations. The excellent photo-bleaching resistance and biocompatibility of the P, N-CDs are taken for directly mapping the intracellular endogenous ALP via turned-on fluorescence imaging, as well as real-time monitoring the polarity fluctuation in cells through ratiometric fluorescence imaging. The present work offers a new way to design and synthesize functional CDs for direct imaging of intracellular enzymes.

Barbaloin Promotes Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells: Involvement of Wnt/β-catenin Signaling Pathway.

Barbaloin, found in Aloe vera, exerts broad pharmacological activities, including antioxidant, anti-inflammatory, and anti-cancer. This study aims to investigate the effects of barbaloin on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Osteogenic induction of hBMSCs was performed in the presence or absence of barbaloin. Cell viability was determined by using the CCK-8 assay. The characteristic of hBMSCs was identified by using flow cytometry. Intracellular alkaline phosphatase (ALP) staining was performed to evaluate the ALP activity in hBMSCs. Alizarin Red S staining was performed to evaluate the matrix mineralization. The mRNA and protein levels of target genes were determined using qRT-PCR and western blotting, respectively. Treatment with barbaloin (10 and 20 μg/mL) significantly increased cell viability of hBMSCs after 72 hours. In addition, treatment with barbaloin increased the mRNA expression levels of ALP and its activities. Treatment with barbaloin also increased matrix mineralization and the mRNA and protein levels of late-differentiated osteoblast marker genes BMP2, RUNX2, and SP7 in hBMSCs. The underlying mechanisms revealed that barbaloin increased the protein expressions of Wnt/β-catenin pathway-related biomarkers. Barbaloin promotes osteogenic differentiation of hBMSCs by the regulation of the Wnt/β-catenin signaling pathway.

Maternal high-fat diet promotes calcified atherosclerotic plaque formation in adult offspring by enhancing transformation of VSMCs to osteochondrocytic-like phenotype

AimMaternal high-fat diet (HFD) is associated with the development of cardiovascular disease (CVD) in adult offspring. Atherosclerotic vascular calcification is well documented in patients with CVD. We examined the effect of maternal HFD on calcified plaque formation. Methods and resultsSeven-week-old female apo-E−/− mice (C57BL6/J) were nourished either an HFD or a normal diet (ND) a week before mating, and during gestation and lactation. Offspring of both the groups were fed a high-cholesterol diet (HCD) from 8 weeks of age. Osteogenic activity of the thoracic aorta, assessed using an ex vivo imaging system, was significantly increased after 3 months of HCD in male offspring of HFD-fed dams (O-HFD) as compared with those of ND-fed dams (O-ND). Alizarin-red-positive area in the aortic root was significantly increased after 6 months of HCD in male O-HFD as compared to that of O-ND. Plaque size and Oil Red O-positive staining were comparable between the two groups. Primary cultured vascular smooth muscle cells (VSMCs) of the thoracic aorta were treated with phosphate and interleukinL-1β (IL-1β) to transform them into an osteochondrocytic-like phenotype. Intracellular calcium content and alkaline phosphatase activity were markedly higher in the VSMCs of O-HFD than in O-ND. IL-1β concentration in the supernatant of bone marrow-derived macrophages was markedly higher in O-HFD than in O-ND. ConclusionOur findings indicate that maternal HFD accelerates the expansion of atherogenic calcification independent of plaque progression. In vitro phosphate- and IL-1β-induced osteochondrocytic transformation of VSMCs was augmented in O-HFD. Inhibition of VSMCs, skewing toward osteochondrocytic-like cells, might be a potential therapeutic strategy for preventing maternal HFD-associated CVD development.

Tannin-reinforced iron substituted hydroxyapatite nanorods functionalized collagen-based composite nanofibrous coating as a cell-instructive bone-implant interface scaffold

Bone defects account for a significant proportion of surgeries, increasing healthcare costs and complicating therapies. These factors necessitated cell-instructive bioengineering procedures to support and restore pre-existing bone function. Herein, iron-reinforced hydroxyapatite nanorod was used as an inorganic component, collagen/polycaprolactone was used as an organic component, and tannic acid was used as a surface modifier (onto nanorods), crosslinker, and functional agent. The inorganic–organic nanofibers were coated onto an implant surface using electrospinning. The nanocomposite nanostructural, contact angle, surface chemistries, thermomechanical, physicochemical, cytocompatibility, antioxidant potential, aspects of osseointegration ranging from cell adhesion, proliferation, differentiation, and bone remodeling at bone-implant zone in a rat model were studied. The results support that metal-phenolic networks on nanorods regulate interfacial interactions, nanorods cytocompatibility, antioxidant potential, and phase compatibility between organic and inorganic materials of scaffolds. The organic/inorganic nanocomposite better mimicked the bone matrix and better-supported MC3T3-E1 cell attachment, proliferation, matrix mineralization (intracellular alkaline phosphatase and calcium accumulation), and osteogenic gene expression (OPN, OCN, COL1A2, and RUNX2) to direct cell fate for bone reconstruction than its counterpart organic scaffold. The active bone formation and rebuilding of the bone-implant zone in rat models confirmed the bioactive potential of the studied nanocomposite, and it thus could be used as an interface scaffold to boost osteointegration.

Induction of mesenchymal stem cell differentiation by co-culturing with mature cells in double-layered 2-methacryloyloxyethyl phosphorylcholine polymer hydrogel matrices.

The effects of differentiated cells on stem cell differentiation were analyzed via co-culturing using a cell-encapsulated double-layered hydrogel system. As a polymer hydrogel matrix, a water-soluble zwitterionic polymer having both a 2-methacryloyloxyethyl phosphorylcholine unit and a p-vinylphenylboronic acid unit (PMBV), was complexed spontaneously with poly(vinyl alcohol) (PVA) under mild cell culture conditions. The creep modulus of the hydrogel was controlled by changing the composition of the polymer in the solution. Mouse mesenchymal stem cells (MSCs), C3H10T1/2 cells, were encapsulated into PMBV/PVA hydrogels and cultured. In the PMBV/PVA hydrogel with a lower creep modulus (0.40 kPa), proliferation of C3H10T1/2 cells occurred, and the formation of cell aggregates was observed. On the other hand, a higher creep modulus (1.7 kPa) of the hydrogel matrix prevented cell proliferation. Culturing C3H10T1/2 cells encapsulated in the PMBV/PVA hydrogel in the presence of bone morphogenetic protein-2 increased the activity of intracellular alkaline phosphatase (ALP). This indicated that C3H10T1/2 cells differentiated into mature osteoblasts. When the C3H10T1/2 cells encapsulated in the PMBV/PVA hydrogel were cultured in combination with the mature osteoblasts in the hydrogel by a close contacting double-layered hydrogel structure, higher ALP activity was observed compared with the cells cultured separately. It was considered that the differentiation of C3H10T1/2 cells in the hydrogel layer was induced by cytokines diffused from mature osteoblasts encapsulated in another hydrogel layer. It could be concluded that the PMBV/PVA hydrogel system provides a good way to observe the effects of the surrounding cells on cell function in three-dimensional culture.

Preparation and study of 3D printed dipyridamole/β-tricalcium phosphate/ polyvinyl alcohol composite scaffolds in bone tissue engineering

Critical-sized bone defects caused by trauma or infection require timely and effective treatment to restore the shape and function of the damaged area as soon as possible. We have prepared β-tricalcium phosphate (β-TCP)/polyvinyl alcohol (PVA)/dipyridamole by composite scaffolds 3D printing technique to improve the osteoinductive potential of bone substitutes, where dipyridamole can increase the extracellular adenosine concentration by hindering cells to uptake it to promote osteogenesis. For the evaluation of these prepared scaffolds, the experimental design includes mechanical strength test, the porosity measurement, the microscopic morphology observation by SEM, the release profile of loaded drugs, the biocompatibility of scaffolds with osteogenic stem cells, and the intracellular alkaline phosphatase (ALP) assay to verify the ability in osteogenesis. The results have shown that the composite scaffolds' mechanical strength, porosity, and microscopic morphology do not change significantly before and after drug addition. The reaction of cells proves that the new drug-loaded scaffolds have good biocompatibility. Moreover, the higher ALP activity of the cells seeded on the drug-loaded group has confirmed the truth of active osteogenic activities. So, we conclude that β-TCP/PVA/dipyridamole composite scaffolds have brilliant potential in new bone formation as a suitable alternative.

Ligustrazine activate the PPAR-γ pathway and play a protective role in vascular calcification.

The purpose of this study was to explore the role of ligustrazine in vascular calcification. After β-GP stimulation, vascular smooth muscle cells (VSMCs) were detected by Alizarin Red Staing staining. Calcium content and alkaline phosphatase (ALP) activity were detected by intracellular calcium assay kit and ALP assay kit, respectively. The expression of peroxisome proliferation-activated receptor (PPAR-γ) pathway-related proteins was detected by Western blot. PPAR-γ, MSX2, osteopontin (OPN), sclerostin, and BGP were detected by RT-PCR. β-GP induced the decreased activity and expression of PPAR-γ and ALP in VSMCs, while ligustrazine activated the expression of PPAR-γ. Through activation of PPAR-γ, ligustrazine decreased β-GP-induced VSMC calcification, decreased the expression of markers of osteogenesis and chondrogenic differentiation, and increased the expression of VSMC markers. Ligustrazine activates the PPAR-γ pathway and plays a protective role in vascular calcification.

Bax inhibitor-1 inhibits calcification of vascular smooth muscle cells in vitro

To investigate the effect of Bax inhibitor-1(BI-1)on calcification of vascular smooth muscle cells(VSMCs). VSMCs were isolated from the thoracic aorta of SD rats.VSMCs or BI-1-overexpressing VSMCs(transfected with a BI-1-overexpressing plasmid) were cultured in normal medium or calcified medium containing β-glycerophosphate and calcium chloride, and the cell calcification was examined with Alizarin red staining.Enzyme-linked immunosorbent assay was used to determine the intracellular calcium content and alkaline phosphatase activity.The expression levels of Runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP-2) and caspase-3 were detected with Western blotting. After 14 days of culture in the calcified medium, the VSMCs showed significantly reduced expression of BI-1 protein(P=0.001).BI-1 overexpression in the VSMCs caused a significant reduction of calcium level and alkaline phosphatase activities(P=0.0006) and lowered the expression levels of RUNX2 and BMP-2 (P=0.0001) in the cells.The VSMCs with induced calcification exhibited a significantly increased apoptosis rate, but BI-1 overexpression obviously inhibited VSMC apoptosis in the calcified medium (P=0.0003). BI-1 may attenuate vascular calcification by inhibiting calcium deposition, osteogenic differentiation and apoptosis.

Effects of iRoot SP on osteogenic differentiation of human stem cells from apical papilla

BackgroundResearch shows that nano-bioceramics can modulate the differentiation of dental stem cells. The novel ready-to-use calcium-silicate-based root-canal sealer iRoot SP is widely used in root filling. Accordingly, the aim of this study was to evaluate the effects of iRoot SP on proliferation and osteogenic differentiation in human stem cells from the apical papilla (hSCAPs).MethodshSCAPs were isolated and characterized in vitro, then cultured with various concentrations of iRoot SP extract. Cell proliferation was assessed by CCK-8 assay, and scratch-wound-healing assays were performed to evaluate cell-migration capacity. hSCAPs were then cultured in osteogenic medium supplemented with iRoot SP extracts. Alkaline phosphatase (ALP) activity assay was used to evaluate ALP enzyme levels. Alizarin red staining and cetylpyridinium chloride (CPC) assays were performed to assess calcified-nodule formation and matrix-calcium accumulation of hSCAPs. The mRNA and protein expression levels of the osteogenic markers OCN, OSX, Runx2, and DSPP were determined by qRT-PCR and Western blotting. The data were analyzed using one-way ANOVA and LSD-t tests.ResultsiRoot SP at low concentrations (2, 0.2, and 0.02 mg/mL) is nontoxic to hSCAPs. iRoot SP at concentrations of 0.02 and 0.2 mg/mL significantly increases cell-migration capacity. In terms of osteogenic differentiation, 0.2 mg/mL iRoot SP promotes intracellular ALP activity and the formation of mineralized nodules. Moreover, the expression of osteogenic markers at the mRNA and protein levels are upregulated by iRoot SP.ConclusioniRoot SP is an effective filling material for periapical bone regeneration.

Expression of the developmental important candidate genes in oocytes, embryos, embryonic stem cells, cumulus cells, and fibroblast cells of buffalo (Bubalus bubalis)

The present study was undertaken to study the expression of the developmental important gene transcripts in immature oocytes, mature oocytes, different stages of IVF produced embryos, embryonic stem (ES), cumulus (BCC), fetal fibroblast (BFF), newborn fibroblast (NBF) and adult fibroblast (BAF) cells of buffalo by semi-quantitative RT-PCR. The expression of GLUT1, HSP70.1, POL A Polymerase, GDF9, BMP15, and SURVIVIN transcripts was found in immature oocytes, mature oocytes, 2-cell, 4-cell, 8–16 cell, morula, and the blastocyst. Interestingly, the CX43 expression was found in oocytes, embryos, and other cell types, but it was not detected in the blastocyst. However, the IFNT expression was found in the blastocyst only, but not in other cells. The buffalo ES cells showed the expression of intracellular and cell surface markers (NANOG, OCT4, SOX2, FOXD3, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) and alkaline phosphatase activity. Two ES cell lines (S-line and M-line-II) were continued to survive up to 98th passages (~630 days) and 97th passages (~624 days), respectively. It was interesting to note that GLUT1, CX43, HSP70.1, POL A Polymerase, GDF9, BMP15, and SURVIVIN transcripts (except the IFNT) were expressed in buffalo ES, BCC, BFF, NBF and BAF cells. This is the first preliminary report that the buffalo ES, BCC, BFF, NBF, and BAF cells expressed the several developmental important candidate genes. It is concluded that the expression of the major developmental important genes was not only expressed in the oocytes and embryos but also expressed in the ES, BCC, BFF, NBF, and BAF cells of buffalo.

Ratiometric fluorescent sensor based on MoS2 QDs and AuNCs for determination and bioimaging of alkaline phosphatase

Herein, a ratiometric fluorescent sensor based on MoS2 quantum dots (QDs) and glutathione-capped gold nanoclusters (AuNCs) was developed for determination and imaging of alkaline phosphatase (ALP). The sensor was developed by covalently linking QDs with AuNCs to form stable MoS2@AuNCs nanohybrids that exhibited the blue fluorescence of MoS2 QDs and the red fluorescence of AuNCs. In the presence of Ce3+, the fluorescence intensity of AuNCs was increased due to the aggregation-induced emission enhancement (AIEE), while that of MoS2 QDs remained unchanged, thus could be used as a reference signal. After adenosine 5′-monophosphate (AMP) and ALP were introduced into the system, AMP was hydrolyzed to adenosine and phosphate ions (PO43−). Owing to higher affinity between Ce3+ and PO43−, the AIEE effect was inhibited, in turn resulting in the decrease of AuNCs fluorescence. The developed ratiometric fluorescent sensor had a linear response to ALP concentration ranging from 0.5 to 50 U L−1 with a detection limit (LOD) of 0.08 U L−1. Moreover, the sensor had low cytotoxicity and was successfully employed in lysosome localization and bioimaging of intracellular ALP in living cells.

Cancer cells targeted visible light and alkaline Phosphatase-Responsive TiO2/Cu2+ carbon Dots-Coated wireless electrochemical biosensor

The utilization of visible light and pyrophosphates (PPi) accompanied by alkaline phosphatase (ALP) activity-responsive cancer cells-targeting carbon dots using carbonized hyaluronic acid (CD(HA)) by means of electrochemical signals generated from CD(HA)/TiO2/Cu2+-coated surfaces was investigated. The stimuli-responsive surface provided an effective method for wireless sensors under intracellular conditions with an antifouling effect to visualize the cell adhesion interaction with the surface as well as the detection of cancer cells. The highly distinctive intracellular PPi and ALP levels of cancer cells compared to normal cells and CD44 receptor-mediated endocytosis by the CD(HA) chain into cancer cells induced an electrochemical signal change in cell-cultured biosensors detected by electrochemical methods including EIS and a wireless sensing system. The antifouling properties of CD(HA)/TiO2/Cu2+ verified by the time-dependent cell detachment occurring on the modified surfaces in the presence of visible light were employed for the detection of cancer. The resistance of cancer cell-cultured CD(HA)/TiO2/Cu2+ biosensors considerably increased (248 kΩ) as detachment time triggered by visible light irradiation increased compared to normal cells (152 kΩ), which was ascribed to the dissociation of Cu2+ in the system by PPi and ALP in cancer cells and the cellular internalization of CD(HA). With high sensitivity and accuracy for cancer detection by the visible light and enzyme-responsiveness with low LOD value (2.31 cells/mL) on basis of electrochemical approach and 70.05 cells/mL for optical approach, the CD(HA)/TiO2/Cu2+ wireless biosensor offers a promising approach that facilitates cancer diagnosis with antifouling effects to investigate the interaction between cells and modified surfaces.