Mineralization Assay Research Articles

Total Phosphate Influences the Rate of Hydrocarbon Degradation but Phosphate Mineralogy Shapes Microbial Community Composition in Cold-Region Calcareous Soils.

Managing phosphorus bioaccessibility is critical for the bioremediation of hydrocarbons in calcareous soils. This paper explores how soil mineralogy interacts with a novel biostimulatory solution to both control phosphorus bioavailability and influence bioremediation. Two large bore infiltrators (1 m diameter) were installed at a PHC contaminated site and continuously supplied with a solution containing nutrients and an electron acceptor. Soils from eight contaminated sites were prepared and pretreated, analyzed pretrial, spiked with diesel, placed into nylon bags into the infiltrators, and removed after 3 months. From XAS, we learned that three principal phosphate phases had formed: adsorbed phosphate, brushite, and newberyite. All measures of biodegradation in the samples (in situ degradation estimates, mineralization assays, culturable bacteria, catabolic genes) varied depending upon the soil's phosphate speciation. Notably, adsorbed phosphate increased anaerobic phenanthrene degradation and bzdN catabolic gene prevalence. The dominant mineralogical constraints on community composition were the relative amounts of adsorbed phosphate, brushite, and newberyite. Overall, this study finds that total phosphate influences microbial community phenotypes whereas relative percentages of phosphate minerals influences microbial community genotype composition.

Effects of a novel light‐curable material on odontoblastic differentiation of human dental pulp cells

To assess the biological effects, including odontoblastic differentiation of a novel light-curable material (TheraCal), on human dental pulp cells (hDPCs). The hDPCs were isolated from freshly extracted, caries-free third molars. Ten discs of TheraCal and MTA (8mm in diameter and 3mm in height) were incubated in α-minimum essential medium (α-MEM) and the supernatant collected. Viability of hDPCs in response to TheraCal and MTA was measured using the WST-1 assay. RT-PCR and real-time PCR were used to detect the gene expression of dentine sialophosphoprotein (DSPP) and dentine matrix protein-1 (DMP-1). ALP staining and Alizarin red S staining were used to evaluate the expression of alkaline phosphatase (ALP) and mineralization behaviour. One-way analysis of variance and Tukey's post hoc test were used to determine the statistically significant differences as a result of the variation in test materials (P<0.05). The effects of TheraCal and MTA on cell viability were similar except at the highest concentration. The mRNA level of DSPP increased significantly in the MTA group relative to the control at day 1 and 3 (P<0.05). Also, the mRNA level of DSPP increased significantly in the TheraCal group relative to the control at day 3 (P<0.05). The increased mRNA level of DMP-1 was 2.5-fold and 2.3-fold each in the MTA and TheraCal groups relative to the control (P<0.05). Cells exposed to MTA exhibited a 1.4-fold increase of ALP staining relative to control (P<0.05). In the mineralization assay, increased calcium nodule formation was twofold and 1.3-fold each in the MTA and TheraCal groups compared to the control (P<0.05). TheraCal and MTA had the ability to induce odontoblastic differentiation and mineralization of hDPCs.

Epigallocatechin-3-gallate Promotes Osteoblastic Activity in Human Osteoblast-like Cells

Purpose: To investigate the effect of epigallocatechin-3-gallate (EGCG) on bone metabolism and osteoblastic activity. Methods: MG-63 human osteoblast-like cells were treated with varied concentrations of EGCG. Alkaline phosphatase (ALP) activity and matrix mineralization assays were carried out on the treated and untreated MG-63 human osteoblast-like cells. Beta-catenin mRNA level was determined by quantitative real-time polymerase chain reaction (q-PCR). Results: The results showed that EGCG treatment significantly increased ALP and mineralization activities at concentrations of 15 and 30 μM, in a dose-dependent manner. Furthermore, EGCG treatment significantly increased beta-catenin mRNA expression by 70.7 ± 11.0 and 126.7 ± 35.1 %, respectively, at EGCG concentration of 15 and 30 μM. In addition, the stimulating effect of EGCG treatment on ALP activity was abolished by co-treatment with ICI 182,780, an antagonist of estrogen receptor (ER). Again, the increase in beta-catenin MRNA, when treated with EGCG, was inhibited by co-treatment with ICI 182,780. Conclusion: EGCG promotes osteoblastic activity in human osteoblast-like cells, by Wnt signaling through estrogen receptor (ER) pathway. Keywords: Epigallocatechin-3-gallate, Osteoporosis, Osteoclast, Proliferation, Estrogen receptor

Actinorhizal Alder Phytostabilization Alters Microbial Community Dynamics in Gold Mine Waste Rock from Northern Quebec: A Greenhouse Study.

Phytotechnologies are rapidly replacing conventional ex-situ remediation techniques as they have the added benefit of restoring aesthetic value, important in the reclamation of mine sites. Alders are pioneer species that can tolerate and proliferate in nutrient-poor, contaminated environments, largely due to symbiotic root associations with the N2-fixing bacteria, Frankia and ectomycorrhizal (ECM) fungi. In this study, we investigated the growth of two Frankia-inoculated (actinorhizal) alder species, A. crispa and A. glutinosa, in gold mine waste rock from northern Quebec. Alder species had similar survival rates and positively impacted soil quality and physico-chemical properties in similar ways, restoring soil pH to neutrality and reducing extractable metals up to two-fold, while not hyperaccumulating them into above-ground plant biomass. A. glutinosa outperformed A. crispa in terms of growth, as estimated by the seedling volume index (SVI), and root length. Pyrosequencing of the bacterial 16S rRNA gene for bacteria and the ribosomal internal transcribed spacer (ITS) region for fungi provided a comprehensive, direct characterization of microbial communities in gold mine waste rock and fine tailings. Plant- and treatment-specific shifts in soil microbial community compositions were observed in planted mine residues. Shannon diversity and the abundance of microbes involved in key ecosystem processes such as contaminant degradation (Sphingomonas, Sphingobium and Pseudomonas), metal sequestration (Brevundimonas and Caulobacter) and N2-fixation (Azotobacter, Mesorhizobium, Rhizobium and Pseudomonas) increased over time, i.e., as plants established in mine waste rock. Acetate mineralization and most probable number (MPN) assays showed that revegetation positively stimulated both bulk and rhizosphere communities, increasing microbial density (biomass increase of 2 orders of magnitude) and mineralization (five-fold). Genomic techniques proved useful in investigating tripartite (plant-bacteria-fungi) interactions during phytostabilization, contributing to our knowledge in this field of study.

In vivo bone regeneration on titanium devices using serum-free grown adipose-derived stem cells, in a sheep femur model.

The aim of this study was to investigate the capacity of adipose-derived stem cells (ADSC), grown in serum-free conditions, to regenerate bone around titanium discs with different titanium surfaces. Ovine ADSC (oADSC) were isolated from seven sheep and cultured using serum-free and osteogenic conditions. Prior to invivo testing, the growth and osteogenic behaviour of these cells were analysed invitro using cell proliferation and extracellular matrix mineralisation assays. The bone regenerative capacity of autologous oADSC was evaluated invivo on titanium discs in a sheep femur epicondyle model. Machined (MTi) and alumina-blasted (ABTi) titanium discs were used. Bone regeneration within the defects was evaluated after 1month using histology and histomorphometry. PKH26 cell-tracking dye was used to verify the persistence of oADSC in the surgical wound. oADSC sourced from five of seven sheep differentiated into osteoblast-like cells. Cellular proliferation was reduced only for osteogenically induced oADSC (oOS-ADSC) grown on ABTi, compared to non-induced oADSC grown on ABTi and tissue culture polystyrene (P=0.03 and 0.02 respectively). There was no significant difference for invitro mineralisation assays comparing oADSC with oOS-ADSC, regardless of implant surface type. oADSC labelled with PKH26 were detected 1month after surgery within the defect. There was no difference in bone regeneration between the bone defects treated with oADSC vs. just blood clot. After 1-month healing, the use of autologous oADSC did not improve bone regeneration in defects containing titanium devices with different surfaces.

Osteogenic Potential of Multipotent Adult Progenitor Cells for Calvaria Bone Regeneration.

Osteogenic cells derived from rat multipotent adult progenitor cells (rMAPCs) were investigated for their potential use in bone regeneration. rMAPCs are adult stem cells derived from bone marrow that have a high proliferation capacity and the differentiation potential to multiple lineages. They may also offer immunomodulatory properties favorable for applications for regenerative medicine. rMAPCs were cultivated as single cells or as 3D aggregates in osteogenic media for up to 38 days, and their differentiation to bone lineage was then assessed by immunostaining of osteocalcin and collagen type I and by mineralization assays. The capability of rMAPCs in facilitating bone regeneration was evaluated in vivo by the direct implantation of multipotent adult progenitor cell (MAPC) aggregates in rat calvarial defects. Bone regeneration was examined radiographically, histologically, and histomorphometrically. Results showed that rMAPCs successfully differentiated into osteogenic lineage by demonstrating mineralized extracellular matrix formation in vitro and induced new bone formation by the effect of rMAPC aggregates in vivo. These outcomes confirm that rMAPCs have a good osteogenic potential and provide insights into rMAPCs as a novel adult stem cell source for bone regeneration.

Dual Effects of Bisphosphonates on Ectopic Skin and Vascular Soft Tissue Mineralization versus Bone Microarchitecture in a Mouse Model of Generalized Arterial Calcification of Infancy

Generalized arterial calcification of infancy (GACI) is an intractable ectopic mineralization disorder caused by mutations in the ENPP1 gene resulting in reduced plasma inorganic pyrophosphate levels. We previously characterized the Enpp1asj mutant mouse as a model of GACI, and we have now explored the potential efficacy of bisphosphonates, non-hydrolyzable PPi analogs, in preventing ectopic mineralization in these mice. These mice were maintained on either basic diet (control) or diets containing etidronate or alendronate in three different concentrations (experimental). Considering low bioavailability of bisphosphonates when administered orally, subsequent studies tested the mice with subcutaneous injections of etidronate. The treatments were initiated at 4 weeks of age, and the degree of mineralization was assessed at 12 weeks of age by quantitation of calcium deposits in the muzzle skin containing dermal sheath of vibrissae and in aorta. We found that bisphosphonate treatments significantly reduced mineralization in skin and aorta. These changes in treated mice were accompanied with restoration of their bone microarchitecture, determined bymicrocomputed tomography. The inhibitory capacity of bisphosphonates, with mechanistic implications, was confirmed in a cell-based mineralization assay in vitro. Collectively, these results suggest that bisphosphonate treatment may be beneficial by a dual effect for preventing ectopic soft tissue mineralization while correcting decreased bone mineralization in GACI caused by ENPP1 mutations.

A nacre protein forms mesoscale hydrogels that “hijack” the biomineralization process within a seawater environment

We examined the mineralization performance of a nacre protein, AP7, within seawater mineralization assays that form aragonite and magnesium calcite. Under these conditions AP7 forms hydrogel particles that vary in size and complexity depending upon ionic conditions. These hydrogels “hijack” the mineralization process by limiting nucleation in bulk solution and promoting nucleation within the hydrogels.

Increased acellular and cellular surface mineralization induced by nanogrooves in combination with a calcium-phosphate coating

The current work evaluated the influence of nanoscale surface-topographies in combination with a calcium phosphate (CaP) coating on acellular and cellular surface mineralization. Four groups of substrates were produced, including smooth, grooved (940nm pitch, 430nm groove width, 185nm depth), smooth coated, and grooved coated. The substrates were characterized by scanning/transmission electron microscopy and atomic force microscopy. Osteoblast-like MC3T3 cells were cultured on the substrates for a period up to 35days under osteogenic conditions. Differentiation was observed by alkaline phosphatase assay and PCR of collagen I (COLI), osteopontin (OPN), osteocalcin (OC), bone-morphogenic protein 2 (BMP2), and bone sialoprotein (BSP). Mineralization was quantified by a calcium assay and Alizarin Red staining. In addition, acellular mineralization was determined after incubation of substrates in just cell culture medium without cells. Results showed that a reproducible nano-metric (∼50nm) CaP-layer could be applied on the substrates, without losing the integrity of the topographical features. While no relevant differences were found for cell viability, cells on smooth surfaces proliferated for a longer period than cells on grooved substrates. In addition, differentiation was affected by topographies, as indicated by an increased expression of OC, OPN and ALP activity. Deposition of a CaP coating significantly increased the acellular mineralization of smooth as well grooved substrate-surfaces. However, this mineralizing effect was strongly reduced in the presence of cells. In the cell seeded situation, mineralization was significantly increased by the substrate topography, while only a minor additive effect of the coating was observed. In conclusion, the model presented herein can be exploited for experimental evaluation of cell–surface interaction processes and optimization of bone-anchoring capability of implants. The model showed that substrates modified with CaP-coated coated nanogrooves display enhanced in vitro mineralization as compared to unmodified controls or substrates modified with either nanogrooves or CaP coatings. However, our results also indicated that acellular mineralization assays are not necessarily predictive for biological performance. Statement of significanceThe manuscript describes the possibility to combine the mechanical properties of nanosized topographies with the biochemical properties of a calcium phosphate based coating for improvement of surface mineralization.Interestingly, our results demonstrate that further incubation of our surfaces in SBF type media allowed all surfaces to mineralize rapidly to a high extent. Moreover we prove that nanotexture be used to can stimulate and organize mineralization and that the combination surface of a CaP coating and a nanotexture has the potential to be effective as a bone-implant surface. Such experiments will be of considerable interest to those in the research community and industry, who are focusing on bio-mineralization processes and optimization of modern bone-implants.

Concurrent reconciliation of chemical and mineral assays for mineral processing circuits

The steady-state data reconciliation of mineral processing plants has traditionally depended on chemical assays of samples collected from various streams in the plant. In some cases, additional information in the form of size assays and size-wise chemical assays have been employed to improve the reconciliation problem. Reconciliation algorithms, in addition to making the measured data consistent with the basic conservation principles, also help in predicting the material flow rates in different streams. This information helps the process engineer to identify and troubleshoot problem areas, and further use it to optimize the plant's performance. If all the minerals present in the ore system are known (established through X-Ray Diffraction), the chemical formulae of the minerals provide the relationship between the chemical assays and mineral composition. In this communication, first we propose an approach to estimate the mineral composition from the measured chemical assays for a given sample or stream. It is then extended to a plant-wide concurrent data reconciliation methodology, involving both the chemical assays and mineral composition. This concurrent data reconciliation procedure provides estimates of mineral composition around the plant even when mineral composition measurements are not available. This additional information will help the plant engineers to track and improve their plant's behavior more intuitively. When mineral compositions are measured (for example, through QEMSCAN) they can be reconciled along with chemical assays. The methodology is demonstrated with data from batch and semi-batch laboratory studies on selective dispersion-flocculation of iron ore slimes.

Effects of heterodimeric bone morphogenetic protein‐2/7 on osteogenesis of human adipose‐derived stem cells

Roles of bone morphogenetic proteins (BMPs) on osteogenesis of human adipose-derived stem cells (hASCs) remain ambiguous. In this study, we evaluated in vitro and in vivo functional characteristics of BMPs of different dimerization types, with the aim of determining osteoinductive efficiency of heterodimeric BMP-2/7 on osteogenesis of hASCs. We explored osteoinductive effects of three different BMPs by using cell DNA assay, alkaline phosphatase (ALP) activity assay, alizarin red staining and mineralization assay, and real-time PCR, in vitro. Also, we examined ectopic bone formation in nude mice by using soft X-ray, histomorphometric and immunohistochemical analyses in vivo. In our dose-response study, we showed that BMPs with both dimerization types did not induce in vitro osteogenesis of hASCs without osteogenic medium (OM). In the presence of OM, BMPs significantly enhanced hASC osteogenesis in a dose-dependent manner. In in vivo experiments, our analyses indicated that BMPs promoted osteogenesis of hASCs without in vitro osteogenic induction. However, both in vitro and in vivo, there were no significant differences in hASC osteogenic induction between heterodimeric and homodimeric BMPs. Heterodimeric BMP-2/7 significantly promoted osteogenesis of hASCs in vitro and in vivo. However, BMP-2/7 was not found to be a stronger inducer of osteogenesis compared to homodimeric either BMP-2 or BMP-7.

High glucose-associated osmolality promotes adipocytogenic differentiation of primary rat osteoblasts in a protein kinase A and phosphatidylinositol 3-kinase/Akt-dependent manner

AbstractIncreased risk of osteoporosis in patients with diabetes mellitus may be related to hyperglycemia. However, the potential mechanisms accounting for diabetic bone disorder remain unresolved. The present study investigated the effects of high glucose-associated osmolality on differentiation of primary rat calvarial osteoblasts. Osteoblastogenic differentiation was determined by bone nodule staining for mineralization assay, enzyme-linked immunosorbent assay for type I collagen production and real-time polymerase chain reaction (PCR) for osteoblastogenic marker gene expression. Adipocytogenic differentiation was assessed by oil red O staining for lipid accumulation and real-time PCR for adipocytogenic marker gene expression. The phosphorylations of protein kinase A (PKA) and Akt were measured with or without specific inhibitors to confirm osmolality involved signalling pathways. The results showed that high glucose-associated osmolality significantly promoted adipocytogenic differentiation, manifested by increased lipid droplet formation and gene expression of adipocytogenic markers including adipocyte fatty acid binding protein (aP2), adipsin and peroxisome proliferator-activated receptor gamma (PPARγ). Meanwhile, high glucose-associated osmolality inhibited osteoblastogenic differentiation, characterized by decreased collagen I protein production and cell mineralization, as well as gene expression of osteoblastogenic markers including collagen I, osteocalcin and runt-related transcription factor 2 (Runx2). More importantly, we demonstrated for the first time that high glucose-associated osmolality induced adipocytogenic differentiation and suppressed osteoblastogenic differentiation in a PKA and phosphatidylinositol 3-kinase (PI3K)/Akt-dependent manner. These results indicated that osmolality was involved in high glucose-induced osteoblast trans-differentiation into adipocyte-like cell and suppression of cellular osmolality could provide novel therapeutic approach for diabetic osteopenia.

Tetracycline-grafted PLGA nanoparticles as bone-targeting drug delivery system.

PurposeNanoparticles (NPs) that target bone tissue were developed using poly(lactic-co-glycolic acid) (PLGA) copolymers and tetracycline (TC)-based bone-targeting moieties. These NPs are expected to enable the transport of drugs, such as simvastatin (SIM), for the treatment of osteoporosis.MethodsThe molecular structures of TC–PLGA were validated by 1H-NMR, and the SIM-loaded NPs were prepared using the solvent emulsification method. The surface properties, cytotoxicity, cellular uptake, cell mineralization, bone targeting potential, and animal pharmacodynamics of the TC–PLGA NPs were evaluated and compared to those of PLGA NPs.ResultsIt was confirmed that the average particle size of the NPs was approximately 220 nm. In phosphate-buffered saline (PBS, pH 7.4), the SIM-loaded NPs exhibited a cumulative release of up to 80% within 72 hours. An in vitro cell evaluation indicated that the NPs had an excellent cellular uptake capacity and showed great biocompatibility with MC3T3-E1 cells, thereby reducing the cytotoxic effects of SIM. The cell mineralization assay showed that the SIM-loaded NPs induced osteogenic differentiation and mineralized nodule formation in MC3T3-E1 cells, thereby achieving the same effect as SIM. Preliminary findings from in vitro and in vivo bone affinity assays indicated that the TC–PLGA NPs may display increased bone-targeting efficiency compared to PLGA NPs lacking a TC moiety. The use of SIM-loaded TC–PLGA NPs in treating osteoporosis was tested through animal pharmacodynamics analyses performed in ovariectomized rats, and the results suggested that the SIM-loaded TC–PLGA NPs can improve the curative effects of SIM on the recovery of bone mineral density compared to either SIM-loaded PLGA NPs or SIM alone.ConclusionBone-targeting NPs, which were based on the conjugation of TC to PLGA copolymers, have the ability to target bone. These NPs may be developed as a delivery system for hydrophobic drugs, and they are expected to improve the curative effects of drugs, reduce the administered drug doses, and reduce side effects in other organs.

Production, Characterization and Bioactivity of 46S6 Bioactive Glass from Rice Hull Ash Silica

In the present study, 46S6 bioactive glasses with composition 46 SiO2% –24% Na2O – 24% CaO – 6% P2O5in weight percent were synthesized through melt-derived process using alkali extracted silica from rice hull ash and commercial silica. Bioactive glasses were incubated in SBF (simulated body fluid), before and after immersing in SBF, characterized by SEM to investigate degradation and apatite formation. Also bioactivity tests such as cell culture, cell viability and cytotoxicity, differentiation, mineralization and alkaline phosphatase assay have been performed. Results showed that 46S6 bioactive glasses from rice hull ash silica had better metabolic activity and viability than that obtained from commercial silica.

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

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

Microbial Community Composition, Functions, and Activities in the Gulf of Mexico 1 Year after the Deepwater Horizon Accident.

Several studies have assessed the effects of the released oil on microbes, either during or immediately after the Deepwater Horizon accident. However, little is known about the potential longer-term persistent effects on microbial communities and their functions. In this study, one water column station near the wellhead (3.78 km southwest of the wellhead), one water column reference station outside the affected area (37.77 km southeast of the wellhead), and deep-sea sediments near the wellhead (3.66 km southeast of the wellhead) were sampled 1 year after the capping of the well. In order to analyze microbial community composition, function, and activity, we used metagenomics, metatranscriptomics, and mineralization assays. Mineralization of hexadecane was significantly higher at the wellhead station at a depth of ∼1,200 m than at the reference station. Community composition based on taxonomical or functional data showed that the samples taken at a depth of ∼1,200 m were significantly more dissimilar between the stations than at other depths (surface, 100 m, 750 m, and >1,500 m). Both Bacteria and Archaea showed reduced activity at depths of ∼1,200 m when the wellhead station was compared to the reference station, and their activity was significantly higher in surficial sediments than in 10-cm sediments. Surficial sediments also harbored significantly different active genera than did 5- and 10-cm sediments. For the remaining microbial parameters assessed, no significant differences could be observed between the wellhead and reference stations and between surface and 5- to 10-cm-deep sediments.

Potential osteogenic activity of ethanolic extract and oxoflavidin isolated from Pholidota articulata Lindley

Ethnopharmacological relevancePholidota articulata Lindley (PA) locally known as Hadjojen (bone jointer) belongs to family Orchidaceae is used for healing fractures in folklore tradition of Kumaon region of Uttarakhand, Himalaya, India. Bone is a dynamic organ and is constantly being remodeled in order to facilitate growth and repair. This process requires the involvement of bone forming osteoblast and bone resorbing osteoclast cells, which function in generating and mineralizing bone, giving strength and rigidity to the skeletal system. Present study was aimed to determine the therapeutic potential of ethanolic extract of PA and its isolated compound oxoflavidin, by characterizing their fracture healing properties. Materials and methodsOvariectomized (Ovx) estrogen deficient adult female Balb/c mice were used for in vivo evaluation of osteogenic or bone healing potential of ethanolic extract of PA. Further, its isolated compounds were tested for their osteogenic efficacy using alkaline phosphatase assay and mineralization assay in vitro in mice calvarial osteoblasts. ResultsThe ethanolic extract of PA exhibited significant restoration of trabecular micro-architecture in both femoral and tibial bones. Additionally, treatment with PA extract led to better bone quality and devoid of any uterine estrogenicity in ovariectomized estrogen deficient mice. One of the isolated compound, oxoflavidin enhanced ALP activity (a marker of osteoblast differentiation), mineral nodule formation and mRNA levels of osteogenic markers like BMP-2, Type 1 Collagen, RUNX-2 and osteocalcin. ConclusionThese results warrant that ethanolic extract of PA and it’s pure compound oxoflavidin have fracture healing properties. The extract and oxoflavidin exhibit a strong threapeutical potential for the treatment and management of postmenopausal osteoporosis.

Identification of C-terminal Hsp70-interacting protein as a mediator of tumour necrosis factor action in osteoblast differentiation by targeting osterix for degradation.

In patients with inflammatory arthritis, tumour necrosis factor (TNF)-α are overproduced in inflamed joints. This leads to local erosion of cartilage and bone, periarticular osteopenia, as well as osteoporosis. But less is known regarding the molecular mechanisms that mediate the effect of TNF-α on osteoblast function. The purpose of this study was to test that C terminus of Hsc70-interacting protein (CHIP) has a specific role in suppressing the osteogenic activity of osteoblasts under inflammatory conditions. C2C12, MC3T3-E1 and HEK293T cell lines were cultured and cotransfected with related plasmids. After transfection, the cells were cultured further in the presence or absence of murine TNF-α and subjected to real time RT-PCR, Western blot, Ubiquitination assay, Co-immunoprecipitation, Luciferase reporter assay, Small interfering RNAs and Mineralization assay. The expression levels of TNF-α-induced CHIP and Osx were examined by RT-PCR and Western blot analysis. Co-immunoprecipitation and ubiquitination assays revealed ubiquitinated Osx, confirmed that CHIP indeed interacted with Osx and identified K55 and K386 residues as the ubiquitination sites in Osx, Luciferase reporter assay and Small interfering RNAs examined whether TNF-α target the bone morphogenetic protein signalling through CHIP. We established stable cell lines with the overexpression of HA-CHIP, Mineralization assay and CHIP siRNA demonstrated the important roles of CHIP on osteoblast function in conditions in which TNF-α is overexpressed. We found that the K55 and K386 residues are ubiquitination site(s) in Osx, and that TNF-α inhibits osteoblast differentiation by promoting Osx degradation through up-regulation of E3 ubiquitin ligase CHIP in osteoblast. Thus, CHIP targets Osx for ubiquitination and degradation in osteoblasts after chronic exposure to TNF-α, and inhibition of CHIP expression in osteoblasts may be a new mechanism to limit inflammation-mediated osteoporosis by promoting their differentiation into osteoblasts.

Mecoprop mineralization potential at oxygen-reduced conditions in subsoil with phenoxy acid contamination history

Historic phenoxy acid waste disposal and spillage at an industrial site close to Copenhagen (Denmark) has contaminated the groundwater with especially mecoprop (RS)-2-(4-chloro-2-methylphenoxy)propionic acid. The contamination is very persistent, probably because it is situated in the subsoil where biological degradation of the contaminant is hindered by lack of oxygen or alternative electron acceptors like nitrate. To examine this, we sampled soil at 3, 4.5 and 6 m depth at the contaminated site and measured the microbial mineralization of mecoprop as affected by nitrate and glucose at nitrate-reducing conditions.The results showed that at these conditions, glucose always had a clear negative impact on mecoprop mineralization. Mineralization of mecoprop was significantly enhanced in soil inoculated with phenoxy acid degraders (p < 0.001), in comparison to the non-inoculated controls. After a lag-phase of ∼7 weeks, up to 50% of initially added mecoprop was mineralized. However, the genomic DNA band pattern of numerically dominant isolates at the end of the experiment did not match with the inoculated strains. A mineralization assay confirmed that mecoprop was degraded by an indigenous microbial consortium.The findings in this study suggest that stimulation of indigenous microbes is a promising approach for removing persistent mecoprop contamination in subsoils.

NF-κB decoy oligodeoxynucleotide enhanced osteogenesis in mesenchymal stem cells exposed to polyethylene particle.

Excessive generation of wear particles after total joint replacement may lead to local inflammation and periprosthetic osteolysis. Modulation of the key transcription factor NF-κB in immune cells could potentially mitigate the osteolytic process. We previously showed that local delivery of ultrahigh-molecular-weight polyethylene (UHMWPE) particles recruited osteoprogenitor cells and reduced osteolysis. However, the biological effects of modulating the NF-κB signaling pathway on osteoprogenitor/mesenchymal stem cells (MSCs) remain unclear. Here we showed that decoy oligodeoxynucleotide (ODN) increased cell viability when primary murine MSCs were exposed to UHMWPE particles, but had no effects on cellular apoptosis. Decoy ODN increased transforming growth factor-beta 1 (TGF-β1) and osteoprotegerin (OPG) in MSCs exposed to UHMWPE particles. Mechanistic studies showed that decoy ODN upregulated OPG expression through a TGF-β1-dependent pathway. By measuring the alkaline phosphatase activity, osteocalcin levels, Runx2 and osteopontin expression, and performing a bone mineralization assay, we found that decoy ODN increased MSC osteogenic ability when the cells were exposed to UHMWPE particles. Furthermore, the cellular response to decoy ODN and UHMWPE particles with regard to cell phenotype, cell viability, and osteogenic ability was confirmed using primary human MSCs. Our results suggest that modulation of wear particle-induced inflammation by NF-κB decoy ODN had no adverse effects on MSCs and may potentially further mitigate periprosthetic osteolysis by protecting MSC viability and osteogenic ability.