Elevated Alkaline Phosphatase Activity Research Articles

High calcium to phosphorus ratio impairs growth and bone mineralization in Pekin ducklings

Two experiments were conducted to investigate the effect of high dietary calcium (Ca) level on growth performance, Ca and phosphorus (P) metabolism, and nutrient utilization in ducklings subjected to normal and low P levels in diets. A completely randomized design was used with a factorial arrangement of 2 total dietary P levels [normal-P (0.60%) and low-P (0.45%) groups] × 4 dietary Ca levels [low-Ca (0.55%), normal-Ca (0.75%), medium-Ca (0.95%) and high-Ca (1.15%) groups)]. Compared to normal-P group, low-P group had lower (P <0.05) final body weight (BW), average daily gain (ADG), and average daily feed intake (ADFI) and reduced (P <0.05) serum Ca and P levels, bone Ca, P, and ash content, and bone mineral density in ducklings during the starter period. Under the low-P group, birds from high-Ca group had lower (P <0.05) final BW, ADG, ADFI, bone ash content, bone mineral density, and the utilization of energy, Ca, and P than those from low-Ca, normal-Ca, and medium-Ca groups. Our results indicate that high-Ca diet induced greater growth suppression and bone mineralization loss in ducklings fed a low-P diet. The aggravated negative effect of high dietary Ca level with a low P level might be related to the elevated serum alkaline phosphatase activity and the reduced utilization of energy, Ca, and P.

Anti-inflammatory and hepatoprotective potentials of the aerial parts of &lt;i&gt;Silene villosa&lt;/i&gt; Caryophyllaceae methanol extract in rats

Purpose: To explore the anti-inflammatory and hepatoprotective potentials of Silene villosa Caryophyllaceae methanol extract in rats.Methods: Toxicity of S. villosa extract was evaluated in rats. Inflammation was induced in rats by injection of 0.1 mL carrageenan (1 %) in the left hind paws. Carbon tetrachloride (CCl4) was used to induce liver damage. Five groups of rat were used. The 1st (normal control) and 2nd (hepatotoxic) groups received the vehicle. The 3rd, 4th, and 5th groups received silymarin, 250 and 500 mg/kg of S. villosa extract, respectively, for 7 days. Liver injury was induced on the 7th day by intraperitoneal administration of 1 mL/kg of CCl4 to rats in groups 2 - 5.Results: The results showed that S. villosa is safe. It significantly reduced carrageenan-induced edema compared to normal (p &lt; 0.01) and standard (p &lt; 0.01) groups. The extract protected (p &lt; 0.01) rats against the deleterious effect of CCl4. It decreased (p &lt; 0.01) the elevated serum activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transferase (γ-GT) and alkaline phosphatase (ALP) as well as elevated serum levels of bilirubin (BRN), compared to CCl4 control rats. Reduced activities of the antioxidant enzymes were significantly increased (p &lt; 0.01) in rat liver, compared with CCl4 control group. The results were confirmed by histological findings in rat liver as the extract reduced necrosis and hydropic degeneration of hepatic tissue compared to CCl4 control group.Conclusion: The results suggest that S. villosa possesses anti-inflammatory and hepatoprotective activities in rats, and therefore, has therapeutic potentials in humans.Keywords: S. villosa, anti-inflammatory, carrageenan, CCl4, antioxidant, hepatotoxicity.

Characterization of glycosylphosphatidylinositol biosynthesis defects by clinical features, flow cytometry, and automated image analysis

BackgroundGlycosylphosphatidylinositol biosynthesis defects (GPIBDs) cause a group of phenotypically overlapping recessive syndromes with intellectual disability, for which pathogenic mutations have been described in 16 genes of the corresponding molecular pathway. An elevated serum activity of alkaline phosphatase (AP), a GPI-linked enzyme, has been used to assign GPIBDs to the phenotypic series of hyperphosphatasia with mental retardation syndrome (HPMRS) and to distinguish them from another subset of GPIBDs, termed multiple congenital anomalies hypotonia seizures syndrome (MCAHS). However, the increasing number of individuals with a GPIBD shows that hyperphosphatasia is a variable feature that is not ideal for a clinical classification.MethodsWe studied the discriminatory power of multiple GPI-linked substrates that were assessed by flow cytometry in blood cells and fibroblasts of 39 and 14 individuals with a GPIBD, respectively. On the phenotypic level, we evaluated the frequency of occurrence of clinical symptoms and analyzed the performance of computer-assisted image analysis of the facial gestalt in 91 individuals.ResultsWe found that certain malformations such as Morbus Hirschsprung and diaphragmatic defects are more likely to be associated with particular gene defects (PIGV, PGAP3, PIGN). However, especially at the severe end of the clinical spectrum of HPMRS, there is a high phenotypic overlap with MCAHS. Elevation of AP has also been documented in some of the individuals with MCAHS, namely those with PIGA mutations. Although the impairment of GPI-linked substrates is supposed to play the key role in the pathophysiology of GPIBDs, we could not observe gene-specific profiles for flow cytometric markers or a correlation between their cell surface levels and the severity of the phenotype. In contrast, it was facial recognition software that achieved the highest accuracy in predicting the disease-causing gene in a GPIBD.ConclusionsDue to the overlapping clinical spectrum of both HPMRS and MCAHS in the majority of affected individuals, the elevation of AP and the reduced surface levels of GPI-linked markers in both groups, a common classification as GPIBDs is recommended. The effectiveness of computer-assisted gestalt analysis for the correct gene inference in a GPIBD and probably beyond is remarkable and illustrates how the information contained in human faces is pivotal in the delineation of genetic entities.

Klf10 silencing inhibited mechanical force induced osteogenic differentiation of human periodontal ligament cells

To investigate the effect of Klf10 silence on human periodontal ligament cells (HPDLCs) under mechanical force. HPDLCs were isolated and transfected with Klf10 siRNA, and then exposed to centrifugal force for 6 h at 631 r/min. Purmorphamine, an hedgehog signaling pathway agonist, was used for intervention. The activity of alkaline phosphatase (ALP) was detected by ELISA. RT-PCR and Western blot were performed to detect the mRNA and protein expression of Klf10, Runt related transcription factor 2 (Runx2), osteopontin (OPN) and osteocalcin (OCN). The protein expression of glioma associated oncogene homolog 1 (Gli1) and patched-1 (PTCH1) was detected by Western blot. SPSS 20.0 software package was used to analyze the data. Mechanical force increased mRNA and protein level of Klf10, Runx2, OPN and OCN, and elevated ALP activity significantly (P<0.05). Mechanical force also upregulated the protein expression of GLI1 and Ptch1 significantly (P<0.05). Compared with the control group, Klf10 siRNA transfection significantly decreased mRNA and protein level of Klf10 (P<0.05). Klf10 siRNA significantly inhibited the activity of ALP, and downregulated mRNA and protein expression of Runx2, OPN and OCN (P<0.05). Moreover, Klf10 siRNA significantly inhibited protein expression of GLI1 and Ptch1 (P<0.05), and purmorphamine obviously inhibited the effect of Klf10 siRNA (P<0.05). Klf10 silencing could inhibit bone differentiation of human periodontal ligament cells under mechanical force, which may be through regulation of hedgehog signaling pathway.

Enhancement of Bone Marrow-Derived Mesenchymal Stem Cell Osteogenesis and New Bone Formation in Rats by Obtusilactone A.

The natural pure compound obtusilactone A (OA) was identified in Cinnamomum kotoense Kanehira & Sasaki, and shows effective anti-cancer activity. We studied the effect of OA on osteogenesis of bone marrow-derived mesenchymal stem cells (BMSCs). OA possesses biocompatibility, stimulates Alkaline Phosphatase (ALP) activity and facilitates mineralization of BMSCs. Expression of osteogenesis markers BMP2, Runx2, Collagen I, and Osteocalcin was enhanced in OA-treated BMSCs. An in vivo rat model with local administration of OA via needle implantation to bone marrow-residing BMSCs revealed that OA increased the new bone formation and trabecular bone volume in tibias. Micro-CT images and H&E staining showed more trabecular bone at the needle-implanted site in the OA group than the normal saline group. Thus, OA confers an osteoinductive effect on BMSCs via induction of osteogenic marker gene expression, such as BMP2 and Runx2 expression and subsequently elevates ALP activity and mineralization, followed by enhanced trabecular bone formation in rat tibias. Therefore, OA is a potential osteoinductive drug to stimulate new bone formation by BMSCs.

Emodin promotes the osteogenesis of MC3T3-E1 cells via BMP-9/Smad pathway and exerts a preventive effect in ovariectomized rats.

Emodin, a natural anthraquinone extracted from the Chinese herbs rhubarb and giant knotweed rhizome, has been reported to enhance osteoblast differentiation. However, the mechanisms underlying its ability to regulate osteogenesis are unclear. The objective of this study was to determine the role of emodin in osteoblast function in vitro and its osteoprotective effect in vivo. Emodin enhanced the differentiation and mineralization of MC3T3-E1 cells, as evidenced by elevated alkaline phosphatase activity and increased number of mineralized nodules. In cultured osteoblasts, emodin significantly induced the mRNA expression of BMP-9 which is one of the least studied but most osteogenic bone morphogenetic proteins (BMPs). Furthermore, the bone morphogenetic protein receptor-Smad (BMPR-Smad) signaling axis and p38 mitogen activated protein kinase (p38 MAPK) were activated. The in vivo function of emodin were evaluated by assessing bone histomorphology, trabecular bone microarchitecture, mechanical properties of the skeleton, and serum parameters of bone turnover in ovariectomized (OVX) rats. Emodin combined with low-dose of estrogen improved trabecular bone microarchitecture in the fourth lumbar vertebra compared with low-dose estrogen alone and enhanced vertebral body strength. Moreover, emodin suppressed the OVX-induced elevation of serum osteocalcin (OC). In addition, there were fewer side effects on uterine hypertrophy with the combination therapy than with high-dose estrogen alone. However, emodin alone did not exert any osteoprotective effect. These results suggest that emodin may be a promising alternative agent for osteoporosis in combination therapy.

DNA METHYLATION OF AN INTRONIC ENHANCER DYSREGULATES PHOSPHOLIPID PHOSPHATASE 3 AND PROMOTES OSTEOGENESIS IN THE AORTIC VALVE

Calcific aortic valve disease (CAVD) is characterized by the transition of valve interstitial cells (VICs) into osteoblast-like cells. Lysophosphatidic acid (LPA), which is produced in mineralized aortic valves (AVs), is a potent driver of osteogenic reprograming. LPA is degraded by the integral membrane protein phospholipid phosphatase 3, encoded by the PLPP3 gene. We examined the expression of PLPP3 in surgically explanted CAVD and in control nonmineralized AVs. The epigenetic regulation of PLPP3 was investigated in control and CAVD. The impact of PLPP3 on the osteogenic transition was examined in human VICs. The mRNA and enzymatic activity of PLPP3 were significantly decreased in CAVD compared to control AVs. Multidimensional gene profiling showed that intronic (intron 1 of PLPP3) DNA methylation was associated with PLPP3 expression. Bisulfite pyrosequencing in 52 AVs showed that intronic methylation of a CpG site was increased by 2.3-fold in CAVD compared to control AVs. In AVs, intronic CpG methylation level was inversely related to PLPP3 expression. The intronic site is highly conserved and showed histone marks of enhancer (H3K4me1). In reporter assay, the cloned intronic sequence with a PLPP3 minimal promoter induced a high level of activity in an orientation-independent manner. Co-transfection of ubiquitous transcription factors increased by several-fold the reporter activity. Valves with lower mRNA level of PLPP3 had higher content of LPA. In VICs treated with small interfering RNA (siRNA) for PLPP3, we observed that LPA-induced osteogenic transition was exacerbated as shown by higher expression of RUNX2, BGLAP and BMP2 as well as elevated alkaline phosphatase (ALP) activity. DNA methylation of an intronic enhancer decreases the expression of PLPP3 during CAVD. A lower expression of PLPP3 promotes the osteogenic transition of VICs.

Hydrogen Sulfide Facilitates the Impaired Sensitivity of Carotid Sinus Baroreflex in Rats with Vascular Calcification.

Arterial baroreflex is a general mechanism maintaining cardiovascular homeostasis; its sensitivity is reduced in vascular calcification (VC). Hydrogen sulfide (H2S) treatment facilitates baroreflexive sensitivity in normal and hypertensive rats. Here, we aimed to detect the effect of H2S on baroreflexive sensitivity in rats with VC. The rat VC model was induced by vitamin D3 plus nicotine for 4 weeks. The sensitivity of baroreflex was detected by perfusing the isolated carotid sinus. VC was assessed by hematoxylin and eosin (H&E) staining, Ca2+ content and alkaline phosphatase (ALP) activity. Protein levels were detected by western blot analysis. Vitamin D3 plus nicotine induced structural disorder and elevated Ca2+ content in the aortic and carotid arterial wall and increased plasma ALP activity. In the calcified aorta and carotid artery, protein levels of contractile phenotype markers of vascular smooth muscle cells (VSMCs) were downregulated and that of osteoblast-like phenotype markers and endoplasmic reticulum stress (ERS) markers were upregulated. NaHS treatment ameliorated the histologic disorder and Ca2+ content in the calcified aorta and carotid artery, inhibited the elevated plasma ALP activity, and prevented the transformation of the VSMC phenotype and activation of ERS in rats with VC. Chronic NaHS treatment prevented the impairment of the baroreflex sensitivity and acute NaHS treatment dose-dependently improved the sensitivity in rats with VC. Our results suggested that H2S could directly facilitate the impairment of baroreflex in rats with VC and ameliorate VC, which might provide new target and strategy for regulation of the baroreflex and therapy of VC.

Hepatoprotective effect of 10% ethanolic extract from Curdrania tricuspidata leaves against ethanol-induced oxidative stress through suppression of CYP2E1

The hepatoprotective effect of 10% ethanolic extract of Curdrania tricuspidata (CTE) was investigated in HepG2/2E1 cells and C57BL/6 J mice. When compared ethanol-only treated HepG2/2E1 cells, pretreatment of CTE prevented increased intra-cellular reactive oxygen species levels and decreased antioxidant activities by ethanol-induced oxidative stress. In C57BL/6 J mice, CTE at a dose of 250 mg/kg/day was administered for 10 days, with ethanol (5 g/kg/day) administered for the final 3 days. Pretreatment with CTE prevented the elevated activities of serum aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase caused by ethanol-induced hepatic damage. CTE-treated mice displayed a reduced level of malondialdehyde and increased antioxidant activities of catalase, glutathione S-transferase, glutathione peroxidase, and superoxide dismutase, as well as a reduced level of glutathione as compared with ethanol-only-treated mice. CTE-treated mice exhibited significant inhibition of CYP2E1 activities and expression. These results suggest that CTE could be a useful agent for the prevention of ethanol-induced oxidative damage in the liver, elevating antioxidative potentials and alleviating oxidative stress by suppressing CYP2El.

Anti-Inflammatory Effect of Hostacortin (Steroidal) or Vioxx (Non-Steroidal) on Mice Infected with Schistosoma Mansoni and Treated with Praziquantel

Background: Schistosoma mansoni, a helminthic parasite induces granulomatous inflammation following deposition of the eggs in the liver. The present study was conducted to evaluate the efficacy of two anti-inflammatory drugs; Hostacortin (steroidal) and Vioxx (non-steroidal) in ameliorating the damaging effects of S. mansoni infection in mice. The effects of the two anti-inflammatory drugs after treatment of schistosomiasis mansoni with praziquantel (PZQ) in mice were assessed for management of S. mansoni infection. Materials and Methods: The PZQ drug was administered to 6-weeks S. mansoni infected mice at one oral dose of 685 mg/kg body weight. The anti-inflammatory activity of the two drugs was evaluated at dose levels of 10, 50,100 and 200 mg/kg body weight in mice infected with 80 S.mansoni cercariae / mouse and treated for 10 consecutive days after 6-weeks of infection. Some biochemical parameters indicating the hepatic function as enzymatic activity of transaminases; alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT) and alkaline phosphatase (ALP) in liver as well as serum albumin and liver total protein were performed to evaluate the possible anti-inflammatory effect of any of the two used anti-inflammatory drugs in ameliorating the severity of the disease. In addition, some parasitological parameters as worm burden, liver egg count, hepatic granuloma size and hepato-somatic index were performed to evaluate the possible anti-inflammatory effect of any of the two used anti-inflammatory drugs in ameliorating the severity of the disease. Results: The results indicated that Hostacortin had no marked effect on the parasite burden and liver egg count. However, it caused a percentage decrease by 14.9 in the elevated ALP activity and pronounced increases by 33.2% and 11.3% in ALAT and ASAT activities, respectively, in liver tissue homogenate. Also, a significant reduction in granuloma size by 22.5% and 31.6% for doses 100 and 200 mg/kg, respectively, was recorded. Hostacortin treatment with PZQ improved the liver status as indicated by a significant reduction in number of worms, eggs count and size of the liver granuloma. However, Vioxx did not affect the parasite burden and liver egg count, it caused high reduction in the enzymatic activities of ALAT, ASAT, and ALP in liver tissue homogenate. In contrast with Hostacortin, Vioxx significantly increased the granuloma size by 27.6% at a dose level of 200 mg/kg. Conclusion: the treatment with Hostacortin, after PZQ treatment, ameliorates to some extent the severity of the disease, but Vioxx treatment causes additional hepatotoxicity in the S. mansoni infected mice after treatment with PZQ.

Beneficial effect of resveratrol on phenotypic features and activity of osteoarthritic osteoblasts

BackgroundOsteoarthritis (OA) is a complex disease, which affects multiple tissues, namely the subchondral bone, articular cartilage and synovial membrane. Alterations of the subchondral bone include an increased, yet under mineralized osteoid matrix, abnormal osteoblast cell phenotype including elevated alkaline phosphatase (ALP) activity, increased release of osteocalcin (OC) and transforming growth factor β-1 (TGF-β1). Previous studies have demonstrated an inhibition of the canonical Wnt signaling (cWnt) pathway in OA osteoblasts (Ob). As resveratrol (RSV) has been shown to upregulate the Wnt signaling pathway in different cell systems, we hypothesized that RSV could be beneficial for OA Ob.MethodWe prepared primary human Ob using the subchondral bone plate of tibial plateaus of OA patients undergoing total knee arthroplasty, or tibial plateaus of normal individuals at autopsy. Sirtuin 1 (Sirt1) expression in normal and OA subchondral bone tissue was evaluated by immunohistochemical analysis. Expression of genes was evaluated by qRT-PCR and protein production by western blot analysis. ALP activity and osteocalcin secretion were evaluated respectively with substrate hydrolysis and enzyme immunoassay. Mineralization levels were evaluated with alizarin red staining. Wnt/β-catenin signaling was evaluated by target gene expression using the TOPflash TCF/lef luciferase reporter assay and intracellular signaling using β-catenin levels in western blot analysis. Extracellular signal-regulated kinase (Erk)1/2 and the Smad1/5/8 pathways were evaluated by western blot analysis.ResultsSirt1 expression and production were reduced in OA subchondral bone tissue compared to normal tissue. RSV upregulated Sirt1 and its activity, and reduced the expression of leptin. RSV increased Erk1/2 phosphorylation in OA Ob; however, it had no effect on Smad 1/5/8 phosphorylation. RSV had little effect on cell proliferation and only slightly affected the Bax/Bcl2 ratio. The expression of Runx2/Cbfa1 and peroxisome proliferator-activated receptor (PPAR)γ were not affected by increasing doses of RSV. The endogenous increased ALP activity and OC release observed in OA Ob compared to normal Ob were partly corrected only for ALP at high RSV levels but not for OC release. In contrast, RSV increased the mineralization of OA Ob. Moreover, whereas Wnt3a stimulates the Wnt/β-catenin pathway in these cells, RSV further increased the response to Wnt3a.ConclusionThese data indicate that RSV promotes Sirt1 levels, inhibits the endogenous expression of leptin by OA osteoblasts and can promote the Wnt/β-catenin and Erk1/2 signaling pathways, which are altered in these cells.

Mieloma múltiplo em uma cadela de 10 anos

Background: The malignant neoplasms, in general, constitute one of the main causes of death in dogs and cats. The multiple myeloma is a neoplasm characterized by the monoclonal proliferation of plasma cells in the bone marrow, causing several systemic symptoms and it is rare in pets, representing less than 1% of the malignant neoplasms in these animals. It usually occurs in older dogs. Thus, the purpose of this report was to describe a case of multiple myeloma in a 10-year-old poodle dog, occurred in the Veterinary Hospital Unit of the State University of Ceará.Case: A 10-year-old Poodle bitch was admitted in the Veterinary Hospital Unit of the State University of Ceará presenting lump erythematosus, alopecic, soft and ulcers in the region of the digits of the left front leg, pain and lameness. The blood count evidenced normocytic and normochromic anemia, presence of rouleaux, lymphopenia and monocytopenia, and the biochemical exams have demonstrated an elevated value of alanine aminotransferase (ALT). The cytology of the nodule indicated probable diagnosis of plasma cell neoplasm and radiographic examination showed the presence of bone lysis, and therefore the patient underwent amputation of the affected limb surgery. A biopsy of the nodule indicated neoplastic proliferation, with an arrange compatible to plasmacytoma of cleaved type. A follow-up consultation was realized 30 days after surgery, and 2 new nodules appeared in the superior lip region and close to the surgery site. A new blood count indicated anemia with presence of rouleaux, thrombocytopenia, leucopenia, lymphopenia and monocytopenia, and the biochemical results showed a discrete diminution of aspartate aminotransferase(AST) and a considerable increase on the alkaline phosphatase levels. The cytology of the new nodules indicated plasmacytoma, and was realized a myelogram, that showed dysplastic marrow plasma cells, with more than 20% of plasma cells.Discussion: In the present case, the patient’s lump grew rapidly with bleeding and ulcer formation. The animal presented bone involvement, which characterizes the most suggestive sign of multiple myeloma. Justified by the stimulating factor of osteoclasts, promoting bone lysis, which can cause pathological fractures and pain. The amputation was a partial treatment to attenuates the discomfort and pain presented by the patient. The appearance of new nodules stimulated a more detailed investigation for multiple myeloma. The enzyme AST presented a discrete diminution, and ALT initially was higher than normal, but then returned to normal values. So, the elevated activity of alkaline phosphatase directed the suspects to multiple myeloma, since the activity of AST and ALT was normal, the alkaline phosphatase level was ligated to bone diseases. The rouleaux was constant at the hemograms, this is justified by the excess of immunoglobulins produced in marrow. Based on the chart presented by the patient, the bone marrow was punctured for myelogram. The symptoms displayed by the animal along with the cytological analysis, histopathological, hematological, biochemical and with myelogram indicated diagnosis of multiple myeloma. Other exams could be investigated, for example, detection of Bence Jones proteinuria, serum calcium dosage and inmunoelectroforesis for determination of the type of immunoglobulin produced by the plasma cells in the bone marrow. The definitive diagnostic was established approximately three months after the first consult, emphasizing the importance of a multiple myeloma differential diagnostic in case of plasma cell neoplasm.

Enamel matrix derivative enhances the proliferation and osteogenic differentiation of human periodontal ligament stem cells on the titanium implant surface

ABSTRACTPeriodontal ligament stem cells (PDLSCs) have mesenchymal-stem-cells-like qualities, and are considered as one of the candidates of future clinical application in periodontal regeneration therapy. Enamel matrix derivative (EMD) is widely used in promoting periodontal regeneration. However, the effects of EMD on the proliferation and osteogenic differentiation of human PDLSCs grown on the Ti implant surface are still no clear. Therefore, this study examined the effects of EMD on human PDLSCs in vitro. Human PDLSCs were isolated from healthy participants, and seeded on the surface of Ti implant disks and stimulated with various concentrations of EMD. Cell proliferation was determined with Cell Counting Kit-8 (CCK-8). The osteogenic differentiation of PDLSCs was evaluated by the measurement of alkaline phosphatase (ALP) activity, Alizarin red staining, and real-time polymerase chain reaction (qRT-PCR) and Western blotting, respectively. The results indicated that EMD at concentrations (5–60 µg/ml) increased the viability and proliferation of PDLSCs. The treatment with 30 and 60 µg/ml of EMD significantly elevated ALP activity, augmented mineralized nodule formation and calcium deposition, and upregulated the mRNA and protein levels of Runx-2 and osteocalcin (OCN) in the PDLSCs grown on the Ti surface. Further investigation found that EMD treatment did not change the protein levels of phosphatidylinositol-3-kinase (PI3K), p-PI3K, Akt and mTOR, but significantly upregulated the phosphorylated levels of Akt and mTOR. Collectively, these results suggest that EMD stimulation can promote the proliferation and osteogenic differentiation of PDLSCs grown on Ti surface, which is possibly associated with the activation of Akt/mTOR signaling pathway.

Accelerated osteogenic differentiation of human bone-derived cells in ankylosing spondylitis.

Ankylosing spondylitis (AS) is characterized by excessive bone formation with syndesmophytes, leading to bony ankylosis. The contribution of osteoblasts to the pathogenesis of ankylosis is poorly understood. The aim of this study was to determine molecular differences between disease controls (Ct) and AS bone-derived cells (BdCs) during osteogenic differentiation with or without inflammation using AS patient serum. We confirmed osteoblastic differentiation of Ct and AS BdCs under osteogenic medium by observing morphological changes and measuring osteoblastic differentiation markers. Osteoblast differentiation was detected by alkaline phosphatase (ALP) staining and activity, and alizarin red and hydroxyapatite staining. Osteoblast-specific markers were analyzed by quantitative reverse-transcriptase-polymerase chain reaction, immunoblotting, and immunostaining. To examine the effects of inflammation, we added AS and healthy control serum to Ct and AS BdCs, and then analyzed osteoblast-specific markers. AS BdCs showed elevated basal intercellular and extracellular ALP activity compared to Ct. When osteoblast differentiation was induced, AS BdCs exhibited higher expression of osteoblast-specific marker genes and faster mineralization than Ct, indicating that these cells differentiated more rapidly into osteoblasts. ALP activity and mineralization accelerated when serum from AS patients was added to Ct and AS BdCs. Our results revealed that AS BdCs showed significantly increased osteoblastic activity and differentiation capacity by regulating osteoblast-specific transcription factors and proteins compared to Ct BdCs. Active inflammation of AS serum accelerated osteoblastic activity. Our study could provide useful basic data for understanding the molecular mechanism of ankylosis in AS.

Prognostic value of alkaline phosphatase in patients with acute coronary syndromes

ObjectivesThe objective of the study was to investigate the association between alkaline phosphatase (AP) activity and prognosis of patients with acute coronary syndrome (ACS). Design and methodsThe study included 2134 patients with ACS undergoing percutaneous coronary intervention. All included patients had baseline AP measurements available. The receiver operating characteristic curve analysis showed that the best cut-off of AP for mortality prediction was 98.0U/L. Using this cut-off, patients were divided into two groups: a group with AP>98.0U/L (n=493) and a group with AP≤98.0U/L (n=1641). The primary endpoint was 3-year mortality. ResultsOverall, there were 229 deaths over the follow-up: 90 deaths among patients with an AP >98.0U/L and 139 deaths among patients with an AP≤98.0U/L (Kaplan-Meier estimates of 3-year total mortality, 19.5% and 9.3%, respectively; adjusted hazard ratio [HR]=1.37, 95% confidence interval [CI] 1.10–1.70, P=0.004 for each unit higher log AP). Cardiac deaths occurred in 157 patients: 66 deaths among patients with an AP>98.0U/L and 91 deaths among patients with an AP≤98.0U/L (Kaplan-Meier estimates of 3-year cardiac mortality, 14.3% and 6.0%, respectively; adjusted HR=1.32 [1.02–1.70], P=0.033, for each unit higher log AP). The C-statistic of the multivariable model with baseline variables was 0.836 [0.807–0.866] and it increased to 0.842 [0.814–0.874] after inclusion of AP (P=0.045). ConclusionsIn patients presenting with an ACS and treated with percutaneous coronary intervention, elevated AP activity is associated with increased risk of subsequent mortality.

Elevated alkaline phosphatase activity in a phosphate‐replete environment: Influence of sinking particles

AbstractAlkaline phosphatase activity (APA) is traditionally a proxy for phosphate (DIP)‐limitation because it is induced by DIP‐limited microbes to access the labile ester fraction of the organic phosphorus (OP) pool. Here, we present multi‐year summertime depth distributions of APA and enzyme kinetics in the DIP‐replete Celtic Sea. Our findings support the cumulating body of evidence that APA has a potentially widespread role in OP remineralization through the water column. APA and Vmax were positively correlated with depth and DIP, with total APA being threefold higher below (0.93 ± 0.32 nM P h−1) compared to above the thermocline (0.30 ± 0.24 nM P h−1, p &lt; 0.001). Separation of particles by sinking speed demonstrated that APA was eightfold higher on fast sinking (Ffast) particles compared to slow sinking particles (Fslow; p &lt; 0.05). When normalized to particulate organic carbon (POC) and bacterial production (BP), APAPOC and APABP associated with Ffast (0.76 ± 0.10 nmol P μmol C−1 h−1, 21.13 ± 2.2 nmol P nmol C−1, respectively) were fourfold and 25‐fold higher compared to the combined APA associated with dissolved plus suspended (Fsusp) and Fslow fractions (0.19 ± 0.06 nmol P μmol C−1 h−1 and 0.84 ± 0.23 nmol P nmol C−1, respectively). We postulate that this may reflect enhanced ectoenzyme activity associated with bacteria colonizing particle surfaces and/or release by zooplankton via faecal pellet excretion. Knowledge of the disparity between APA and BP associated with particle and dissolved phases is required to accurately define the O2 : P ratio of regenerated P derived from sinking particles as a result of AP‐facilitated remineralization.

Alkaline phosphatase and prognosis in patients with coronary artery disease.

The evidence on the association between alkaline phosphatase (ALP) and prognosis of patients with coronary artery disease (CAD) is limited. The aim of this study was to assess the association of ALP with the risk of mortality or coronary events in patients with CAD. The study included 5540 patients with angiography-proven CAD treated with catheter-based coronary revascularization. Baseline ALP measurements were available for analysis in all patients. Patients were divided into three groups: a group with an ALP activity in the 1st tertile (ALP ≤ 65·5 U/L; n = 1855), a group with an ALP activity in the 2nd tertile (ALP > 65·5 to 85·7 U/L; n = 1839) and a group with an ALP in the 3rd tertile (ALP > 85·7 U/L; n = 1846). The primary outcome was all-cause mortality at 3-year follow-up. All-cause deaths (number [Kaplan-Meier estimates]) occurred in 443 patients: 117 (7·2%), 130 (8·1%) and 196 deaths (11·8%) among patients of the 1st, 2nd and 3rd ALP tertiles (unadjusted hazard ratio [HR] = 1·33, 95% confidence interval [CI]: 1·19 to 1·50; P < 0·001, calculated per tertile increment in the ALP activity). After adjustment in multivariable Cox proportional hazards model, ALP was independently associated with the risk of all-cause mortality (adjusted HR = 1·33 [1·18-1·51], P < 0·001, calculated per unit increment in log ALP). The multivariable model for all-cause mortality with baseline variables without ALP had a C statistic of 0·820 [0·797-0·843] which increased to 0·825 [0·804-0·849] after ALP inclusion; delta C statistic 0·005 [0·001-0·011]; P < 0·001. In patients with CAD, elevated ALP activity was independently associated with the risk of 3-year all-cause mortality.

Cyclical compressive stress induces differentiation of rat primary mandibular condylar chondrocytes through phosphorylated myosin light chain II

The role of myosin light chainII (MLC‑II) in cellular differentiation of rat mandibular condylar chondrocytes (MCCs) induced by cyclical uniaxial compressive stress (CUCS) remains unclear. In the current study, a four‑point bending system was used to apply CUCS to primary cultured MCCs from rats. It was identified that CUCS stimulated features of cellular differentiation including morphological alterations, cytoskeleton rearrangement and overproduction of proteoglycans. Furthermore, CUCS promoted runt‑related transcription factor‑2 (RUNX2) expression at mRNA (P<0.01) and protein levels (P<0.05) and elevated alkaline phosphatase (ALP) activity (P<0.01), which are both markers of osteogenic differentiation. Under conditions of stress, western blotting indicated that the ratio of phosphorylated MLC‑II to total MLC‑II was increased significantly (P<0.05). Silencing MLC‑II by RNA interference reduced ALP activity (P<0.01), and eliminated RUNX2 mRNA expression (P<0.01). Addition of the MLC kinase inhibitor, ML‑7, reduced the CUCS‑associated upregulation of RUNX2 expression (P<0.01) and ALP activity (P<0.01). The data indicated that CUCS promoted cellular differentiation of rat primary MCCs, and this was suggested to be via the phosphorylation of MLC‑II.

Fe3O4/BSA particles induce osteogenic differentiation of mesenchymal stem cells under static magnetic field.

Differentiation of stem cells is influenced by many factors, yet uptake of the magnetic particles with or without magnetic field is rarely tackled. In this study, iron oxide nanoparticles-loaded bovine serum albumin (BSA) (Fe3O4/BSA) particles with a diameter below 200nm, negatively charged surface, tunable Fe3O4 content and subsequently adjustable magnetic property were prepared. The particles could be internalized into bone marrow mesenchymal stem cells (MSCs), and their release from the cells was significantly retarded under external magnetic field. Uptake of the Fe3O4/BSA particles enhanced significantly the osteogenic differentiation of MSCs under a constant static magnetic field, while the magnetic particles and external magnetic field alone do not influence significantly the osteogenic differentiation potential of MSCs regardless of the uptake amount. The results demonstrate a potential magnetic manipulation method for stem cell differentiation, and also convey the significance of careful evaluation of the safety issue of magnetic particles in real an application situation.

Osteogenic differentiation of preosteoblasts on a hemostatic gelatin sponge.

Bone tissue engineering provides many advantages for repairing skeletal defects. Although many different kinds of biomaterials have been used for bone tissue engineering, safety issues must be considered when using them in a clinical setting. In this study, we examined the effects of using a common clinical item, a hemostatic gelatin sponge, as a scaffold for bone tissue engineering. The use of such a clinically acceptable item may hasten the translational lag from laboratory to clinical studies. We performed both degradation and biocompatibility studies on the hemostatic gelatin sponge, and cultured preosteoblasts within the sponge scaffold to demonstrate its osteogenic differentiation potential. In degradation assays, the gelatin sponge demonstrated good stability after being immersed in PBS for 8 weeks (losing only about 10% of its net weight and about 54% decrease of mechanical strength), but pepsin and collagenases readily biodegraded it. The gelatin sponge demonstrated good biocompatibility to preosteoblasts as demonstrated by MTT assay, confocal microscopy, and scanning electron microscopy. Furthermore, osteogenic differentiation and the migration of preosteoblasts, elevated alkaline phosphatase activity, and in vitro mineralization were observed within the scaffold structure. Each of these results indicates that the hemostatic gelatin sponge is a suitable scaffold for bone tissue engineering.