Stimulated Alkaline Phosphatase Activity Research Articles

Autonomous cortisol secretion promotes vascular calcification in vivo and in vitro under hyperaldosteronism.

Autonomous cortisol secretion (ACS) is not uncommon in patients with primary aldosteronism (PA). However, the cardiovascular burden of ACS due to its dysregulated cortisol secretion remains poorly understood. Thus, we examined the effects of ACS on vascular calcification in a hyperaldosteronism environment in vitro and in vivo. A total of 339 patients with PA with adrenal incidentaloma and low-dose dexamethasone suppression test data (cutoff level: cortisol > 1.8 μg/dL) from a prospectively maintained database were enrolled; abdominal aortic calcification (AAC) scores were quantitatively estimated. Human aortic smooth muscle cells (HAOSMCs) were used as in vitro model of vascular calcification. In this study, 65 of the 339 patients with PA had ACS; 274 did not. Patients with PA/ACS had a higher AAC score (1171.0 ± 2434.0 vs. 489.5 ± 1085.3, P = 0.012) than patients without ACS. ACS was independently associated with AAC score (β = 0.139, P = 0.004) in multivariate analysis, and post-suppression cortisol level was significantly correlated with the AAC score (P = 0.004). In the HAOSMC model, co-treatment with cortisol synergistically stimulated alkaline phosphatase activity and calcium deposition in a hyperaldosteronism environment. The stimulatory effect of cortisol was abolished by the mineralocorticoid receptor (MR) antagonist eplerenone, but not glucocorticoid receptor antagonist mifepristone, indicating a MR-dependent mechanism. In conclusion, the presence of ACS is associated with heavier vascular calcification in patients with PA. Aldosterone and cortisol synergistically activate HAOSMC calcification via MR signaling, via a process that can be attenuated by eplerenone.

Impact of varying dissolved organic carbon load on sediment phosphorus release and its periodic mechanisms

Phosphorus (P) release from sediment poses a severe challenge for eutrophication management in the aquatic environment. The dissolved organic carbon (DOC) concentrations in riverine ecosystems have shown an increasing trend due to intensified climate change and anthropogenic activities, while their impact on sediment P cycling remains unclear. To investigate the effects of different DOC loads on sediment P release and the underlying mechanisms, we conducted a two-month experiment in 15 plexiglass tanks, with five gradient-increasing target DOC concentrations set according to reality: control (S0), 5 mg/L (S5), 10 mg/L (S10), 15 mg/L (S15), and 20 mg/L (S20). The results demonstrated that: i) DOC enrichment promoted the sediment P mobilization and release, with the underlying mechanisms exhibited periodic characteristics. ii) reduced dissolved oxygen (DO) concentration and stimulated alkaline phosphatase activity (APA) were likely the primary and sustained facilitating mechanisms. While after the termination of DOC load, elevated pH level was also considered a contributing factor when chlorophyll a (Chl a) ranged between 5.9 μg/L and 7.7 μg/L iii) ultimate concentration of total P (TP) in the overlying water depended on DOC load. After DOC addition was terminated, decreased TP concentrations were observed when DOC concentration was in the range of 5–15 mg/L, which may be attributed to the direct uptake of P by phytoplankton counteracting the minor promotion of P release induced by anoxic conditions. However, when DOC concentrations exceeded 15–20 mg/L, there were notable increments in TP concentrations. Our findings provide further insight into the response mechanisms of sediment P release to the increasing organic C load in natural ecosystems. The impact of broader C forms or C loads on sediment P cycling needs to be fully elucidated and even quantified in future studies, especially through large-scale field investigations to further clarify the coupled roles between C and P.

Self-Assembled Multilayered Coatings with Multiple Cyclic Self-Healing Capability, Bacteria Killing, Osteogenesis, and Angiogenesis Properties on Magnesium Alloys.

Self-healing coatings improve the durability of magnesium (Mg) implants, but rapid corrosion still poses a challenge in the healing stage. Moreover, Mg-based materials with acceptable bacteria killing, osteogenic and angiogenic properties are challenging in biomedical applications. Herein, the self-healing polymeric coatings are fabricated on Mg alloys using the spin-assisted layer-by-layer (SLbL) assembly of hyaluronic acid (HA) and branched polyethyleneimine (bPEI) followed by chemical crosslinking treatment. The self-healing coatings show excellent adhesion strength and structure stability. The corrosion resistance is improved due to the physical barrier of polymer coatings, which also promotes the formation of hydroxyapatite (HAp) during degradation for further protection of Mg substrate. Owing to the dynamic reversible hydrogen bonds existing between HA and bPEI, the crosslinked multilayered coatings possess fast, substantial, and cyclic self-healing capabilities leading to restoration of the original structure and functions. Invitro investigations reveal that the self-healing coatings have multiple functionalities pertaining to bacteria killing, cytocompatibility, osteogenesis, as well as angiogenesis. In addition, the self-healing coatings stimulate alkaline phosphatase activity (ALP), extracellular matrix (ECM) mineralization, and the expression of osteogenesis-related genes of mBMSCs and HUVECs. This study reveals a feasible strategy to design and prepare versatile self-healing coatings on Mg implants for biomedical applications.

Mir-4699 promotes the osteogenic differentiation of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are drawing considerable attention in the field of regenerative medicine due to their differentiation capabilities. The miRNAs are among the most important epigenetic regulators of MSC differentiation. Our previous study identified miR-4699 as a direct suppressor of the DKK1 and TNSF11 gene expression. However, the precise osteogenic-related phenotype or mechanism caused by miR-4699 change has yet to be dealt with in depth. In the present study, miR-4699 mimics were transfected into human Adipose tissue-derived mesenchymal stem cells (hAd-MSCs) and osteoblast marker gene expression (RUNX2, ALP, and OCN), was analyzed to investigate whether miR-4699 promotes osteoblast differentiation of hAd-MSCs through targeting the DKK-1 and TNFSF11. We further examined and compared the effects of recombinant human BMP2 with miR-4699 on cell differentiation. In addition to quantitative PCR, analysis of alkaline phosphatase activity, calcium content assay, and Alizarin red staining were used to explore osteogenic differentiation. To evaluate the effect of miR-4699 on its target gene (on protein level) we utilized the western blotting technique. The overexpression of miR-4699 in hAd-MSCs resulted in the stimulation of alkaline phosphatase activity, osteoblast mineralization, and the expression of RUNX2, ALP, and OCN osteoblast marker genes. Our findings indicated that miR-4699 supported and synergized the BMP2-induced osteoblast differentiation of mesenchymal stem cells. We suggest, thereof, the utilization of hsa-miR-4699 for further in vivo experimental investigation to reveal the potential therapeutic impact of regenerative medicine for different types of bone defects.

Cytotoxicity and Biomineralization Potential of Flavonoids Incorporated into PNVCL Hydrogels.

This study aimed to evaluate the effects of flavonoids incorporated into poly(N-vinylcaprolactam) (PNVCL) hydrogel on cell viability and mineralization markers of odontoblast-like cells. MDPC-23 cells were exposed to ampelopsin (AMP), isoquercitrin (ISO), rutin (RUT) and control calcium hydroxide (CH) for evaluation of cell viability, total protein (TP) production, alkaline phosphatase (ALP) activity and mineralized nodule deposition by colorimetric assays. Based on an initial screening, AMP and CH were loaded into PNVCL hydrogels and had their cytotoxicity and effect on mineralization markers determined. Cell viability was above 70% when MDPC-23 cells were treated with AMP, ISO and RUT. AMP showed the highest ALP activity and mineralized nodule deposition. Extracts of PNVCL+AMP and PNVCL+CH in culture medium (at the dilutions of 1/16 and 1/32) did not affect cell viability and stimulated ALP activity and mineralized nodules' deposition, which were statistically higher than the control in osteogenic medium. In conclusion, AMP and AMP-loaded PNVCL hydrogels were cytocompatible and able to induce bio-mineralization markers in odontoblast-cells.

Vancomycin Containing PDLLA and PLGA/β-TCP Inhibit Biofilm Formation but Do Not Stimulate Osteogenic Transformation of Human Mesenchymal Stem Cells.

AimsChronic osteomyelitis, including implant-related prosthetic joint infection, is extremely difficult to cure. We develop vancomycin containing release systems from poly(d,l-lactide) (PDLLA) and poly(d,l-lactide-co-glycolide) (PLGA) composites with beta-tricalcium phosphate (β-TCP) to treat methicillin-resistant Staphylococcus aureus osteomyelitis. We ask whether vancomycin containing PDLLA/β-TCP and PLGA/β-TCP composites will prevent early biofilm formation, allow cell proliferation and osteogenic differentiation, and stimulate osteogenic signaling molecules in the absence of an osteogenic medium.MethodsComposites were produced and characterized with scanning electron microscopy. In vitro vancomycin release was assessed for 6 weeks. Biofilm prevention was calculated by crystal violet staining. Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and osteosarcoma cell (SaOS-2) proliferation and differentiation were assessed with water soluble tetrazolium salt and alkaline phosphatase (ALP) staining. Real-time quantitative polymerase chain reaction defined osteogenic signaling molecules for hBM-MSCs.ResultsTotally, 3.1 ± 0.2 mg and 3.4 ± 0.4 mg vancomycin released from PDLLA/β-TCP and the PLGA/β-TCP composites, respectively, and inhibited early biofilm formation. hBM-MSCs and SaOS-2 cells proliferated on the composites and stimulated ALP activity of cells. Runt-related transcription factor 2 (RUNX2) and SRY-Box transcription Factor 9 (SOX9) expressions were, however, lower with composites when compared with control.ConclusionVancomycin containing PDLLA/β-TCP and PLGA/β-TCP composites inhibited early biofilm formation and proliferated and differentiated hBM-MSCs and SaOS-2 cells, but osteogenesis-related RUNX2 and SOX9 transcription factors were not strongly expressed in the absence of an osteogenic medium for 14 days.

Dissolved Organic Carbon Loading Stimulates Sediment Phosphorus Mobilization and Release: Preliminary Evidence From Xiangshan Port, East China Sea

Increasing concentrations of dissolved organic carbon (DOC) have been observed in coastal ecosystems worldwide over the past decade, and tight coupling of the carbon (C) and phosphorus (P) biogeochemical cycle has been recognized in aquatic ecosystems. However, there is still no consensus regarding the potential effects of DOC loading on sediment P release. In a 2-month mesocosm experiment, we tested the effects of DOC enrichment on sediment P release in six glass aquariums. Two treatments were set: Control (without sodium acetate (Na(CH3COO)) addition) and Na(CH3COO) addition (equivalent to 5 mg C L−1). The results showed the following: 1) DOC loading stimulated sediment P mobilization and release, as indicated by increases in the labile P recorded for 7-cm-deep sediment using diffusive gradients in thin films, the flux of P across the sediment–water interface, and the total P concentrations in the overlying water; and 2) stimulated alkaline phosphatase activity, increased P-solubilizing bacteria proportion, and decreased dissolved oxygen concentration were likely the primary mechanisms behind the DOC-stimulated sediment P mobilization and release. These results provide insight into the promotion of sediment P release induced by C addition. Further studies investigating the quantitative relationships between DOC loadings and P release are needed to fully elucidate the coupled roles of C and P, especially those based on large-scale field investigations with broader C forms and loadings.

Simulated root exudates stimulate the abundance of Saccharimonadales to improve the alkaline phosphatase activity in maize rhizosphere

Plants promote the mineralization of soil organic phosphorus by secreting phosphatase into the rhizosphere, in addition they promote the growth of bacteria which also release phosphatase to the rhizosphere. Sugars are the main component of root exudates and a key driver of soil microbial activity. In this study, we added different concentrations of a range of sugars found in root exudates to the soil during maize growth to investigate whether sugars could stimulate alkaline phosphatase activity. Sugars increased alkaline phosphatase activity in soil, while different sugars increased this activity to different extents. Small concentration of sugars did not stimulate alkaline phosphatase activity in the rhizosphere, change the bacterial community structure or select phosphatase producing species such as Saccharimonadales. However, alkaline phosphatase activity was enhanced by larger concentration of added sugars, which increased indigenous soil Saccharimonadales abundance. Notwithstanding this, Saccharimonadales were rare in the microbial taxa at the order level and their relative abundance was only 2.1%–4.9% at different sugar concentration levels. Our results suggest that addition of sugars selected for indigenous soil Saccharimonadales, enhancing alkaline phosphatase activity in the rhizosphere. These rare microbial taxa may be a key species in soil phosphorus cycling.

Gallium (Ga)-strontium (Sr) layered double hydroxide composite coating on titanium substrates for enhanced osteogenic and antibacterial abilities.

Bacterial infection and poor osteogenic capacity can result in the loosing or failure of titanium (Ti)-based implants in the clinic. Therefore, it is urgent to design an effective approach to enhance the osteogenic property and restrict bacterial activity. In this study, a layered double hydroxide (LDH) composed of Ga and Sr ions on Ti substrates by a hydrothermal method, then calcined in 250°C and denoted as LDH250. The scanning electron microscopy (SEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) were confirmed that the LDH films were successfully formed on the Ti substrates. Importantly, the obtained LDH films can induce an alkaline microenvironment around the Ti surface and regulate the behaviors of osteogenic cells and bacteria. In vitro cellular experiments, the LDH250 can enhance the differentiation of both MC3T3-E1 cells and osteoblasts, stimulate alkaline phosphatase activity (ALP), collagen secretion, and mineralization levels. Meanwhile, antimicrobial assay against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) demonstrated that the LDH250 samples had strong antibacterial abilities, which attributed to the release profile of Ga3+ could act as a "Trojan horse" to destroy the bacterial iron metabolism, inducing of local alkaline environment, and producing reactive oxygen species. Hence, this study provides an effective method for reducing antibacterial infection and enhancing the bone integrative capacity of Ti-based implants for orthopedic applications.

Concentrated Growth Factors (CGF) Induce Osteogenic Differentiation in Human Bone Marrow Stem Cells.

Simple SummaryOsteogenesis is a complex physiologic process that occurs during development as well as during damaged bone regeneration. This process requires several growth factors that act on stem cell populations, including Bone Marrow Stem Cells (BMSC). The present study fits into the research field for safe improvement of cell osteogenesis induction. In this context there is a great interest on an autologous and biocompatible blood derived product, named Concentrated Growth Factor (CGF). In particular, the ability of CGF to induce osteogenic differentiation of human BMSC (hBMSC) in vitro was here investigated. The osteogenic differentiation was evaluated measuring typical osteogenic markers such as alkaline phosphatase enzyme activity, matrix mineralization of hBMSC, and expression of some osteogenic-related genes. The results show that CGF alone is able to induce osteogenic differentiation of hBMSC. This finding opens up further, interesting perspectives in the biotechnological use of CGF in the tissue regeneration field.Bone regeneration is a complex process regulated by several factors that control overlapping biological processes, coordinating interactions among distinct cell populations. There is a great interest in identifying new strategies for inducing osteogenesis in a safe and efficient manner. Concentrated Growth Factor (CGF) is an autologous blood derived product obtained by centrifugation of venous blood following the procedure set on the Silfradent device. In this study the effects of CGF on osteogenic differentiation of human Bone Marrow Stem Cells (hBMSC) in vitro have been investigated; hBMSC were cultured with CGF or osteogenic medium, for 21 days. The osteogenic differentiation was evaluated measuring alkaline phosphatase (ALP) enzyme activity, matrix mineralization by alizarin red staining and through mRNA and protein quantification of osteogenic differentiation markers by Real-time PCR and Western blotting, respectively. The treatment with CGF stimulated ALP activity and promoted matrix mineralization compared to control and seems to be more effective than osteogenic medium. Also, hBMSC lost mesenchymal markers and showed other osteogenic features. Our study showed for the first time that CGF alone is able to induce osteogenic differentiation in hBMSC. The application of CGF on hBMSC osteoinduction might offer new clinical and biotechnological strategies in the tissue regeneration field.

Anti-Osteoporotic Effects of Antioxidant Peptides PIISVYWK and FSVVPSPK from Mytilus edulis on Ovariectomized Mice.

Numerous amounts of evidence suggest that bioactive peptides with diverse physiological activities can be nutraceuticals or potential drug candidates. In this study, blue mussel-derived antioxidant peptides PIISVYWK and FSVVPSPK were subjected to evaluate their osteogenic effect in mouse bone marrow mesenchymal stem cells (mBMMSCs) followed by an in vivo anti-osteoporotic effect. Treatment of PIISVYWK and FSVVPSPK on mBMMSCs stimulated alkaline phosphatase activity and calcification. Western blot results revealed that PIISVYWK and FSVVPSPK increased the expression of bone morphogenetic protein-2/4 (BMP-2/4) followed by upregulating p-Smad1/5, type I collagen, and transcription factors including Runx2 and osterix in mBMMSCs. Two peptides also activated the phosphorylation of MAPKs (p-p38, p-ERK, and p-JNK). Treatment of MAPK inhibitors significantly inhibited the BMP signaling pathway, indicating that PIISVYWK and FSVVPSPK stimulated osteoblast differentiation of mBMMSCs through the MAPK-dependent BMP signaling pathway. The anti-osteoporotic effect of PIISVYWK and FSVVPSPK in ovariectomized (OVX) mice was investigated. Treatment of PIISVYWK and FSVVPSPK for ten weeks showed a notable anti-osteoporotic effect in OVX mice via increasing bone mineral density and other bone parameters compared to OVX mice without peptides. Serum analysis also showed that treatment of PIISVYWK and FSVVPSPK completely reduced osteocalcin and ALP (alkAline phosphatase) activity. Taken together, these results suggest that PIISVYWK and FSVVPSPK could be health-promoting functional food ingredients against osteoporosis.

Amine Derivative from the Aerial Part of Spilanthes acmella Murr. and their Alkaline Phosphatase Activity

Background: Spilanthes acmella Murr. is included in Asteraceae family which is used as a traditional remedy for tooth-aches, and originated from Africa, America, Borneo, India, Sri Lanka, Bangladesh, China, Japan, Thailand, and Indonesia. The present research aims to isolate the amine derivative from the ethyl acetate layer of this plant and to evaluate the isolated compounds of alkaline phosphatase activity as a sign of bone formation. Methods: The air-dried plants of Spilanthes acmella Murr. were extracted with methanol, then partitioned with n-hexane and ethyl acetate successively by using liquid-liquid extraction, and then the chromatographic techniques were repeated, such as silica gel, octadecyl silylated silica gel, and HPLC. The isolated compounds were determined by spectrometric analysis using ultraviolet, infrared, high-resolution electrospray ionization mass spectrometry, 1D and 2D NMR. Results: Benzenepropanoic acid, 4 hydroxy-2-oxo-3 piperidinyl ester (1), was isolated from the ethyl acetate layer of the whole plants of Spilanthes acmella Murr. together with dendranthemoside A (2), uridine (3), icariside B2 (4), chicoriin (5), dendranthemoside B (6), and ampelopsisionoside (7) from their butanol layer. Conclusion: An amine derivative, a benzenepropanoic acid that determined as 4 hydroxy-2-oxo-3 piperidinyl ester (1) was isolated and reported for the first time from the ethyl acetate layer of Spilanthes acmella naturally. All the isolated compounds from this plant stimulated alkaline phosphatase activity as a mark of bone formation up to 128%.

Collagen Extract Derived from Yeonsan Ogye Chicken Increases Bone Microarchitecture by Suppressing the RANKL/OPG Ratio via the JNK Signaling Pathway.

Yeonsan Ogye is a traditional Korean chicken breed (Gallus domesticus, GD), with a dominant gene for fibromelanosis, showing entirely black fluffy head feathers, ear lobes, and pupils. GD collagen extract (78.6 g per 100 g total protein) was derived from the flesh of Yeonsan Ogye. The effects of GD collagen on bone mass, microarchitecture, osteogenic, osteoclastogenic differentiations, and function factor expression were investigated in ovariectomized (OVX) rats. GD collagen stimulated osteogenesis in OVX rats and increased tibial bone strength and calcium content. Micro-computed tomography analysis of tibia cross-sections revealed that GD collagen attenuated the OVX-induced changes in trabecular thickness, spacing, and number. GD collagen stimulated alkaline phosphatase activity, bone-specific matrix proteins (alkaline phosphatase (ALP), osteocalcin, collagen type I (COL-I)) and mineralization by activating bone morphogenetic protein 2 (BMP-2)/mothers against decapentaplegic homolog 5 (SMAD5)/runt-related transcription factor 2 (Runx2). GD collagen inhibited osteoclast differentiation and function gene markers (TRAP, cathepsin K) by interfering with the Wnt signaling, increasing OPG production, and reducing the expression of RANKL, TRAP, and cathepsin K. GD collagen promoted osteogenesis by activating the p38 signal pathway and prevented osteoclastogenesis by lowering the RANKL/OPG ratio and blocking the JNK signaling pathway. Dietary supplementation with GD collagen might inhibit osteoclastogenesis, stimulate osteoblastogenesis, and regulate bone metabolism.

Effects of water-soluble extract from cactus pear fruit on proliferation, differentiation, and mineralization of new bone rat osteoblasts

Objective To investigate the effects of the water-soluble extract from cactus pear fruit (CPFWE) on differentiation of obsteoblasts. Methods Rat osteoblasts were isolated as an in vitro model. MTT assay was used to evaluate the growth and proliferation of rat osteoblasts. The spectrophotometer was used to determine alkaline phosphatase (ALP) activity and hydroxyproline (Hyp) content. By alizarin red staining, bone nodule formation was observed. Results In the middle stage of differentiation, CPFWE observably stimulated ALP activity and Hyp content in osteoblasts. Especially, CPFWE increased the ALP activity by (57.748±4.071)% at the concentration of 50.000 μg/ml for 9 days and Hyp content by (26.444±0.990)% at the concentration of 25.000 μg/ml for 22 days as compared with the control group. In the later stage, the CPFWE significantly increased the area of nodules by (43.840±1.200)% at 50.000 μg/ml concentration and increased IOD by (48.241±1.760)% at 6.250 μg/ml for 18 days. Conclusion The effects of CPFWE on bone formation by rat osteoblasts suggest that these phenolics may have beneficial effects on bone health. Key words: Cactus pear fruit; Water-soluble extract; Osteoblast cell; Bone formation

High ammonium loading can increase alkaline phosphatase activity and promote sediment phosphorus release: A two-month mesocosm experiment

In aquatic ecosystems, ammonium is one of the dominant substances in the effluent discharge from wastewater treatment plants and its impact has been widely explored as it is thought, in its toxic form (NH3), to cause stress on organisms. Little is, however, known about its potential effect on the release of phosphorus (P) from the sediment. In a two-month mesocosm (150 L) experiment, we tested if high loading of ammonium promotes sediment P release and investigated the dominant underlying mechanisms. A gradient of five target ammonium loading levels was used by adding NH4Cl fertilizer: no addition/control (N0), 3 (N1), 5 (N2), 10 (N3), and 21 (N4) mg NH4Cl L−1 (NH4Cl expressed as nitrogen). We found that: 1) significant sediment P release for N3 and N4 but minor release or retention for N0, N1, and N2 were detected both by the total phosphorus concentration (TP) in the overlying water and in situ measurements of diffusive gradients in thin-films (DGT) at the sediment-water interface; 2) overall, TP correlated significantly and positively with total nitrogen (TN) concentrations in the water. Further correlation and path analyses suggested that stimulated alkaline phosphatase activity (APA) was likely the dominant mechanisms behind the ammonium-induced sediment P release and decreased dissolved oxygen (DO) levels (an approximate reduction from 9.2 to 6.6 mg O2 L−1) was likely a contributing factor, particularly in the beginning of the experiment.

Metformin protects bone mass in ultra-high-molecular-weight polyethylene particle-induced osteolysis by regulating osteocyte secretion.

Metformin, an anti-hyperglycemic agent used for type 2 diabetes, has recently been found to have more effects apart from glucose regulation. We found that, in ultra-high-molecular-weight polyethylene particle-induced osteolysis mouse models, metformin had bone protect property and reduced the negative regulator of bone formation sclerostin (SOST) and Dickkopf-related protein 1 (DKK1), and increased osteoprotegerin (OPG) secretion and the ratio of OPG/Receptor Activator for Nuclear Factor-κB Ligand (RANKL). In vitro, we established a 3D co-culture system in which metformin affects osteoblasts and osteoclasts through mature osteocytes secretion. Metformin (50μM) significantly decreased SOST and DKK1 mRNA expression, stimulating alkaline phosphatase activity and proliferation of osteoblast, and increased OPG secretion and the ratio of OPG/RANKL, inhibiting osteoclastogenesis. Moreover, the effect on OPG was reversed by adenosine 5'-monophosphate-activated protein kinase inhibitor, Compound C. Our finding suggests that metformin induces differentiation and mineralization of osteoblasts, while inhibits osteoclastogenesis via mature osteocytes secretion. Therefore, the drug might be beneficial for not only diabetes but also in other bone disorders by acting on mature osteocytes.

Metformin Enhances the Differentiation of Dental Pulp Cells into Odontoblasts by Activating AMPK Signaling

IntroductionMetformin is a first-line drug for treating type 2 diabetes that regulates the differentiation of mesenchymal stem cells. Its effects on human dental pulp cells (DPCs) remain unknown. This study aimed to investigate the effects of metformin on the proliferation and differentiation of DPCs. MethodsA live/dead viability assay kit was used to examine the effects of metformin on the cell viability of DPCs. Cell proliferation was analyzed using a cell counting kit (CCK-8; Dojindo, Tokyo, Japan). Levels of phosphorylated and unphosphorylated adenosine 5′-monophosphate-activated protein kinase (AMPK) were quantified by Western blot analysis in response to metformin and the AMPK signaling inhibitor Compound C (EMD Chemicals, San Diego, CA). The effects of Compound C on the metformin-induced odontoblast differentiation of DPCs were determined by alkaline phosphatase activity assay and von Kossa staining, and the expression of odontoblastic markers was evaluated by reverse-transcription polymerase chain reaction analysis. ResultsDPCs exhibited mesenchymal stem cell characteristics using flow cytometry. Different doses of metformin were shown to be cytocompatible with DPCs, yielding >90% cell viability. None of the concentrations of metformin up to 50 μmol/L affected cell proliferation. The Western blot assay showed that DPCs express functional organic cation transporter 1, a transmembrane protein that mediates the intracellular uptake of metformin. Metformin significantly activated the AMPK pathway in a dose-dependent manner. In addition, it stimulated alkaline phosphatase activity; enhanced mineralized nodule formation; and increased the expression of odontoblastic markers including dentin sialophosphoprotein, dentin matrix protein 1, runt-related transcription factor 2, and osteocalcin. Moreover, pretreatment with Compound C, a specific AMPK inhibitor, markedly reversed metformin-induced odontoblastic differentiation and cell mineralization. ConclusionsThis study shows that metformin can induce DPC differentiation and mineralization in an AMPK-dependent manner and that this well-tolerated antidiabetic drug has potential in regenerative endodontics as well as in other regenerative applications.

Parthenolide Promotes Differentiation of Osteoblasts Through the Wnt/β-Catenin Signaling Pathway in Inflammatory Environments.

Periodontitis is a progressive inflammatory disease initiated by bacterial biofilm adhering to the tooth surface. If left untreated, periodontitis may lead to tooth loss and destruction of the alveolar bone. Regaining the lost alveolar bone is a clinical challenge because of the limited differentiation ability of osteoblasts in inflammatory environments. We have previously shown the anti-inflammatory and antiosteoclastogenic activities of parthenolide (PTL) in human periodontal ligament-derived cells by inhibiting nuclear factor kappa B (NF-κB) signaling, indicating its potential for periodontitis treatment. In this study, we further examined whether PTL could stimulate differentiation of osteoblasts from human alveolar bone in inflammatory conditions and investigated the involvement of the Wnt/β-catenin signaling pathway during this process. The results showed that PTL significantly stimulated alkaline phosphatase activity, mineralization nodule formation, and osteogenesis-related gene/protein expression of osteoblasts under the stimulation of tumor necrosis factor-α (TNF-α). In addition, PTL inhibited the NF-κB/p50 pathway and resisted the inhibition of Wnt/β-catenin signaling induced by TNF-α. Our results indicate that the stimulatory effect of PTL on the differentiation of osteoblasts in inflammatory environments may involve the activation of the Wnt/β-catenin signaling pathway, and PTL may be a promising component for bone regeneration in periodontitis treatment.

Metformin induces osteoblastic differentiation of human induced pluripotent stem cell-derived mesenchymal stem cells.

Metformin, a first-line antidiabetic drug used by millions of patients, has been shown to have potential osteogenic properties. The present study was performed to test the hypothesis that clinically relevant doses of metformin promote the osteogenic differentiation and mineralization of induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs). iPSC-MSCs were treated with metformin (10 μm) to assess cell viability, osteogenic differentiation, mineralization and activation of the LKB1/AMP-activated protein kinase (AMPK) signalling pathway, a surrogate marker of metformin action. To determine its potential application in MSC-based bone and periodontal tissue engineering, iPSC-MSCs were also treated with metformin when seeded on to calcium phosphate cement (CPC) scaffolds. Immunoblotting and cellular uptake assays showed that iPSC-MSCs express functional organic cation transporter-1 (OCT-1), a transmembrane protein that mediates the intracellular uptake of metformin. Although metformin treatment did not impair iPSC-MSC viability, it significantly stimulated alkaline phosphatase activity, enhanced mineralized nodule formation and increased expression of osteogenic markers, including Runt-related transcription factor 2 (RUNX2) and osterix. Inhibition of LKB1 activity, a common upstream AMPK kinase, markedly reversed metformin-induced AMPK activation, RUNX2 expression and nuclear localization. Moreover, metformin substantially increased mineralized nodule formation of iPSC-MSC seeded on CPC scaffolds. Collectively, functional OCT-expressing iPSC-MSCs responded to metformin by inducing an osteogenic effect in part mediated by the LKB1/AMPK pathway. Considering the widespread use of metformin in diabetics, this work may lead to novel tissue-engineering platforms where autogenous OCT-expressing iPSC-MSCs might be used to enhance bone and periodontal regeneration in diabetic patients prescribed with daily doses of metformin.

MiR-203 and miR-320 Regulate Bone Morphogenetic Protein-2-Induced Osteoblast Differentiation by Targeting Distal-Less Homeobox 5 (Dlx5).

MicroRNAs (miRNAs) are a family of small, non-coding RNAs (17–24 nucleotides), which regulate gene expression either by the degradation of the target mRNAs or inhibiting the translation of genes. Recent studies have indicated that miRNA plays an important role in regulating osteoblast differentiation. In this study, we identified miR-203 and miR-320b as important miRNAs modulating osteoblast differentiation. We identified Dlx5 as potential common target by prediction algorithms and confirmed this by knock-down and over expression of the miRNAs and assessing Dlx5 at mRNA and protein levels and specificity was verified by luciferase reporter assays. We examined the effect of miR-203 and miR-320b on osteoblast differentiation by transfecting with pre- and anti-miRs. Over-expression of miR-203 and miR-320b inhibited osteoblast differentiation, whereas inhibition of miR-203 and miR-320b stimulated alkaline phosphatase activity and matrix mineralization. We show that miR-203 and miR-320b negatively regulate BMP-2-induced osteoblast differentiation by suppressing Dlx5, which in turn suppresses the downstream osteogenic master transcription factor Runx2 and Osx and together they suppress osteoblast differentiation. Taken together, we propose a role for miR-203 and miR-320b in modulating bone metabolism.