Affect Alkaline Phosphatase Research Articles

Effects of emodin on the physiological responses and antioxidant gene expression of Wuchang bream infected with Aeromonas hydrophila

This experiment aimed to investigate the effects of emodin on the physiological responses and antioxidant gene expression of Wuchang bream infected with Aeromonas hydrophila. The experimental diets were prepared with supplementing 0, 30, 100 and 150 mg kg−1 emodin to basal (control) diet respectively, and fed to fish with initial weight of 50.4 ± 2.35 g. All fish were divided into five experimental groups: uninfected fish fed with basal control diet (negative control, NC), infected fish fed with the diet supplemented with 0 (positive control group, PC), 30, 100, and 150 mg kg-1 emodin. The fish were reared for 14 days, sampled at different time points and then analyzed. The results showed that the physiological responses and related antioxidant gene expression of infected Wuchang bream were significantly influenced by the dosage of added emodin and the feeding duration (P < 0.05). Comparing to the positive control group, emodin could inhibit the levels of cortisol (COR), triiodothyronine (T3), and thyroxine (T4) in infected Wuchang bream, with hormone levels reaching equilibrium in the shortest time at 30 and 100 mg kg-1 emodin supplementation. Meanwhile, emodin significantly affected alkaline phosphatase (AKP) activity, glucose (Glu) and triglyceride (TG) contents, and related antioxidant gene expression in infected Wuchang bream (P < 0.05), with the best effect observed at 100 mg kg-1 emodin supplementation in the diet. In conclusion, the supplementation of 100 mg/kg emodin to diet can enhance the resistance of Wuchang bream to A. hydrophila infection via promoting physiological metabolism and antioxidant capacity.

Assessing the effects of a commercial fungicide and an herbicide, alone and in combination, on Apis mellifera: Insights from biomarkers and cognitive analysis

Agrochemicals play a vital role in protecting crops and enhancing agricultural production by reducing threats from pests, pathogens and weeds. The toxicological status of honey bees can be influenced by a number of factors, including pesticides. While extensive research has focused on the lethal and sublethal effects of insecticides on individual bees and colonies, it is important to recognise that fungicides and herbicides can also affect bees' health. Unfortunately, in the field, honey bees are exposed to mixtures of compounds rather than single substances. This study aimed to evaluate the effects of a commercial fungicide and a commercial herbicide, both individually and in combination, on honey bees. Mortality assays, biomarkers and learning and memory tests were performed, and the results were integrated to assess the toxicological status of honey bees. Neurotoxicity (acetylcholinesterase and carboxylesterase activities), detoxification and metabolic processes (glutathione S-transferase and alkaline phosphatase activities), immune system function (lysozyme activity and haemocytes count) and genotoxicity biomarkers (Nuclear Abnormalities assay) were assessed. The fungicide Sakura® was found to activate detoxification enzymes and affect alkaline phosphatase activity. The herbicide Elegant 2FD and the combination of both pesticides showed neurotoxic effects and induced detoxification processes. Exposure to the herbicide/fungicide mixture impaired learning and memory in honey bees. This study represents a significant advance in understanding the toxicological effects of commonly used commercial pesticides in agriculture and contributes to the development of effective strategies to mitigate their adverse effects on non-target insects.

Evaluating the effect of optimal zinc amino-acid complex supplementation in laying pullets on performance and zinc retention.

This study investigated the optimal dietary intake of zinc amino acid complex (Zn-AAC) for white-layer pullets, focusing on their productive performance, biochemical profile, organ biometry, and body zinc retention. The study involved 360 Dekalb White pullets (average weight: 433 ± 4.42 g) aged from 6 to 16 weeks and distributed into 6 treatments with 6 replications each. The Zn-AAC inclusion levels ranged from 5 to 75 mg kg-1. Zinc intake was modeled using a nonlinear equation, Y = ∝ *1- e-βX, where α is the maximum response, β is the rate at which the response approaches the maximum (P < 0.05). The Zn-AAC supplementation had significant effects on average daily gain (ADG), average daily feed intake (ADFI), and feed convention ratio (FCR) (P < 0.01). Optimal intake levels of Zn-AAC were estimated at 0.234, 0.340, and 0.315 mg bird-1 day-1 (5.42, 7.87, and 7.30 mg kg-1) for ADG, ADFI, and FCR, respectively. The Zn-AAC supplementation affected the Zn body retention in pullets (P < 0.01), with an optimal ingestion at 1.86 mg bird -1 day-1, corresponding to a dietary supplementation of 43.10 mg kg-1. Additionally, supplementation affected alkaline phosphatase (ALP) activity (P < 0.01) without significant changes in aspartate aminotransferase, albumin, and globulin levels. The optimal Zn-AAC intake level for ALP activity was 1.45 mg bird -1 day-1, corresponding to dietary supplementation of 33.60 mg kg-1. Based on Zn body retention, we recommend up to 1.86 mg bird -1 day-1 of Zn-AAC, which is equivalent to 43.10 mg kg-1.

Copper-flavonoid family of complexes involved in alkaline phosphatase activation.

The flavonoid naringenin and a family of naringenin derivative Cu(II) complexes having phenanthroline-based second ligands were selected to study alkaline phosphatase activation. This enzyme plays a critical role in tissue formation, increasing the inorganic phosphate formation, favoring mineralization, and being essential to producing bone mineralization. The effects of those compounds on the function and structure of the enzyme were evaluated by kinetic measurements, fluorescence, FTIR, and UV-Vis spectroscopies. The results showed that naringenin did not affect alkaline phosphatase activity, having a value of the Michaelis-Menten-constant close to the enzyme (Km = 3.07 × 10-6). The binary complex, Cu(II)-naringenin, and the ternary complex Cu(II)-naringenin-phenanthroline behaved as an enzyme activator in all the concentrations range used in this study. Those complexes increased in c.a. 1.9% the catalytic efficiency concerning enzyme and naringenin. The ternary complex Cu(II)-naringenin-bathophenanthroline, provokes an activator mixed effect, dependent on the substrate concentrations. The different kinetic behavior can be correlated with different conformational changes observed under the interaction with ALP. Fluorescence experiments showed a raising of the binding constant with temperature. FTIR determinations showed that the complex with bathophenanthroline modifies the ALP structure but maintains the helical structure. The other copper complexes provoked a structural unfolding, decreasing the α-helix content. None of them affect the dephosphorylation enzyme ability. Even though the interactions and structural modifications on ALP are different, it is evident that the presence of copper favors enzymatic activity. The observed electrostatic interactions probably benefit the dissociation of the bound phosphate. The results suggest potential biological applications for the studied compounds.

Dietary calcium and non-phytate phosphorus levels affect the performance, serum biochemical indices, and lipid metabolism in growing pullets

This experiment aimed to study the effects of dietary calcium (Ca) and non-phytate phosphorus (NPP) levels on performance, serum biochemical indices, and lipid metabolism in Beijing You Chicken (BYC), a local chicken. A 3×3 factorial design was adopted, dietary Ca levels were 0.66, 0.71, and 0.76%, NPP levels were 0.25, 0.30, and 0.35%. A total of 648 ten-wk-old BYC growing pullets were randomly divided into 9 groups with 6 replicates per group, and 12 birds per replicate. Growth performance, serum biochemical indices, and lipid metabolism indicators from 10 to 16 wk were measured. The results showed as follows: 1) Dietary Ca and NPP alone did not affect growth performance, but the interaction of dietary Ca and NPP affected average feed intake (AFI) of growing pullets (P < 0.05). The AFI was the lowest for the group with 0.71% Ca and 0.25% NPP (3,550.0 g, P=0.036). 2) Dietary Ca level significantly affected serum P content (P < 0.05); dietary NPP had an influence trend on serum Ca content (P=0.054). Dietary NPP levels and the interaction of Ca and NPP significantly affected alkaline phosphatase (AKP) activity. 3) Dietary Ca levels significantly affected TC content and HDL-C content (P < 0.05). Dietary NPP level significantly affected TG content (P < 0.05), the TG content in 0.25% and 0.30% NPP groups was significantly lower than that in 0.35% NPP group (P < 0.05). The interaction of dietary Ca and NPP significantly affected TG, TC and HDL-C contents (P < 0.05). TG, TC, and LDL-C levels were lower and HDL-C levels were the highest in the group with 0.66% Ca and 0.25% NPP. In summary, appropriate dietary Ca level can regulate serum TG, TC, and HDL-C content. Dietary Ca and NPP levels can be adjusted in pullet phase to avoid excessive obesity during the egg-laying period. This study recommended that dietary 0.66% Ca and 0.25% NPP benefit for the lipid metabolism of BYC growing pullets without affecting the performance.

The lysine methyltransferases SET and MYND domain containing 2 (Smyd2) and Enhancer of Zeste 2 (Ezh2) co-regulate osteoblast proliferation and mineralization

Bone formation is controlled by histone modifying enzymes that regulate post-translational modifications on nucleosomal histone proteins and control accessibility of transcription factors to gene promoters required for osteogenesis. Enhancer of Zeste homolog 2 (EZH2/Ezh2), a histone H3 lysine 27 (H3K27) methyl transferase, is a suppressor of osteoblast differentiation. Ezh2 is regulated by SET and MYND domain-containing protein 2 (SMYD2/Smyd2), a lysine methyltransferase that modifies both histone and non-histone proteins. Here, we examined whether Smyd2 modulates Ezh2 suppression of osteoblast differentiation. Musculoskeletal RNA-seq data show that SMYD2/Smyd2 is the most highly expressed SMYD/Smyd member in human bone tissues and mouse osteoblasts. Smyd2 loss of function analysis in mouse MC3T3 osteoblasts using siRNA depletion enhances proliferation and calcium deposition. Loss of Smyd2 protein does not affect alkaline phosphatase activity nor does it result in a unified expression response for standard osteoblast-related mRNA markers (e.g., Bglap, Ibsp, Spp1, Sp7), indicating that Smyd2 does not directly control osteoblast differentiation. Smyd2 protein depletion enhances levels of the osteo-suppressive Ezh2 protein and H3K27 trimethylation (H3K27me3), as expected from increased cell proliferation, while elevating the osteo-inductive Runx2 protein. Combined siRNA depletion of both Smyd2 and Ezh2 protein is more effective in promoting calcium deposition when compared to loss of either protein. Collectively, our results indicate that Smyd2 inhibits proliferation and indirectly the subsequent mineral deposition by osteoblasts. Mechanistically, Smyd2 represents a functional epigenetic regulator that operates in parallel to the suppressive effects of Ezh2 and H3K27 trimethylation on osteoblast differentiation.

Protein kinase A is activated during bone morphogenetic protein 2-induced osteogenic differentiation of dental follicle stem cells via endogenous parathyroid hormone-related protein

ObjectiveThe aim of this study was to investigate the mechanisms of how protein kinase A (PKA) is activated during bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation in dental follicle stem cells. DesignHuman dental follicle stem cells were cultured and treated with a BMP2-containing osteogenic differentiation medium or differentiation medium without BMP2. Specific siRNAs and substances/proteins were used to modulate pathways. PKA activity and activity of alkaline phosphatase were determined. Expression of targets was measured by Western Blots and reverse transcription-quantitative polymerase chain reaction, while protein interactions were investigated by immunoprecipitation. Immunofluorescence staining was used for subcellular target localization. ResultsPKA activity is stimulated after osteogenic induction by BMP2. Differentiation medium without BMP2 strongly induces BMP2 gene expression, which correlates with downstream target expression. Elevation of cAMP levels does not affect alkaline phosphatase activity and PKA does not directly interact with Smad 4. However, PKA activation requires expression of parathyroid hormone-related protein (PTHrP), which is stimulated after BMP2-induced differentiation. Furthermore, neither supplementation with PTHrP nor with the receptor antagonist parathyroid hormone (7−34) affects PKA activity. Thus, endogenous PTHrP expression is required for PKA activation and immunofluorescence staining shows that PTHrP is mainly located in the nucleus of dental follicle stem cells. Beyond, knockdown of PKA stimulates the BMP2 signaling pathway and down-stream expression of PTHrP. ConclusionsBMP2-induced osteogenic differentiation activates PKA in dental follicle stem cells via endogenous expression of PTHrP. Additionally, PKA inhibits BMP2 signaling and expression of PTHrP in a negative feedback loop.

Pulsating fluid flow affects pre-osteoblast behavior and osteogenic differentiation through production of soluble factors.

Bone mass increases after error‐loading, even in the absence of osteocytes. Loaded osteoblasts may produce a combination of growth factors affecting adjacent osteoblast differentiation. We hypothesized that osteoblasts respond to a single load in the short‐term (minutes) by changing F‐actin stress fiber distribution, in the intermediate‐term (hours) by signaling molecule production, and in the long‐term (days) by differentiation. Furthermore, growth factors produced during and after mechanical loading by pulsating fluid flow (PFF) will affect osteogenic differentiation. MC3T3‐E1 pre‐osteoblasts were either/not stimulated by 60 min PFF (amplitude, 1.0 Pa; frequency, 1 Hz; peak shear stress rate, 6.5 Pa/s) followed by 0–6 h, or 21/28 days of post‐incubation without PFF. Computational analysis revealed that PFF immediately changed distribution and magnitude of fluid dynamics over an adherent pre‐osteoblast inside a parallel‐plate flow chamber (immediate impact). Within 60 min, PFF increased nitric oxide production (5.3‐fold), altered actin distribution, but did not affect cell pseudopodia length and cell orientation (initial downstream impact). PFF transiently stimulated Fgf2, Runx2, Ocn, Dmp1, and Col1⍺1 gene expression between 0 and 6 h after PFF cessation. PFF did not affect alkaline phosphatase nor collagen production after 21 days, but altered mineralization after 28 days. In conclusion, a single bout of PFF with indirect associated release of biochemical factors, stimulates osteoblast differentiation in the long‐term, which may explain enhanced bone formation resulting from mechanical stimuli.

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.

Oxygen-releasing nanofibers for breathable bone tissue engineering application.

Oxygen is a vital molecule for cell and tissue processes. Electrospun fibers have been extensively used as drug loading carriers due to possibility of well control over drug release with modulating fiber properties. However, they have not been used as depots for oxygen release. In the present study, an oxygen-releasing nanofibrous scaffold has been developed by electrospinning of polylactic acid/nano-calcium peroxide suspension with different polylactic acid concentrations (6.5 and 13% w/v). The electrospun fibers with calcium peroxide cargo provided oxygen content of 30-94 mmHg in a period of 14 days which lies well within the oxygen level of osseous tissue. The release profile of 13% polylactic acid fibers was different with that of 6.5% fibers with respects to the initial content of released oxygen and the release rate. Not only did 13% fibers supply oxygen with a slower rate, but also they resulted in a lower burst release of oxygen. Cell culture studies in hypoxia corroborated that 13% polylactic acid fibers better preserve cell viability comparing 6.5% counterparts as perceived by MTT assay. Moreover, they endowed more favored milieu for adherence, arrangement and migration of mesenchymal stem cells as confirmed by microscopy images. The oxygen-releasing fibers equally affected alkaline phosphatase, osteocalcin, and calcium deposition by mesenchymal stem cells most likely due to interplay between topographical and metabolic cues offered by 6.5 and 13% formulations.

Regulation of the activity and kinetic properties of alkaline phosphatase in eutrophic water bodies by phosphorus and heavy metals

Phosphatase-catalyzed hydrolysis of organic phosphorus is an important pathway for providing bioavailable phosphorus to aquatic organisms. In this paper, a eutrophic water body was used to investigate how phosphorus and heavy metal ions affect alkaline phosphatase activity (APA) and its kinetic properties. The results show that phosphate (PO43-), hexametaphosphate ((PO3)(6)(6-)), pyrophosphate (P2O74-), and beta-glycerophosphate (C3H4O6P2-) inhibit APA and the inhibition effects increased with increasing concentration at the range of 0.01-1 mmol L-1. Addition of heavy metal ions, that is, Co2+, Pb2+, and Cr6+ at concentrations of 1 mmol L-1, increased the APA 3.1-fold, 2.3-fold, and 2.7-fold, respectively. The Cu2+, Zn2+, and Ni2+ exhibited an inhibitory effect on APA in water. For 1 mmol L-1 Cu2+, the APA deceased by 37.7% relative to the control. As the concentrations of Cu2+, PO43-, (PO3)(6)(6-), and P2O74- increased, the V-max of the alkaline phosphatase (AP) remained stable and K-m increased, indicating that these ions are competitive inhibitors of AP. In addition, V-max/K-m decreased with increasing concentrations of Cu2+, PO43-, (PO3)(6)(6-), P2O74-, and C3H4O6P2-, demonstrating the decreased catalytic efficiency of the AP. In summary, phosphorus and heavy metal significantly affect conversion of organic phosphorus into bioavailable inorganic phosphorous by changing the APA in eutrophic water bodies.

3D printing of silk fibroin-based hybrid scaffold treated with platelet rich plasma for bone tissue engineering

3D printing/bioprinting are promising techniques to fabricate scaffolds with well controlled and patient-specific structures and architectures for bone tissue engineering. In this study, we developed a composite bioink consisting of silk fibroin (SF), gelatin (GEL), hyaluronic acid (HA), and tricalcium phosphate (TCP) and 3D bioprinted the silk fibroin-based hybrid scaffolds. The 3D bioprinted scaffolds with dual crosslinking were further treated with human platelet-rich plasma (PRP) to generate PRP coated scaffolds. Live/Dead and MTT assays demonstrated that PRP treatment could obviously promote the cell growth and proliferation of human adipose derived mesenchymal stem cells (HADMSC). In addition, the treatment of PRP did not significantly affect alkaline phosphatase (ALP) activity and expression, but significantly upregulated the gene expression levels of late osteogenic markers. This study demonstrated that the 3D printing of silk fibroin-based hybrid scaffolds, in combination with PRP post-treatment, might be a more efficient strategy to promote osteogenic differentiation of adult stem cells and has significant potential to be used for bone tissue engineering.

Effects of Anogeissus leiocarpus Root Barks on Rat Liver, Kidney and Haematological Parameters

This study investigated the safety of aqueous root bark extract of Anogeissus leiocarpus (DC.) Guill and Perr. in albino rats. Male rats were orally administered the extract (250, 500 and 1000 mg/kg body weight) for a seven-day period. Malondialdehyde concentration and enzyme activities were monitored in liver and kidney as well as haematological, hepatic and renal function parameters. Administration of the extract significantly (p 0.05) affect kidney alkaline phosphatase activity, serum albumin, creatinine, and haematological parameters. Overall, the findings in this study revealed that the extract is not completely safe at 250, 500 and 1000 mg/kg body weight when consumed repeatedly for a seven-day period, though the adverse effects were mild, selective and not systemic.

Interaction between Nitrogen and Phosphate Stress Responses in Sinorhizobium meliloti.

Bacteria have developed various stress response pathways to improve their assimilation and allocation of limited nutrients, such as nitrogen and phosphate. While both the nitrogen stress response (NSR) and phosphate stress response (PSR) have been studied individually, there are few experiments reported that characterize effects of multiple stresses on one or more pathways in Sinorhizobium meliloti, a facultatively symbiotic, nitrogen-fixing bacteria. The PII proteins, GlnB and GlnK, regulate the NSR activity, but analysis of global transcription changes in a PII deficient mutant suggest that the S. meliloti PII proteins may also regulate the PSR. PII double deletion mutants grow very slowly and pseudoreversion of the slow growth phenotype is common. To understand this phenomenon better, transposon mutants were isolated that had a faster growing phenotype. One mutation was in phoB, the response regulator for a two component regulatory system that is important in the PSR. phoB::Tn5 mutants had different phenotypes in the wild type compared to a PII deficient background. This led to the hypothesis that phosphate stress affects the NSR and conversely, that nitrogen stress affects the PSR. Our results show that phosphate availability affects glutamine synthetase activity and expression, which are often used as indicators of NSR activity, but that nitrogen availability did not affect alkaline phosphatase activity and expression, which are indicators of PSR activity. We conclude that the NSR is co-regulated by nitrogen and phosphate, whereas the PSR does not appear to be co-regulated by nitrogen in addition to its known phosphate regulation.

Prebiotics and β‐Glucan Affect Jejunal and Serum Alkaline Phosphatase Affinity in Weanling Pigs

Biological mechanisms of dietary supplementation of prebiotics and β‐glucan in modulation of health status need further clarification. Given the recognized role of alkaline phosphatases (AP) in detoxification of endotoxin lipopolysaccharides (LPS), we hypothesized that dietary supplementation of three prebiotics and β‐glucan in modulation of health status was mediated through affecting alkaline phosphatase functionality such as maximal enzyme activity (Vmax) and enzyme affinity (Km). This study was conducted to determine effects of dietary supplementation of three prebiotics and β‐glucan at 0.75% instead of feed antibiotics on growth performance, blood urea concentration, fecal scores, jejunal and serum AP kinetics and large intestinal fermentation in weanling piglets fed corn and soybean meal (SBM)‐based diets. Six experimental diets were formulated with corn, SBM and fishmeal as the bulk ingredients. This includes Diet 1, a negative control (NC) of the basal diet with no supplemental feed antibiotics and no tested supplements; Diet 2, a positive control (PC) of the basal diet with supplemental feed antibiotics but no other tested supplements; Diet 3, a retrograded resistant corn starch supplementation of the basal diet with no supplemental feed antibiotics; Diet 4, an oat β‐glucan supplementation of the basal diet with no supplemental feed antibiotics; Diet 5, a Fibersol‐2 supplementation of the basal diet with no supplemental feed antibiotics; and Diet 6, an inulin supplementation of the basal diet with no supplemental feed antibiotics. A total of 216 piglets, 21 d old, with an initial body weight of 7.0 kg, were fed 1 of the 6 diets for 21 d in three study blocks according to a completely randomized block design. Dietary supplementation of the three prebiotics and β‐glucan did not significantly affect growth performance and main intestinal fermentation endpoints. Dietary supplementation of β‐glucan in Diet 4 increased (P&lt;0.05) the wk 1–3 overall fecal score compared with the NC diet. Fibersol‐2 in Diet 5 and inulin in Diet 6 reduced (P&lt;0.05) the Vmax of the proximal jejunal AP compared with the NC diet. The Vmax of serum AP was different (P&lt;0.05) between the β‐glucan Diet 4 and the PC diet. Dietary supplementation of Fibersol‐2 in Diet 5 and β‐glucan in Diet 4 increased the AP affinity by about 1 fold compared with the NC diet. Serum AP affinity Km values were at least 10‐fold higher than the Km values of the jejunal AP, suggesting that the proximal jejunal AP is potentially much more efficient than the serum AP in detoxification of endotoxin LPS. Dietary supplementation of the three prebiotics and oat β‐glucan at 0.75% had little effects on growth performance but might affect the gut and whole body health status via influencing the AP detoxification kinetics in the weanling pig.Support or Funding InformationSupported by the NSERC Discovery and the OMAFRA‐University Partnership Programs.

FGF-2 induces the proliferation of human periodontal ligament cells and modulates their osteoblastic phenotype by affecting Runx2 expression in the presence and absence of osteogenic inducers.

The exact phenotype of human periodontal ligament cells (hPDLCs) remains a controversial area. Basic fibroblast growth factor (FGF-2) exhibits various functions and its effect on hPDLCs is also controversial. Therefore, the present study examined the effect of FGF-2 on the growth and osteoblastic phenotype of hPDLCs with or without osteogenic inducers (dexamethasone and β-glycerophosphate). FGF-2 was added to defined growth culture medium and osteogenic inductive culture medium. Cell proliferation, osteogenic differentiation and mineralization were measured. The selected differentiation markers, Runx2, collagen type I, α1 (Col1a1), osteocalcin (OCN) and epidermal growth factor receptor (EGFR), were investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Runx2 and OCN protein expression was measured by western blotting. FGF-2 significantly increased the proliferation of hPDLCs, but did not affect alkaline phosphatase activity. RT-qPCR analysis revealed enhanced mRNA expression of Runx2, OCN and EGFR, but suppressed Col1a1 gene expression in the absence of osteogenic inducers, whereas all these gene levels had no clear trend in their presence. The Runx2 protein expression was clearly increased, but the OCN protein level showed no evident trend. The mineralization assay demonstrated that FGF-2 inhibited mineralized matrix deposition with osteogenic inducers. These results suggested that FGF-2 induces the growth of immature hPDLCs, which is a competitive inhibitor of epithelial downgrowth, and suppresses their differentiation into mineralized tissue by affecting Runx2 expression. Therefore, this may lead to the acceleration of periodontal regeneration.

The effects of amlodipine and platelet rich plasma on bone healing in rats.

AimThe aim of this study was to evaluate the effects of calcium channel blocker (CCB) amlodipine (AML), platelet rich plasma (PRP), and a mixture of both materials on bone healing.Materials and methodsFifty-six male Wistar rats were randomly divided into four groups: group A, tibia defect model with no treatment; group B, tibia defect model treated with AML, 0.04 mg daily by oral gavage; group C, tibia defect model treated with local PRP; group D, tibia defect model treated with local PRP and AML, 0.04 mg daily by oral gavage.ResultsAt day 21, bone healing was significantly better in groups C and D compared to group A (P<0.05), but comparisons showed no statistically significant difference in group B (P>0.05). At day 30, groups B and C showed no statistically significant difference (P>0.05) compared to group A, but bone healing in group D was significantly better than in group A (P<0.05). Statistically, AML did not affect alkaline phosphatase (ALP) activity at 21 and 30 days (P>0.05), but PRP and AML + PRP increased ALP activity statistically (P<0.05).ConclusionIt can be concluded that AML had neither a positive nor a negative effect on bone healing, but when used in combination with PRP, it may be beneficial.

Elucidating the individual effects of calcium and phosphate ions on hMSCs by using composite materials

The biological performance of bone graft substitutes based on calcium phosphate bioceramics is dependent on a number of properties including chemical composition, porosity and surface micro- and nanoscale structure. However, in contemporary bioceramics these properties are interlinked, therefore making it difficult to investigate the individual effects of each property on cell behavior. In this study we have attempted to investigate the effects of calcium and inorganic phosphate ions independent from one another by preparing composite materials with polylactic acid (PLA) as a polymeric matrix and calcium carbonate or sodium phosphate salts as fillers. Clinically relevant bone marrow derived human mesenchymal stromal cells (hMSCs) were cultured on these composites and proliferation, osteogenic differentiation and ECM mineralization were investigated with time and were compared to plain PLA control particles. In parallel, cells were also cultured on conventional cell culture plates in media supplemented with calcium or inorganic phosphate to study the effect of these ions independent of the 3D environment created by the particles. Calcium was shown to increase proliferation of cells, whereas both calcium and phosphate positively affected alkaline phosphatase enzyme production. QPCR analysis revealed positive effects of calcium and of inorganic phosphate on the expression of osteogenic markers, in particular bone morphogenetic protein-2 and osteopontin. Higher levels of mineralization were also observed upon exposure to either ion. Effects were similar for cells cultured on composite materials and those cultured in supplemented media, although ion concentrations in the composite cultures were lower. The approach presented here may be a valuable tool for studying the individual effects of a variety of soluble compounds, including bioinorganics, without interference from other material properties.

Osteoclastogenesis is negatively regulated by D‐serine produced by osteoblasts

We have shown the functional expression by chondrocytes of serine racemase (SR) which is responsible for the synthesis of D-serine (Ser) from L-Ser in cartilage. In this study, we evaluated the possible functional expression of SR by bone-forming osteoblasts and bone-resorbing osteoclasts. Expression of SR mRNA was seen in osteoblasts localized at the cancellous bone surface in neonatal rat tibial sections and in cultured rat calvarial osteoblasts endowed to release D-Ser into extracellular medium, but not in cultured osteoclasts differentiated from murine bone marrow progenitor cells. Sustained exposure to D-Ser failed to significantly affect alkaline phosphatase activity and Ca(2+) accumulation in cultured osteoblasts, but significantly inhibited differentiation and maturation in a concentration-dependent manner at a concentration range of 0.1-1 mM without affecting cellular survival in cultured osteoclasts. By contrast, L-Ser promoted osteoclastic differentiation in a manner sensitive to the inhibition by D-Ser. Matured osteoclasts expressed mRNA for the amino acid transporter B(0,+) (ATB(0,+) ) and the system alanine, serine, and cysteine amino acid transporter-2 (ASCT2), which are individually capable of similarly incorporating extracellular L- and D-Ser. Knockdown of these transporters by siRNA prevented both the promotion by L-Ser and the inhibition by D-Ser of osteoclastic differentiation in pre-osteoclastic RAW264.7 cells. These results suggest that D-Ser may play a pivotal role in osteoclastogenesis through a mechanism related to the incorporation mediated by both ATB(0,+) and ASCT2 of serine enantiomers in osteoclasts after the synthesis and subsequent release from adjacent osteoblasts.

Automated Printing of Electrochemical Immunoassay Microarrays: Studies of Conditions Affecting Alkaline Phosphatase Enzyme Label Activity

Microarray printing of components of an enzyme-linked immunosorbant assay (ELISA) is attractive for constructing miniaturized platforms to simultaneously and quantitatively analyze multiple chemical species in clinical, environmental, and forensic applications. A truncated version of an electrochemical ELISA was used to address a major challenge of this approach, namely the effect of evaporation on assay activity. An antibody-alkaline phosphatase conjugate (Ab-AP) was printed with a microarrayer onto an N-hydroxysuccinimide-ester-terminated self-assembled monolayer on gold. The activity of the enzyme label AP to convert the substrate p-aminophenyl phosphate to electroactive p-aminophenol was followed as a function of different conditions via detection by cyclic voltammetry at microband electrodes. It was found that enzyme activity decreased by 60% from 30 min to 4 h after evaporation. However, printing in ambient humidity ("dry") as opposed to printing in a humidified chamber, resulted in the highest assay signals observed and low nonspecific adsorption.