Levels Of Alkaline Phosphatase Activity Research Articles

Effects of CD4+ T lymphocytes from ovariectomized mice on bone marrow mesenchymal stem cell proliferation and osteogenic differentiation.

The present study was designed to investigate the effects of T cells on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs). BMMSCs were co-cultured with CD4+ T cells that had been pretreated with anti-TNF-α or controls and were derived from ovariectomized (OVX) mice or sham control mice. MTT was used to assess the proliferative ability of BMMSCs and flow cytometry was used to analyze the BMMSC cell cycle. Following the induction of osteogenic differentiation in BMMSCs, calcium nodules were observed using alizarin red staining and alkaline phosphatase (ALP) staining. The expression levels of the osteogenesis-associated genes, runt related transcription factor 2 (Runx2) and osteocalcin (OCN) in BMMSCs were quantified using reverse transcription-quantitative PCR and western blotting. Osteogenesis-related signaling pathways, including ERK, JNK and p38 MAPK were also examined by western blotting. BMMSCs co-cultured with CD4+ T cells from OVX mice exhibited reduced proliferative ability compared with sham mice and the cell cycle was arrested at the G2/M phase. Additionally, BMMSCs co-cultured with CD4+ T cells from OVX mice presented with reduced levels of osteogenic differentiation and lower ALP activity, less calcium deposition and reduced expression of Runx2 and OCN compared with sham mice. The reduced levels of proliferation and osteogenic differentiation of BMMSCs induced by CD4+ T cells were not seen when the T cells were had been pretreated with anti-TNF-α. The results indicated that CD4+ T cells from OVX mice inhibited the proliferation and osteogenic differentiation of BMMSCs by producing high levels of TNF-α and may provide a novel insight into the dysfunction of BMMSCs caused by estrogen deficiency.

Tabersonine ameliorates osteoblast apoptosis in rats with dexamethasone-induced osteoporosis by regulating the Nrf2/ROS/Bax signalling pathway

We explored how tabersonine (Tab) protected against dexamethasone (Dex)-induced osteoporosis. Osteoblasts were treated with Dex (100 µM) with or without Table (5 or 10 µM). We measured cell viability, alkaline phosphatase (ALP) activity, and mitochondrialsuperoxide and reactive oxygen species levels. We used flow cytometry to explore the effects of Tab on mitochondrial membrane potential and osteoblast apoptosis. We used RT-PCR and western blotting to examine the effect of Tab on protein expression. We evaluated the effects of Tab on bone histopathology and bone mineral density in rats with Dex-induced osteoporosis. Tab increased cell viability and ALP activity, and reduced the mitochondrial superoxide, reactive oxygen species and matrix metalloproteinase levels and osteoblast apoptosis. Tab significantly reduced the levels of nuclear factor erythroid 2-related factor 2 (Nrf2), haem oxygenase-1 and NAD(P)H quinone dehydrogenase 1. Moreover, it increased the levels of mRNAs encoding runt-related transcription factor 2, bone morphogenetic protein-2 and osterix. These data suggest that Tab ameliorates Dex-induced osteoporosis by regulating the Nrf2 signalling pathway.

Novel Dietary Proteins Selectively Affect Intestinal Health In Vitro after Clostridium difficile-Secreted Toxin A Exposure.

Bacterial gastroenteritis forms a burden on a global scale, both socially and economically. The Gram-positive bacterium Clostridium difficile is an inducer of gastrointestinal bacterial infections, often triggered following disruption of the microbiota by broad-spectrum antibiotics to treat other conditions. The clinical manifestatiaons, e.g., diarrhea, are driven by its toxins secretion, toxin A (TcdA) and toxin B (TcdB). Current therapies are focused on discontinuing patient medication, including antibiotics. However, relapse rates upon therapy are high (20–25%). Here, eighteen dietary proteins were evaluated for their capacity to restore gut health upon C. difficile-derived TcdA exposure. We used bioengineered intestinal tubules to assess proteins for their beneficial effects by examining the epithelial barrier, cell viability, brush-border enzyme activity, IL-6 secretion, IL-8 secretion and nitric oxide (NO) levels upon TcdA challenge. TcdA effectively disrupted the epithelial barrier, increased mitochondrial activity, but did not affect alkaline phosphatase activity, IL-6, IL-8 and NO levels. Intervention with dietary proteins did not show a protective effect on epithelial barrier integrity or mitochondrial activity. However, bovine plasma and potato protein increased alkaline phosphatase activity, egg-white protein increased IL-6 and IL-8 release and wheat, lesser mealworm and yeast protein increased NO levels after TcdA exposure. Hence, dietary proteins can influence parameters involved in intestinal physiology and immune activation suggesting that supplementation with specific dietary proteins may be of benefit during C. difficile infections.

Photoencapsulated-mesenchymal stromal cells in biodegradable thiol-acrylate hydrogels enhance regeneration of craniofacial bone tissue defects.

Aim: This study investigatedbiodegradable thiol-acrylate hydrogels as stem cell carriers to facilitate cranial bone regeneration. Materials & methods: Two formulations of thiol-acrylate hydrogels (5 and 15wt% Poly[ethylene glycol]-diacrylate [PEGDA] hydrogels) were used as stem cell carriers. Bone marrow mesenchymal stromal cells and dental pulp mesenchymal stromal cells were photoencapsulated and cultured in basal or osteogenic medium 3 days before the surgery. Using New Zealand White Rabbits, four defects (5mm diameter and 2mm thickness) were createdand hydrogel scaffolds were implanted in each rabbit cranium for 6 weeks. Results & Conclusion: AlamarBlue assay showedincreasing metabolic activity levels in 5wt% PEGDA hydrogels than 15wt% PEGDA hydrogels. Photoencapsulated-mesenchymal stromal cells in 15wt% PEGDA hydrogels demonstratedsignificantly increasing alkaline phosphatase activity levels on day 7 compared with days 1 and 3. Histological diagnosis showed5wt% PEGDA hydrogels resulted in lower averaged residual gel areas than 15wt% PEGDA hydrogel specimens and control groups 6weeks postimplantation.

AntagomiR-29b inhibits vascular and valvular calcification and improves heart function in rats.

We aimed to investigate the role of the miR‐29b and its effect on TGF‐β3 pathway in vascular and valvular calcification in a rat model of calcific aortic valve diseases (CAVD). A rat model of CAVD was established by administration of warfarin plus vitamin K. The expression levels of miR‐29b, osteogenic markers and other genes were determined by qRT‐PCR, Western blot and/or immunofluorescence and immunohistochemistry. The calcium content and alkaline phosphatase (ALP) activity were measured. The calcium content, ALP activity and osteogenic markers levels in calcified aorta and aortic valve were augmented compared to controls. The expression of miR‐29b, p‐Smad3, and Wnt3 and β‐catenin was significantly up‐regulated, whereas TGF‐β3 was markedly down‐regulated. However, compared with the CAVD model group, the calcium content and ALP activity in rats treated with antagomiR‐29b were significantly decreased, and antagomiR‐29b administration reversed the effects of CAVD model on the expression of miR‐29b and osteogenic markers. Inhibition of miR‐29b in CAVD rats prevented from vascular and valvular calcification and induced TGF‐β3 expression, suggesting that the miR‐29b/TGF‐β3 axis may play a regulatory role in the pathogenesis of vascular and valvular calcification and could play a significant role in the treatment of CAVD and other cardiovascular diseases.

Enhanced Osteogenic Differentiation of Human Mesenchymal Stem Cells on Amine-Functionalized Titanium Using Humidified Ammonia Supplied Nonthermal Atmospheric Pressure Plasma.

The surface molecular chemistry, such as amine functionality, of biomaterials plays a crucial role in the osteogenic activity of relevant cells and tissues during hard tissue regeneration. Here, we examined the possibilities of creating amine functionalities on the surface of titanium by using the nonthermal atmospheric pressure plasma jet (NTAPPJ) method with humidified ammonia, and the effects on human mesenchymal stem cell (hMSC) were investigated. Titanium samples were subjected to NTAPPJ treatments using nitrogen (N-P), air (A-P), or humidified ammonia (NA-P) as the plasma gas, while control (C-P) samples were not subjected to plasma treatment. After plasma exposure, all treatment groups showed increased hydrophilicity and had more attached cells than the C-P. Among the plasma-treated samples, the A-P and NA-P showed surface oxygen functionalities and exhibited greater cell proliferation than the C-P and N-P. The NA-P additionally showed surface amine-related functionalities and exhibited a higher level of alkaline phosphatase activity and osteocalcin expression than the other samples. The results can be explained by increases in fibronectin absorption and focal adhesion kinase gene expression on the NA-P samples. These findings suggest that NTAPPJ technology with humidified ammonia as a gas source has clinical potential for hard tissue generation.

Soil alkaline phosphatase activity and bacterial phoD gene abundance and diversity under regimes of inorganic fertilizer reduction with straw

Alkaline phosphatase (ALP), which was encoded by bacterial phoD genes, hydrolyzes organic phosphorus (P) into dissolved phosphorus in soils and is therefore vital for absorbing phosphorus (P) in vegetation. However, the unreasonable application of chemical fertilizer will inhibit this process of dissolving phosphorus. Until now, although the fact that straw is the optimal C source has been recognized, its impact on phoD-harboring bacteria under decreasing chemical fertilizer is not clear. A field experiment was established with chemical fertilizer only (F) and the gradient inorganic fertilizer reduction with straw: inorganic fertilizer with straw (FS), 70% inorganic fertilizer with straw (0.7FS), 60% inorganic fertilizer with straw (0.6FS), and 50% inorganic fertilizer with straw (0.5FS). Mixed treatments received equal amounts of straw. Integrated high-throughput absolute abundance quantification (iHAAQ) was used to investigate the phoD bacterial community structure. From the correlation between the plant-total P of three vegetables and microbial variables, only the plant-total P of chili was significantly related to ALP and the abundance of phoD gene. Although the total vegetable yield was significantly increased by the addition of straw, no correlation with ALP and phoD bacterial community was found. In addition, the abundance of the phoD bacterial community and the levels of ALP activity were both higher in a regime featuring 70% inorganic fertilization with straw (0.7FS), but the ability of phoD bacteria ALP release was strongly activated in a regime featuring 50% inorganic fertilization with straw (0.5FS). Distance-based redundancy analysis (db-RDA) indicated that the community of phoD bacteria clustered four groups and suggested that a number of soil factors played key roles in shaping the entire phoD bacterial community, including soil organic carbon, total nitrogen, available phosphorus, Ca2+-Ex, and total potassium. The correlation between the absolute abundance of ALP-regulating bacteria and ALP activity suggests that Amycolatopsis may predominantly account for ALP activity, especially in the 0.5FS regime. In the reduction of chemical fertilizer, the straw substitution did not reshape the structure of the phoD bacterial community but strongly activated phoD bacteria to release ALP.

Effect of autophagy on irradiation‑induced damage in osteoblast‑like MC3T3‑E1 cells.

Autophagy is activated under radiation stress, which serves an important role in maintaining bone homeostasis. However, the underlying mechanisms of irradiation-induced autophagy in bone homeostasis is not well understood. The present study aimed to determine the effects of radiation-activated autophagy on pre-osteoblastic MC3T3-E1 cells. X-ray irradiation activated autophagy in a dose-dependent manner, with an increased fluorescence intensity of monodansylcadaverine staining, increased ratio of microtubule-associated protein 1 light chain 3β (LC3)-II/LC3-I, decreased p62 expression, and increased ATG5 and beclin-1 expression levels in MC3T3-E1 cells 72 h after irradiation compared with those in non-irradiated MC3T3-E1 cells. Irradiation reduced colony formation and mineralization in a dose-dependent manner in MC3T3-E1 cells at 2 and 3 weeks after irradiation, respectively. Decreased levels of alkaline phosphatase activity and runt-related transcription factor 2 expression were observed at 72 h post-irradiation. In addition, irradiation-induced apoptosis was accompanied by a decreased ratio of Bcl-2/BAX protein and increased the activity of caspase-3. By contrast, doxycycline (DOX)-inhibited autophagy attenuated the decreased colony formation and mineralization, and aggravated the increased cell apoptosis in irradiated MC3T3-E1 cells. Furthermore, the ratio of phosphorylated P38/P38 was observed to be higher following DOX treatment within 1 week of irradiation, which was reversed 2 weeks post-irradiation. In conclusion, DOX-inhibited autophagy aggravated X-ray irradiation-induced apoptosis at an early stage, but maintained cell proliferation and mineralization at a late stage in irradiated MC3T3-E1 cells.

The Role of Insulin-Like Growth Factor-2 on the Cellular Viability and Differentiation to the Osteogenic Lineage and Mineralization of Stem Cells Cultured on Deproteinized Bovine Bone Mineral

Insulin-like growth factors (IGFs) plays various roles, including differentiation and mitogenesis, and IGFs are reported to regulate the bone growth and maintenance. This study was performed to analyze the enhancing effects of IGF-2 on osteogenic differentiation and the mineralization of stem cells cultured on deproteinized bovine bone mineral. Stem cell loaded bone graft material was cultured in the presence of the IGF-2 at final concentrations of 10 and 100 ng/mL and the morphology of the cells was observed on Days 1, 3, and 7. The commercially available, two-color assay based on plasma membrane integrity and esterase activity was also used for qualitative analyses on Days 1, 3, and 7. The level of alkaline phosphatase activity and anthraquinone dye assay were used to evaluate osteogenic differentiation on Days 7 and 14. Real-time polymerase chain reaction was applied in order to identify the mRNA expression of BGLAP, Runx2, and β-catenin. The stem cells were well-attached with fibroblast morphology and most of the stem cells produced a high intensity of green fluorescence, indicating that there were live cells on Day 1. The relative cellular viability assay values for IGF-2 groups at 0, 10, and 100 ng/mL on Day 1 were 0.419 ± 0.015, 0.427 ± 0.013, and 0.500 ± 0.030, respectively (p < 0.05). The absorbance values at 405 nm for alkaline phosphatase activity on Day 7 for IGF-2 at 0, 10, and 100 ng/mL were 2.112 ± 0.152, 1.897 ± 0.144, and 2.067 ± 0.128, respectively (p > 0.05). The mineralization assay results at Day 7 showed significantly higher values for IGF-2 groups at 10 and 100 ng/mL concentration when compared to the control (p < 0.05). The application of IGF-2 groups of 10 and 100 ng/mL produced a significant increase of BGLAP. Conclusively, this study indicates that the use of IGF-2 on stem cell loaded bone graft increased cellular viability, Alizarin red staining, and BGLAP expression of stem cells. This report suggests the combined approach of stem cells and IGF-2 with scaffold may have synergistic effects on osteogenesis.

Characterization and Potential of a Bilayered Hydrogel of Gellan Gum and Demineralized Bone Particles for Osteochondral Tissue Engineering.

Osteochondral (OC) tissue engineering (TE) is a promising strategy to regenerate acute or degenerative chondral and OC lesions. However, advancing a proper model for OC TE is still under way. Herein, a bilayer hydrogel (BH) based on gellan gum (GG) hydrogel and demineralized bone particles (DBPs) is suggested as a new model. The BH composite can be fabricated easily with a cell-friendly biomaterial and cross-linker. The BH composite was characterized by a morphological method and physicochemical aspect. The mechanical and rheological characters were further confirmed to verify its applicability in OC TE. The thermodynamic property of the composite was determined to analyze thermal stability and interaction among matrices. The bioactivity of the material was studied by treating simulated body fluid (SBF) solution for 28 days to examine the formation of crystalline structure in the BH construct. In vitro studies were carried out to study the viability and biochemical characters of the developed biomaterial. An in vivo study was performed to analyze the biocompatibility of the material and regeneration of the injured OC region implanted with BH composites. The data displayed stable physicochemical properties and mechanical characters when the DBPs were incorporated with a proper amount. The bioactivity of the DBP-loaded hydrogels displayed a high amount of apatite formation. The cytotoxicity of the fabricated material was low, which allows application in vitro and in vivo. The biochemical studies displayed a high level of alkaline phosphatase (ALP) activity and gene expression, which shows promising application of DBP-loaded GG in the bone layer of the BH model. The long-term in vivo study displayed excellent biocompatibility and great potential in the OC defected region. Overall, these results suggest the significance of combined and innovative approaches to improve the therapeutic strategies for OC regeneration, and the BH model suggested in this study can be a promising biomaterial model for OC TE.

Ultrasmall Superparamagnetic Iron Oxide Labeled Silk Fibroin/Hydroxyapatite Multifunctional Scaffold Loaded With Bone Marrow-Derived Mesenchymal Stem Cells for Bone Regeneration.

Numerous tissue-engineered constructs have been investigated as bone scaffolds in regenerative medicine. However, it remains challenging to non-invasively monitor the biodegradation and remodeling of bone grafts after implantation. Herein, silk fibroin/hydroxyapatite scaffolds incorporated with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles were successfully synthesized, characterized, and implanted subcutaneously into the back of nude mice. The USPIO labeled scaffolds showed good three-dimensional porous structures and mechanical property, thermal stability for bone repair. After loaded with bone marrow-derived mesenchymal stem cells (BMSCs), the multifunctional scaffolds promoted cell adhesion and growth, and facilitated osteogenesis by showing increased levels of alkaline phosphatase activity and up-regulation of osteoblastic genes. Furthermore, in vivo quantitative magnetic resonance imaging (MRI) results provided valuable information on scaffolds degradation and bone formation simultaneously, which was further confirmed by computed tomography and histological examination. These findings demonstrated that the incorporation of USPIO into BMSCs-loaded multifunctional scaffold system could be feasible to noninvasively monitor bone regeneration by quantitative MRI. This tissue engineering strategy provides a promising tool for translational application of bone defect repair in clinical scenarios.

Effects of astaxanthin on the immune response and reproduction of Procambarus clarkii stressed with microcystin-leucine-arginine

The aim of this study was to evaluate the effects of the antioxidant astaxanthin on the immune response, growth, and reproduction of the crayfish Procambarus clarkii (Girard, 1852) stressed with the toxin microcystin-leucine-arginine (MC-LR). P. clarkii exposed to MC-LR were treated with one of four concentrations of dietary astaxanthin supplement (0, 2.5, 5, or 10 mg/g) for 36 consecutive days in tanks. In crayfish exposed to 0.05 mg/l MC-LR, activity levels of superoxide dismutase and alkaline phosphatase in the hepatopancreas significantly increased (P < 0.05) following dietary supplementation with 10 mg/g astaxanthin. The survival and fecundity of P. clarkii decreased significantly with exposure to 0.01 mg/l MC-LR, but astaxanthin supplementation significantly improved survival (P < 0.05). The average daily oxygen consumption rate of P. clarkii stressed with 0.01 mg/l MC-LR was significantly higher than that of unstressed P. clarkii (P < 0.05); astaxanthin supplementation did not significantly affect the average daily oxygen consumption rate of MC-LR-stressed P. clarkii. In conclusion, the antioxidant astaxanthin was found to be a suitable dietary supplement for P. clarkii as it improved immune activity and alleviated MC-LR-induced oxidative stress.

Lead Exposure Influences Serum Biomarkers, Hepatocyte Survival, Bone Marrow Hematopoiesis, and the Reproductive Cycle in Japanese Quails.

Lead toxicity has been a hallmark issue of toxicology over the last decades. However, predictive and non-robust models did not provide complete data on low-dose lead interaction with the organism at different functional levels (e.g., blood-serum-liver-bone marrow-bursa fabricii-reproductive system axis). Japanese quails are an animal model with a strong immune system, making them suitable for the thorough assessment of in vivo chronic lead toxicity. In this study, we have exposed Japanese quails via water ingestion to 0.25 and 0.5 μg/mL lead(II) chloride (PbCl2) for 20 days and assessed blood cells, serum biomarkers, hepatocyte survival, bone marrow hematopoiesis, bursa fabricii, and lead accumulation in eggs. Blood cells passed through morphological alterations (loss and inversion of the erythrocyte nucleus, multiple erythrocyte and thrombocyte aggregation, lymphocyte degradation, and blast cell infiltration). In the serum, PbCl2 increased the activity of creatine kinase (CK) and lactate dehydrogenase (LDH); increased the level of cholesterol, sodium, creatinine, and urea; and reduced the level of proteins, triglycerides, chloride, potassium, calcium, and alkaline phosphatase (ALP) activity (P < 0.05). Liver tissue of the exposed animals exhibited apparent death of hepatocytes. In the bone marrow, macrophages and heterophils contained a vast number of the infiltrated/uptaken granules upon PbCl2 exposure. Ultimately, PbCl2 exposure elicited a series of events observed first in the blood and serum parameters and later translated to the hematopoietic centers.

BIOCHEMICAL INDICES OF ORAL FLUID AS MARKERS FOR THE FEATURES OF THE STRUCTURAL AND FUNCTIONAL STATE OF THE STOMATOGNATIC SYSTEM IN PERSONS WHO WERE BORN WITH MACROSOMIA

Two hundred sixty eight people in the Kharkiv and surrounding Provinces' population have been examined: one hundred sixty eight persons were born with macrosomia, and fifty persons were born with weight and height parameters that correspond to the norm. The macrosomic-at-birth persons were divided into groups, with allowance for the features of their intrauterine development.&#x0D; The levels of total calcium, inorganic phosphorus, and alkaline phosphatase activity were determined in an unstimulated oral fluid.&#x0D; In the macrosomic-at-birth persons, who did not show the symptoms of overweight at the time of birth (Subgroups 1 and 2), the high levels of calcium, phosphorus, and alkaline phosphatase have been revealed in the oral fluid over younger age periods, which may be a consequence of intrauterine hyperthyroidism. The high calcium levels may also be due to the exposure to the elevated concentrations of growth hormone, which stimulated bone growth, and which shifted the peak of jaw growth to a younger age. This is an explanation for the high prevalence of dentoalveolar abnormalities in children.&#x0D; In such individuals, a significant increase in the activity of alkaline phosphatase and in the levels of phosphorus and calcium in the oral fluid is observed to occur with age, which is associated with the formation of dystrophic and inflammatory disorders of periodontal tissues with a predominance of the inflammatory component (periodontitis).&#x0D; In the 4–11-year-old macrosomic-at-birth persons born with the signs of overweight (Subgroups 3 and 4), a decrease in the activity of alkaline phosphatase and in the levels of phosphorus and calcium in the oral fluid has been revealed, which provides evidence for a low level of bone mineralization in such individuals, obviously due to an intrauterine decrease in thyroid activity. This explains the formation of a higher percentage of dentoalveolar abnormalities and agrees well with the information about the high intensity of deciduous and permanent teeth caries.&#x0D; In such individuals, the level of calcium in the oral fluid significantly increased in older age periods, which is consistent with information on the formation of dystrophic and inflammatory disorders of periodontal tissues in most of these individuals with a predominance of dystrophic component (periodontosis). Due to the modulation of the activity of the corresponding enzymes, an increase in the level of calcium is associated with hypercortisolemia and with the effect of cortisol on bone remodeling processes, and it is a consequence of the progression of metabolic inflammation.&#x0D; Conclusions. Changes in the indices of phosphorus-calcium metabolism and in the activity of alkaline phosphatase in the oral fluid can be used as markers for the development of disorders of the stomatognathic system, for various variants of the course of dystrophic-inflammatory diseases of periodontal tissues, and for caries in individuals who were born with macrosomia, taking into account their various anthropometric parameters at birth.

Age-related differences in vitamin D status in Polish centenarians compared with 65-year-olds.

Vitamin D status is known to change with age. However, little is known about vitamin D status in centenarians. The aim of the study was to assess vitamin D status and correlations among the levels of parathyroid hormone (PTH), 25‑hydroxycholecalciferol (25[OH]D), 1,25-dihydroxycholecalciferol (1,25[OH]D), calcium, inorganic phosphorus, and alkaline phosphatase (ALP) activity in centenarians. The study group included 97 participants: 81 women and 16 men (median [interquartile range [IQR]) age, 101.4 [100.5-102.16] years). Centenarians were visited at their homes where examinations were conducted and blood samples collected. The control group consisted of 57 elderly subjects: 35 women and 22 men (median [IQR] age, 65.9 [65.3-66.5] years). The concentrations of PTH, 25(OH)D, and 1,25(OH)D were measured in frozen plasma samples, and calcium, phosphorus, and ALP levels, in serum samples. The median calcium level was 8.88 mg/dl in centenarians versus 9.52 mg/dl in 65‑year-old subjects (P <0.01); ALP, 223 IU versus 190 IU (P = 0.01); phosphorus, 3.01 mg/dl versus 3.23 mg/dl (P = 0.13); PTH, 45.59 pg/ml versus 29.27 pg/ml (P <0.01); 25(OH)D, 7.39 ng/ml versus 19.81 ng/ml (P <0.01); 1,25(OH)D, 57.5 pmol/l versus 78.6 pmol/l (P <0.01). Only centenarians demonstrated correlations among the measured laboratory parameters. Considering lower 25(OH)D, 1,25(OH)D, and calcium concentrations in the majority of centenarians, as well as the negative correlation between vitamin D active metabolites and PTH, vitamin D and calcium should be systematically supplemented in the oldest of the elderly.

Differences in osteogenic and odontogenic differentiation potential of DPSCs and SHED

ObjectiveDental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHED) are types of human dental tissue-derived mesenchymal stem cells (MSCs) that have emerged as an interesting and promising source of stem cells in the field of tissue engineering. The aim of this work is to isolate stem cells from DPSCs and SHED, cultivate them in vitro and compare their odontogenic differentiation potential. MethodsDPSCs and SHED were extracted from molars, premolars and canines of six healthy subjects aged 5–29 years. The cells were characterized, using flow cytometry, for mesenchymal stem cell surface markers. MTT colorimetric assay was applied to assess cell viability. Alizarin red staining, alkaline phosphatase (ALP) activity, quantitative real-time PCR (qRT-PCR) and western blot were carried out to determine DPSCs and SHED osteogenic/odontogenic differentiation. ResultsDPSCs express higher STRO-1 and CD44 levels compared to SHED. Moreover, the cells differentiate and acquire columnar shape with a level of calcium deposition and mineralization that is the same between DPSCs and SHED. ALP activity, ALP, COLI, DMP-1, DSPP, OC, and RUNX2 (osteogenic/odontogenic differentiation markers) expression levels were higher in DPSCs. ConclusionsDPSCs and SHED express MSCs markers. Although both cell types had calcium deposits, DPSCs presented a higher ALP activity level. In addition, DPSCs showed higher levels of osteogenic and odontogenic differentiation markers such as COLI, DSPP, OC, RUNX2, and DMP-1. These results suggest that DPSCs are closer to the phenotype of odontoblasts than SHED and may improve the efficacy of human dental tissue-derived mesenchymal stem cells therapeutic protocols. ‘Clinical significance’DPSCs are closer than t SHED to the phenotype of odontoblasts. This would be helpful to enable better therapeutic decisions when applying MSCs-based therapy in the field of dentistry.

Reduced Membrane-Bound Alkaline Phosphatase Does Not Affect Binding of Vip3Aa in a Heliothis virescens Resistant Colony.

The Vip3Aa insecticidal protein from Bacillus thuringiensis (Bt) is produced by specific transgenic corn and cotton varieties for efficient control of target lepidopteran pests. The main threat to this technology is the evolution of resistance in targeted insect pests and understanding the mechanistic basis of resistance is crucial to deploy the most appropriate strategies for resistance management. In this work, we tested whether alteration of membrane receptors in the insect midgut might explain the >2000-fold Vip3Aa resistance phenotype in a laboratory-selected colony of Heliothis virescens (Vip-Sel). Binding of 125I-labeled Vip3Aa to brush border membrane vesicles (BBMV) from 3rd instar larvae from Vip-Sel was not significantly different from binding in the reference susceptible colony. Interestingly, BBMV from Vip-Sel larvae showed dramatically reduced levels of membrane-bound alkaline phosphatase (mALP) activity, which was further confirmed by a strong downregulation of the membrane-bound alkaline phosphatase 1 (HvmALP1) gene. However, the involvement of HvmALP1 as a receptor for the Vip3Aa protein was not supported by results from ligand blotting and viability assays with insect cells expressing HvmALP1.

Laser Nd:YAG patterning enhance human osteoblast behavior on zirconia implants.

Zirconia has been regarded as a promising material for dental implants, and Nd:YAG laser treatment has been proposed as a potential strategy to improve its bioactivity. The main aim of the present study was to evaluate the in vitro behavior of human fetal osteoblasts in contact with laser-textured zirconia implant surfaces assessing the effect of different texture patterns, spacing between laser passes and number of laser passes. Zirconia discs were produced and treated with Nd:YAG laser according to test group variables: texture (microgrooves and micropillar array), distance between surface features (25μm, 30μm and 35μm), and laser passes [1, 2, 4, and 8]. Untextured sandblasted and acid-etched zirconia discs (SBAE) were used as controls. Human osteoblasts (hFOB 1.19) were cultured for 14days on test and control samples. Morphology and cellular adhesion were observed using scanning electron microscopy (SEM). Cell viability and proliferation were evaluated at 1, 3, 7, and 14days using a commercial resazurin-based method. Collagen type I was evaluated at 3days using ELISA. Alkaline phosphatase (ALP) activity was evaluated at 7days using a colorimetric enzymatic technique. Group comparisons were tested using ANOVA or Mann-Whitney test (Tukey's post hoc) using statistical software, and significance was set at p < 0.05. Cell viability and proliferation increased over time for all groups with statistically higher values for laser-textured groups when compared with control at 7 and 14days in culture (p < 0.05). Collagen type I levels were higher for study groups (p < 0.05) when compared with control group. No statistically differences were detected for ALP activity levels between texture and control groups (p > 0.05). The results suggest that laser-machined zirconia implant surfaces may benefit biological osteoblast response. However, the type of texture, spacing at the range of 25-35μm, and number of laser passes did not seem to be relevant variables.

The Metabolism of Epoxyeicosatrienoic Acids by Soluble Epoxide Hydrolase Is Protective against the Development of Vascular Calcification.

This study addressed the hypothesis that soluble epoxide hydrolase (sEH), which metabolizes endothelium-derived epoxyeicosatrienoic acids, plays a role in vascular calcification. The sEH inhibitor trans-4-(4-(3-adamantan-1-yl-ureido)-cyclohexyloxy)-benzoic acid (t-AUCB) potentiated the increase in calcium deposition of rat aortic rings cultured in high-phosphate conditions. This was associated with increased tissue-nonspecific alkaline phosphatase activity and mRNA expression level of the osteochondrogenic marker Runx2. The procalcifying effect of t-AUCB was prevented by mechanical aortic deendothelialization or inhibition of the production and action of epoxyeicosatrienoic acids using the cytochrome P450 inhibitor fluconazole and the antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE), respectively. Similarly, exogenous epoxyeicosatrienoic acids potentiated the calcification of rat aortic rings through a protein kinase A (PKA)-dependent mechanism and of human aortic vascular smooth muscle cells when sEH was inhibited by t-AUCB. Finally, a global gene expression profiling analysis revealed that the mRNA expression level of sEH was decreased in human carotid calcified plaques compared to adjacent lesion-free sites and was inversely correlated with Runx2 expression. These results show that sEH hydrolase plays a protective role against vascular calcification by reducing the bioavailability of epoxyeicosatrienoic acids.

An easy long-acting BMP7 release system based on biopolymer nanoparticles for inducing osteogenic differentiation of adipose mesenchymal stem cells.

In contrast to the early acting bone morphogenetic protein 2, bone morphogenetic protein 7 (BMP7) plays a decisive role mainly in the late stages of bone formation. To overcome deactivation and degradation of expensive BMP7, we designed a novel long-acting BMP7 release system based on poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB) nanoparticles to enable the induction of osteogenic differentiation in human adipose mesenchymal stem cells (ADSCs). In order to improve the encapsulation efficiency of BMP7 and avoid damage by organic solvents, BMP7 was modified and protected using the biosurfactant soybean lecithin. In an in vitro test, BMP7-soybean lecithin-P34HB nanoparticles (BMP7-SPNPs) showed a short initial burst of BMP7 release during the first 24h, followed by a steady increase to a cumulative 80% release in 20days. Compared with the rapid release of control P34HB nanoparticles without soybean phospholipids loaded with BMP7 without soybean lecithin, BMP7-SPNPs significantly reduced the initial burst of BMP7 release and stabilized the content of BMP7 to allow long-term osteogenic differentiation during the late phase of bone development. Human ADSCs treated with BMP7-SPNPs showed higher alkaline phosphatase activity and higher expression levels of genetic markers of osteogenic differentiation compared with the control group. Thus, the results indicate that BMP7-SPNPs can be used as a rapid and long-acting BMP7 delivery system for osteogenic differentiation.