Increased Alkaline Phosphatase Activity Research Articles

Can phosphorus (P)-releasing bacteria and earthworm (Eisenia fetida L.) co-enhance soil P mobilization and mycorrhizal P uptake by maize (Zea mays L.)?

Both earthworm (Eisenia fetida L.) and phosphorus (P)-releasing (PR) bacteria, including organic P-mineralizing (OPM) and inorganic P-solubilizing (IPS), are able to convert difficult-to-use P into phytoavailable P. However, there is a undesirable gap between soil P mobilization and root P acquisition when mycorrhization is enhanced. The aim of this study was to investigate the combined effects of earthworm and PR bacteria on soil P mobilization, mycorrhizal colonization, and P acquisition of maize (Zea mays L.). This study consisted of three parallel experiments. Firstly, a 90-day microcosm experiment was carried out to test the diversified effects of earthworm, OPM bacterium Bacillus megaterium, and IPS bacterium Kluyvera intermedia, on available P concentration in an autoclaved soil with or without P amendment. Secondly, a 16-week greenhouse pot experiment was conducted to compare the differential effects of earthworm, B. megaterium, and K. intermedia on P mobilization, mycorrhizal colonization, and P acquisition of maize in the same autoclaved soil receiving chemical fertilizers and Funneliformis caledonium. Thirdly, a microplot experiment was established to verify the combined effects of earthworm and PR bacteria on P mobilization and mycorrhizal P acquisition of maize in a field where the soil used in the former two experiments were collected. In the microcosm experiment, earthworm significantly increased soil available P concentration coupled of declined soil pH regardless of P amendment, while B. megaterium and K. intermedia further enhanced P mobilization in P-fertilized soils. In the greenhouse experiment, PR bacteria and earthworm co-increased mycorrhizal colonization and plant biomass, as well as soil alkaline phosphatase (ALP) activity and available P concentration compared to the control, and individual P acquisition compared to the single inoculation treatments. In the field experiment, both PR bacteria and earthworm significantly increased mycorrhizal colonization and tended to increase soil ALP activity, available P concentration, root biomass, and individual P acquisition, while the combined inoculation significantly increased ALP activity and available P concentration, and thereby individual P acquisition by 29% relative to the control. Combined inoculation of PR bacteria and earthworm greatly increased soil available P concentration and maize P acquisition and grain yield. Although the combined inoculation had a lower root mycorrhizal colonization than the single inoculation treatments due to the greatly increased amounts of readily soluble P, it had a higher mycorrhization level than the control. These observations suggest that the combined application could enhance soil P mobilization and be expropriated to elaborate indigenous mycorrhizal fungal function.

Novel bilayer electrospun poly(caprolactone)/ silk fibroin/ strontium carbonate fibrous nanocomposite membrane for guided bone regeneration

AbstractIn this study, a thin layer with a thickness of about 120 μm of poly(caprolactone) (PCL) was fabricated by electrospinning method. Then, a fibrous nanocomposite composed of PCL/silk fibroin/strontium carbonate (PCL/SF/SrCO3) was electrospun on the prepared layer. Then, they were characterized. The mechanical properties, water uptake, degradation rate, wettability, porosity, and bioactivity of the electrospun membrane were scrutinized in vitro. Cytotoxicity of the samples was assessed by using osteoblast‐like cells (SAOS‐2) and L929 fibroblasts. Moreover, the cell adhesion, alkaline phosphatase (ALP) activity, and calcium deposition through alizarin red staining were conducted. Results revealed that the bilayer structure doubled the optimum mechanical properties and the addition of SrCO3 up to 15%–20% increased ALP activity, calcium deposition, and bioactivity. According to the results, the nanofibrous bilayer membrane containing 20 wt% SrCO3, 20 wt% SF, and 60 wt% PCL was chosen as the optimum sample. Therefore, this membrane could be applied in guided bone regeneration (GBR).

Vertebral Bone Marrow-Derived Mesenchymal Stromal Cells from Osteoporotic and Healthy Patients Possess Similar Differentiation Properties In Vitro.

Osteoporosis is a disease characterized by low bone mass and an increased risk of fractures. Although several cellular players leading to osteoporosis have been identified, the role of mesenchymal stromal cells (MSC) is still not fully elaborated. The aim of this study was, therefore, to isolate and characterize MSCs from vertebral body of healthy non-osteoporotic and osteoporotic patients, with a particular focus on their osteogenic differentiation potential. Isolated MSCs were characterized by their osteogenic, adipogenic, and chondrogenic differentiation, as well as surface marker expression, proliferation behavior, and immunomodulatory capacity. The mineralization process was confirmed using Alizarin Red S and alkaline phosphatase (ALP) stains and further evaluated by determining ALP activity, mineral deposition, and free phosphate ion release. MSCs from both healthy and osteoporotic patients showed common fibroblast-like morphology and similar proliferation behavior. They expressed the typical MSC surface markers and possessed immunomodulatory capacity. Both groups demonstrated solid trilineage differentiation potential; osteogenic differentiation was further confirmed by increased ALP activity, deposition of inorganic crystals, phosphate ion release, and expression of osteoblast marker genes. Overall, MSCs from osteoporotic and non-osteoporotic patients showed neither a difference in general MSC features nor in the detailed analysis regarding osteogenic differentiation. These data suggest that vertebral body MSCs from osteoporotic patients were not impaired; rather, they possessed full osteogenic potential compared to MSCs from non-osteoporotic patients.

Aerobic physical training improves skeletal muscle metabolism in heart failure patients

Abstract Purpose To evaluate the efficiency of aerobic physical rehabilitation (PR), selected on the basis of lactate threshold achievement during cardiopulmonary exercising test (CPET), in heart failure patients with normal body mass index (BMI); to study metabolism changes in skeletal muscle fibers (MF) after PR. Methods 100 patients; heart failure with reduced ejection fraction (HFrEF), NYHA III, mean age 52±5.2 years, ejection fraction (EF) 27.3±4.2%, BMI 23.5±2.8 kg/m2. The original estimated CPET, exercise tolerance (ET), quality of life (QOL) and echocardiography (EchoCG). Effects of physical rehabilitation were estimated on the basis of peak oxygen uptake (VO2peak), EF, QOL and ET dynamics after 6 months of training. Shin muscle biopsy was performed at baseline and after 3–6 months of training programm. In muscle samples activity of lactate dehydrogenase (LDH), succinate dehydrogenase (SDG) and alkaline phosphatase (AP) was evaluated with plag-method. Data were statistically processed using software package Statistika, 9.0. Results After 6 months of physical training ET increased by 9.7±0.5 points and QOL improved by 24.8±3.5 points (p1,2 <0.05), EF increased by 10.5±2.3% and VO2 peak increased by 5.2±0.5 ml/min/kg (p3,4 <0.05). After 3–6 months of training AP activity increased by 24.2% (p<0.05). LDH activity in glycolytic MF decreased by 24.4%, in oxidative MF it decreased by only 6.0% (p1 <0.05, p2>0.05). SDG activity in glycolytic MF increased by 30% and in oxidative – by 20% (p1,2 <0.05). There was a positive relation between SDG activity and VO2 peak dynamics (r=0.6, p=0.04), increase in CPET parameters was associated with AP activity (r=0.5, p=0.05). Conclusion Aerobic physical training in stable HFrEF patients with normal BMI was effective in improving values of EF, VO2 peak, QOL and ET; Participating in the rehabilitation program decreased LDH activity in both oxidative and glycolytic muscle fibers; it also significantly increased AP activity and SDG activity in both oxidative and glycolytic MF. This may indicate a decrease in glycolytic metabolism activity and aerobic metabolism activation; The relation between the increase in exercise tolerance and endothelium condition marker (AP activity in muscle tissue), SDG activity and VO2 peak dynamics has been revealed. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): Project was partially funded by the Russian Science Foundation grant 16-15-1017

Gastrointestinal In Vitro Digests of Infant Biscuits Formulated with Bovine Milk Proteins Positively Affect In Vitro Differentiation of Human Osteoblast-Like Cells.

Infant biscuits (IBs) are part of complementary feeding from weaning up to the age of five years. They normally contain bovine milk proteins, which can influence bone development. This potential effect was investigated using experimental baked IBs, which were prepared from doughs containing different type of dairy proteins: milk protein concentrate (IB1), whey protein isolate (IB2), and skimmed milk powder (IB3). Dairy protein-free (IB0) and gluten-free (IB4) biscuits were also formulated. The in vitro gastrointestinal digests of IBs (IBDs) were tested on a co-culture of Caco-2/HT-29 70/30 cells as an in vitro model of human small intestine. None of the IBDs influenced cell viability and monolayer integrity, while IBD0 and IBD4 increased Peptide-YY production. The basolateral contents of Transwell plates seeded with Caco-2/HT-29 70/30 co-culture, mimicking metabolized IBDs (MIBDs), were tested on Saos-2 cells, an in vitro model of human osteoblast-like cells. After incubation, MIBD0, lacking dairy proteins, decreased the cell viability, while MIBD2, containing whey protein isolate, increased both the viability and the number of cells. MIBD2 and MIBD4, the latter containing both casein and whey proteins, increased alkaline phosphatase activity, a bone differentiation marker. These results highlight that IBs containing dairy proteins positively affect bone development.

Nature-inspired topographies on hydroxyapatite surfaces regulate stem cells behaviour

Surface topography is one of the key factors in regulating interactions between materials and cells. While topographies presented to cells in vivo are non-symmetrical and in complex shapes, current fabrication techniques are limited to replicate these complex geometries. In this study, we developed a microcasting technique and successfully produced imprinted hydroxyapatite (HAp) surfaces with nature-inspired (honeycomb, pillars, and isolated islands) topographies. The in vitro biological performance of the developed non-symmetrical topographies was evaluated using adipose-derived stem cells (ADSCs). We demonstrated that ADSCs cultured on all HAp surfaces, except honeycomb patterns, presented well-defined stress fibers and expressed focal adhesion protein (paxillin) molecules. Isolated islands topographies significantly promoted osteogenic differentiation of ADSCs with increased alkaline phosphatase activity and upregulation of key osteogenic markers, compared to the other topographies and the control unmodified (flat) HAp surface. In contrast, honeycomb topographies hampered the ability of the ADSCs to proliferate and differentiate to the osteogenic lineage. This work presents a facile technique to imprint nature-derived topographies on the surface of bioceramics which opens up opportunities for the development of bioresponsive interfaces in tissue engineering and regenerative medicine.

Bone morphogenetic protein 2-enhanced osteogenic differentiation of stem cell spheres by regulation of Runx2 expression.

Bone morphogenetic protein 2 (BMP-2) is a growth factor that is used to induce osteogenic differentiation in stem cells. The present study assessed the effects of BMP-2 on stem cell spheroid morphology, viability and osteogenic differentiation. Stem cell spheres were constructed and treated with BMP-2 at predetermined concentrations (0-100 ng/ml) using concave microwells. Cell viability was qualitatively and quantitatively analyzed via microscopy and a water-soluble tetrazolium salt assay kit, respectively. Alkaline phosphatase activity was assessed and an anthraquinone dye for calcium deposit evaluation was performed to determine osteogenic differentiation. The expressions of (runt-related transcription factor 2) and collagen 1 were also determined via quantitative PCR. Spherical shapes were formed using concave microwells on day 1, which were maintained up to day 21. On day 1, the relative cell viability of 0, 10 and 100 ng/ml BMP-2 treated cells was 100.0±1.9, 97.3±4.4 and 101.3±2.6%, respectively. Significantly higher values for alkaline phosphatase activity were determined in the 100 ng/ml treated group when compared with the control group. Additionally, Runx2 mRNA levels were significantly higher in the 100 ng/ml BMP-2 group compared with the control group, as determined via quantitative PCR. The results of the present study indicated that BMP-2 enhanced the differentiation of stem cell spheres, which was demonstrated by increased alkaline phosphatase activity and Runx2 expression.

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

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

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

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

In vivo and in vitro evaluation of the osteogenic potential of Davallia mariesii T. Moore ex Baker

Ethnopharmacological relevancePostmenopausal osteoporosis is a major bone health issue worldwide. There is an unmet medical need for osteoporosis treatments, a disease which disproportionately impacts women. Exploring botanicals to prevent or treat osteoporosis is currently an interest of investigations. Rhizomes of Davallia mariesii T. Moore ex Baker (Davalliacea) are used an indigenous herbal medicine in Asia for injuries due to fractures, contusions, and strains. Aim of the studyIn the present study, we investigated the osteogenic effect of the water extract of rhizomes of D. mariesii (DMH) on bone loss induced by an ovariectomy (OVX) in mice and also its impact on osteogenesis in primary human osteoblasts (HObs). Additionally, we performed a quantitative analysis of compounds in the DMH extract. Materials and methodsOVX C57BL/6J mice were orally administrated DMH extract for 12 weeks, and microarchitecture parameters were examined by microcomputed tomography. DMH extract was fractionated in a bio-guided manner, and fractions were isolated to obtain active compounds using HObs. Cell viability was evaluated by an MTT assay. Characteristics of early and late osteogenesis were analyzed by alkaline phosphatase activity and a mineralization assay. Molecular mechanisms were explored by a real-time quantitative PCR. Compounds in the DMH extract were identified and quantified using liquid chromatography tandem mass spectroscopy (LC-MS/MS). ResultsDMH improved bone mineral densities of vertebrae and the femur. Through microarchitectural observations, DMH significantly decreased the bone surface/volume ratio and trabecular separation, and also increased the connectivity density in the OVX group. Additionally, DMH inhibited osteoclast differentiation in receptor activator of nuclear factor-κB ligand-induced osteoclasts and increased bone formation in HObs. After bio-guided fractionation and isolation, we found that eriodictyol-7-O-β-d-glucuronide (2) significantly increased alkaline phosphatase activity, and 5-O-β-d-(6-O-vanilloylglucopyranosyl)gentisic acid (3) substantially enhanced mineral deposition. In HObs, compound 3 was more potent in upregulating expressions of bone morphogenetic protein-2, bone sialoprotein, osteopontin, osterix, and estrogen receptor-α. The amount of bioactive compound 3 in DMH was 5.68 ± 0.64 mg/g of dry weight according to LC-MS/MS. ConclusionFor the first time we report that D. mariesii and its isolated compounds demonstrated potent osteogenic activities. Quantitative results of D. mariesii could be a reference for phytochemical analyses.

Magnetic Nanoparticles Synergize with Pulsed Magnetic Fields to Stimulate Osteogenesis In Vitro.

Delayed bone healing is a major challenge in orthopedic clinical practice, highlighting a need for technologies to overcome ineffective cell growth and osteogenic differentiation. The objective of this study was to investigate the synergistic effects of the PhysioStim (PEMF) signal with iron-ion doped tri-calcium phosphate bone substitute on human mesenchymal stem cell (hMSC) osteogenesis in vitro. Intrinsically magnetic nano-bone substitutes (MNBS) were developed with single particles on the order of 100 nm, saturation magnetization of 0.425 emu/g, and remanent magnetization of 0.013 emu/g. MNBS were added to hMSC culture and cell viability, alkaline phosphatase (ALP) activity, mineralization, and osteogenic gene expression in the presence and absence of PEMF were quantified for up to 10 days. MNBS attached to the surface of and were internalized by hMSCs when cultured together for 4 days and had no impact on cell viability with PEMF exposure for up to 7 days. Although total ALP activity was significantly increased with PEMF treatment alone, with a peak at day 5, PEMF combined with MNBS significantly increased ALP activity, with a peak at day 3, compared with all other groups (p < 0.01). The shift can be explained by significantly increased extracellular ALP activity beginning at day 2 (p < 0.01). PEMF combined with MNBS demonstrated continuously increasing mineralization overtime, with significantly greater Alizarin Red S concentration compared with all other groups at day 7 (p < 0.01). Increases in ALP activity and mineral content were in agreement with osteogenic gene expression that demonstrated peak ALP gene expression at day 1, and upregulated BMP-2, BGLAP, and SPP1 gene expression at day 7 (p < 0.05). The results of this study demonstrate the synergistic effects of PEMF and MNBS on osteogenesis and suggest that PEMF and MNBS may provide a method for accelerated bone healing.

Effect of Insulin-Like Growth Factor-1 on Bone Formation of Bone Marrow Mesenchymal Stem Cells Under High Glucose Environment by Regulating Insulin Like Growth Factor Binding Protein 2(IGFBP-2)/p38 Pathway

Diabetes easily affects the biological properties of bone marrow mesenchymal stem cells (BMSCs). Insulin-like growth factor-1 (IGF-1) promotes bone healing during osteoporotic fractures. However, IGF-1's effect on BMSCs high glucose is unclear. Rat BMSCs were assigned into control group, high glucose group, and IGF-1 group in which BMSCs were transfected with pc-DNA 3.1-IGF-1 plasmid on the basis of high glucose followed by analysis of IGF-1, RUNX2 and OPN mRNA level by real time PCR, cell proliferation by MTT assay, alkaline phosphatase (ALP) activity, IGFBP-2 level by ELISA, and p38 phosphorylation by Western blot. High glucose group showed significantly decreased IGF-1, RUNX2 and OPN mRNA level, reduced ALP activity, IGFBP-2 expression and p38 phosphorylation compared to control group (P &lt; 0 05). Transfection of IGF-1 plasmid under high-glucose environment significantly upregulated IGF-1, RUNX2 and OPN mRNA, increased ALP activity, IGFBP-2 expression and p38 phosphorylation compared to high-glucose group (P &lt; 0 05). IGF-1 expression is reduced in high glucose environment. Up-regulation of IGF-1 can promote BMSCs osteogenic differentiation through the IGFBP-2/p38 pathway.

Gallium ions promote osteoinduction of human and mouse osteoblasts via the TRPM7/Akt signaling pathway.

Gallium (Ga) ions have been widely utilized for biomedical applications; however, their role in osteoblast regulation is not completely understood. The aim of the present study was to investigate the potential effect of Ga ions on osteoinduction in two osteoblast cell lines and to explore the underlying mechanisms. Human hFOB1.19 and mouse MC3T3-E1 osteoblasts were treated with Ga nitride (GaN) at different concentrations, and the degree of osteoinduction was assessed. Ga ion treatment was found to increase alkaline phosphatase activity and accelerate calcium nodule formation, as assessed using ALP activity assay and Alizarin red S staining. Moreover, upregulated expression levels of osteogenic proteins in osteoblasts were identified using western blotting and reverse transcription-quantitative PCR. Western blotting was also performed to demonstrate that the biological action of Ga ions was closely associated with the transient receptor potential melastatin 7/Akt signaling pathway. Furthermore, it was found that Ga ions did not cause osteoblast apoptosis, as indicated via flow cytometry, but promoted osteoclast proliferation, migration or invasion. The present study investigated the potential role of Ga ions in regulating osteoinduction of osteoblasts, thereby providing a promising strategy for the treatment of osteoporosis.

Bioassay-guided isolation and evaluation of anti-osteoporotic polysaccharides from Morinda officinalis

Ethnopharmacological relevanceMorinda officinalis is a well-known Chinese tonic herb that has shown clinical efficacy in the treatment of bone disease. However, its anti-osteoporotic potential and the M. officinalis polysaccharides (MOPs) responsible for activity require further investigation. Aim of the studyThis study aimed to investigate the anti-osteoporotic effects of different MOP fractions in ovariectomized (OVX) rats, and to identify the osteoprotective components by bioassay-guided isolation. Materials and methodsMOPs were prepared by hot water and alkali extraction, separated into three fractions (MO50, MO70, and MOB) and evaluated in the classic OVX rat model and in MC3T3-E1 cells for anti-osteoporotic activity. ResultsAdministration of MOPs (400 mg/kg/day) provided significant protection against ovariectomy-induced bone loss and biomechanical dysfunction in rats. Treated animals exhibited reduced deterioration of trabecular microarchitecture and lower levels of bone turnover markers. Bioactivity-guided fractionation led to the isolation of two inulin-type fructans from MO50, MOW50-1 and MOP50-2, with potential anti-osteoporotic activities. These consisted of (2 → 1)-linked β-D-fructosyl residues with degrees of polymerization (DP) of 7 and 13, respectively. Furthermore, MOW50-1 promoted osteogenic differentiation of MC3T3-E1 cells by increasing alkaline phosphatase activity. ConclusionsThese data suggest very strongly that MOPs, especially MO50 and MOW50-1, may play important roles in the prevention and treatment of osteoporosis.

MicroRNA‑204‑5p inhibits the osteogenic differentiation of ankylosing spondylitis fibroblasts by regulating the Notch2 signaling pathway.

Ankylosing spondylitis (AS) is a chronic inflammatory systemic disease and is difficult to detect in the early stages. The present study aimed to investigate the role of microRNA (miR)-204-5p in osteogenic differentiation of AS fibroblasts. Bone morphogenetic protein 2 (BMP-2) was used to induce osteogenic differentiation. Cells were divided into the following groups: AS group, AS + BMP-2 group, AS + BMP-2 + miR-negative control group, AS + BMP-2 + miR-204-5p mimics group and AS + BMP-2 + miR-204-5p mimics + pcDNA-Notch2 group. The expression levels of miR-204-5p, Notch2, runt-related transcription factor 2 (RUNX2) and osteocalcin were detected via reverse transcription-quantitative PCR analysis. The binding site between Notch2 and miR-204-5p was predicted using TargetScan software and verified via the dual-luciferase reporter assay. Alkaline phosphatase (ALP) activity was assessed via the ALP assay, while the mineralized nodules area was determined via the Alizarin Red S staining assay. The results demonstrated that Notch2 is a target gene of miR-204-5p. Furthermore, treatment with BMP-2 significantly decreased miR-204-5p expression, and significantly increased ALP activity, the mineralized nodules area and the expression levels of Notch2, RUNX2 and osteocalcin in ligament fibroblasts (all P<0.05). Conversely, transfection with miR-204-5p mimics significantly increased miR-204-5p expression, and significantly decreased ALP activity, the mineralized nodules area and the expression levels of Notch2, RUNX2 and osteocalcin in ligament fibroblasts (all P<0.05). Notably, transfection with pcDNA-Notch2 significantly reversed the inhibitory effects induced by miR-204-5p mimics on the osteogenic differentiation of ligament fibroblasts (all P<0.05). Furthermore, miR-204-5p inhibited the osteogenic differentiation of ligament fibroblasts in patients with AS by targeting Notch2. Thus, miR-204-5p may negatively regulate Notch2 expression and may be a potential therapeutic target for AS. Collectively, the results of the present study provide a theoretical basis for the effective treatment of patients with AS.

Compounds isolated from Euonymus spraguei Hayata induce ossification through multiple pathways.

The process of bone metabolism includes catabolism of old or mature bone and anabolism of new bone, carried out by osteoclasts and osteoblasts respectively. Any imbalance in this process results in loss of bone mass or osteoporosis. Drugs available to combat osteoporosis have certain adverse effects and are unable to improve bone formation, hence identifying new agents to fulfil these therapeutic gaps is required. To expand the scope of potential agents that enhance bone formation, we identified Euonymus spraguei Hayata as a plant material that possesses robust osteogenic potential using human osteoblast cells. We isolated three compounds, syringaresinol (1), syringin (2), and (−)-epicatechin (3), from E. spraguei. Results demonstrated that syringin (2), and (−)-epicatechin (3), increased alkaline phosphatase activity significantly up to 131.01% and 130.67%, respectively; they also elevated mineral deposition with respective values of up to 139.39% and 138.33%. In addition, 2 and 3 modulated autophagy and the bone morphogenetic protein (BMP)-2 signaling pathway. Our findings demonstrated that 2 and 3 induced osteogenesis by targeting multiple pathways and therefore can be considered as potent multi-targeted drugs for bone formation against osteoporosis.

Long noncoding RNA LINC00314 facilitates osteogenic differentiation of adipose-derived stem cells through the hsa-miR-129-5p/GRM5 axis via the Wnt signaling pathway

BackgroundMany studies have shown that long noncoding RNAs (lncRNAs) are closely related to the stimulation of osteogenic differentiation of adipose-derived stem cells (ADSCs) and the prevention of osteoporosis. Current research aimed to investigate the novel lncRNA and explored the function and molecular mechanism of the LINC00314/miR-129-5p/GRM5 axis in regulating osteogenic differentiation of ADSCs.MethodsLncRNA and miRNA sequencing was performed in normal and osteogenic differentiation-induced ADSCs (osteogenic group). Abnormally expressed lncRNAs and miRNAs were obtained by the R software and the relative expression of LINC00314, miR-129-5p, and GRM5 during osteogenic induction was measured by RT-PCR. ADSCs were then transfected with pcDNA3.1-sh-LINC00314 and agomiR-129-5p. Alizarin red staining (ARS) and alkaline phosphatase (ALP) staining were performed to identify the mechanism of the LINC00314/miR-129-5p/GRM5 axis in regulating osteogenic differentiation of ADSCs.ResultsLINC00314 was significantly upregulated in the group of osteogenic-induced ADSCs. LINC00314 and GRM5 mimics increased the early and late markers of osteogenic differentiation, which manifest in not only the markedly increased ALP activity but also higher calcium deposition, while miR-129-5p mimic had the opposite effects. LINC00314 directly targeted miR-129-5p through luciferase reporter assay, and miR-129-5p suppressed GRM5 expression. Moreover, the LINC00314/miR-129-5p/GRM5 regulatory axis activated the Wnt/β-catenin signaling pathway.ConclusionsLINC00314 confers contributory function in the osteogenic differentiation of ADSCs and thus the LINC00314/miR-129-5p/GRM5 axis may be a novel mechanism for osteogenic-related disease.

Acellular pericardium: A naturally hierarchical, osteoconductive, and osteoinductive biomaterial for guided bone regeneration.

There is great demand for an improved barrier membrane with osteogenic potential for guided bone regeneration (GBR). Natural acellular porcine pericardium (APP) is increasingly used in regenerative medicine as a kind of common extracellular matrix materials. This study aimed to investigate its potential application in GBR, especially its osteoconductive and osteoinductive properties. Bio-Gide (BG), a commercial collagen membrane, was set as the control group. APP samples were characterized by physicochemical analyses and their biological effects on human bone mesenchymal stem cells (hBMSCs) and human gingival fibroblasts (hGFs) were also examined. Additionally, the osteogenic potential of APP was tested on a bilateral critical-sized calvarial defect model. We discovered that the smooth surface of APP tended to recruit more hBMSCs. Moreover, promoted proliferation and osteogenic differentiation of hBMSCs was detected on this side of APP, with increased alkaline phosphatase activity and upregulated expression of bone-specific genes. Besides, the rough side of APP showed good biocompatibility and barrier function with hGFs. Histologic observation and analysis of calvarial defect healing over 4 weeks revealed enhanced bone regeneration under APP compared with BG and the control group. The results of this study indicate that APP is a potential osteoconductive and osteoinductive biomaterial for GBR.

Osteogenesis activity of fractions extracted from &lt;i&gt; Clinacanthus nutans&lt;/i&gt; (Burm. F.) Lindau

Osteogenesis activity of fractions extracted from Clinacanthus nutans was evaluated on an in vitro model using osteoblast MC3T3-E1 cells. The results showed that all fractions, including ethanol (EtOH), n-hexane, ethyl acetate (EtOAc) and butanol (BuOH) were not significantly toxic to the osteoblast cells at the test concentrations of 5, 10, 25, and 50 µg/mL. The EtOH and EtOAc fractions exhibited the highest osteogenesis activity in terms of enhancement of alkaline phosphatase (ALP) and mineralization activity of MC3T3-E1 cells, especially the EtOAc fraction which increased ALP activity up to &gt; 30% and mineralization activity up to &gt; 100%. Thus, the EtOAc fraction shows osteogenesis activity through stimulating activites of the two markers for bone generation including ALP and mineralization in osteoblast cells. The fraction is now under extensive investigation to isolate and fully understand the modes of action of the active compounds.

Icariin Accelerates Fracture Healing via Activation of the WNT1/β-catenin Osteogenic Signaling Pathway.

Icariin has been shown to enhance bone formation. The present study aimed to investigate whether icariin also promotes bone fracture healing and its mechanisms. First, we isolated and cultured rat bone marrow stromal cells (rBMSCs) with icariincontaining serum at various concentrations (0%, 2.5%, 5% and 10%) and then measured alkaline phosphatase (ALP) activity and the expression of Core-binding factor, alpha 1 (Cbfα1), bone morphogenetic protein-2 (BMP-2) and bone morphogenetic protein-4 (BMP-4) in the rBMSCs. Second, we established a model of fracture healing in rats and performed gavage treatment for 20 days. Then, we detected bone biochemical markers (ELISA kits) in the serum, fracture healing (digital radiography, DR), and osteocalcin expression (immunohistochemistry). Icariin treatment increased ALP activity and induced the expression of Cbfα1, BMP-2 and BMP-4 in rBMSCs in a dose-dependent manner. In addition, Icariin increased the serum levels of osteocalcin (OC), bone-specific alkaline phosphatase (BAP), N-terminal telopeptides of type I collagen (NTX-1), C-terminal telopeptide of type I collagen (CTX-1) and tartrate-resistant acid phosphatase 5b (TRACP-5b); promoted osteocalcin secretion at the fracture site; and accelerated fracture healing. Icariin can promote the levels of bone-formation markers, accelerate fracture healing, and activate the WNT1/β-catenin osteogenic signaling pathway.