Effects of genistein, daidzein and glycitein on the osteogenic and adipogenic differentiation of bone marrow stromal cells and on the adipogenic trans-differentiation of osteoblasts

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), alkaline phosphatase (ALP) activity and oil red O assays were used to examine the effects of genistein, daidzein and glycitein on the osteogenic and adipogenic differentiation of primary mouse bone marrow stromal cells (MSCs) and the adipogenic trans-differentiation of primary mouse osteoblasts. The results indicated that daidzein, genistein and glycitein at concentrations from 1×10^-8 mol/L to 1×10^-5 mol/L promoted the proliferation of MSCs and osteoblasts; genistein, daidzein and glycitein promoted osteogenic differentiation and inhibited adipogenic differentiation of MSCs, and inhibited adipocytic transdifferentiation of osteoblasts at appropriate concentrations as 17β-estradiol. It suggests that genistein, daidzein and glycitein regulate a dual differentiational process of MSCs into the osteogenic and adipogenic lineages, and trans-differentiational process of primary osteoblasts into the adipocyte lineages, causing a lineage shift toward osteoblast. Protective effects of them on bone may be mediated by a reversal of adipogenesis which may promote the proliferation, differentiation and mineralization of osteoblasts, and make adipocytes secrete less cytokines which may promote osteoclast formation and activation. In addition, the results also indicated that genistein, daidzein and glycitein may be helpful in preventing the development of steroid induced osteonecrosis.     

Effects of lanthanum and gadolinium on proliferation and differentiation of primary osteoblasts

The effects of La3 and Gd3 on the proliferation, differentiation and adipogenic transdifferentiation of rat calvarial osteoblast-like cells (ROB cells) were evaluated by MTT method, measuring the activity of alkaline phosphatase (ALP) and Oil red O measurement. Both of La3 and Gd3 inhibited the proliferation of ROB cells at all concentrations (1×10-5, 1×10-6, 1×10-7, 1×10-8, 1×10-9 mol·L-1). La3 at concentration of 1×10-5mol·L-1 significantly increased the alkaline phosphatase activity of ROB cells up to 3 folds (P<0.01). However, the effects reversed to inhibit at other concentrations. Gd played a negative role in the alkaline phosphatase activity. La3 inhibited the adipogenic transdifferentiation of ROB cells at all concentrations in a dose-dependent way. However, Gd promoted the adipogenic transdifferentiation of ROB cells at 1×10 and 1×10 mol·L . These findings suggested that the effects of rare earth elements on the proliferation, differentiation and adipogenic transdifferentiation of ROB cells were dependent on their concentrations and species.     

Electrospun Nanofibers of Hydroxyapatite/Collagen/Chitosan Promote Osteogenic Differentiation of BMSCs

Bone tissue engineering, aiming at developing bone substitutes for repair and regeneration of bone defects instead of using autologous bone grafts, has attracted wide attention in the field of tissue engineering and regenerative medicine. Developing biomimetic biomaterial scaffolds able to regulate osteogenic differentiation of stem cells could be a promising strategy to improve the therapeutic efficacy. In this study, clectrospun composite nanofibers of hydroxyapatite/collagen/chitosan （ HAp/Col/CTS ） resembling the fibrous nanostructure and constituents of the hierarchically organized natural bone, were prepared to investigate their capacity for promoting bone mesenchymal stem cells （BMSCs） to differentiate into the osteogenic lineage in the absence and presence of the osteogenlc supplementation, respectively. Call morphology, proliferation and quantified specific osteogenic protein expression on the electrospun HAp/Coi/CTS scaffolds were evaluated in comparison with different controls including dectrospun nanofibrous CTS, HAp/CTS and tissue culture plate. Our remits showed that the nanofibrous HAp/Col/CTS scaffolds supported better spreading and proliferation of the BMSCs than other substrates （ P 〈 0.01 ）. Expressions of osteogenesis protein markers, alkaline phosphatase （ALP） and Col, were significantly upregulated on the HAp/Col/CTS than those on the CTS （P 〈0.01） and HAp/ CTS （P 〈 0. 05 ） scaffolds in the absence of the osteogeulc supplementation. Moreover, presence of osteogeulc supplementation also proved to enhance osteogeule differentiation of BMSCs on HAp/ Col/CTS scaffolds, indicative of a synergistic effect. This study highlights the potential of BMSCs/HAp/Col/CTS cell-scaffold system for functional bone repair and regeneration applications.     

Notch signaling stimulates osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

Notch signaling is one of the most important pathways mediating cell determination and differentiation.In this study, the roles of Notch signal in the regulation of osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs) were investigated. The expression of Notch1, Jaggedl and DTXI detected by reverse transcrip-tion polymerase chain reaction (RT-PCR) suggested that Notch signal might exhibit a physiological regulatory role in the differentiation of MSCs. Constitutive expression of the intracellular domain of Notchl (ICN), the active form of Notchl protein, can activate Notch signal in cells without ligands‘ binding, hMSCs were isolated, expanded, and infected with retrovirus carrying green fluorescent protein (GFP) gene or ICN. Overexpression of ICN in hMSCs resulted in enhanced osteogenic differentiation induced by dexamethasone (Dex), which was characterized by an increase of cellular alkaline phosphatase (ALP) activity and calcium deposition. These results indicate that Notch stimulates differentiation of MSCs into osteoblasts.     

Effects of lanthanum and gadolinium on proliferation and differentiation of primary osteoblasts

The effects of La³⁺ and Gd³⁺ on the proliferation, differentiation and adipogenic transdifferentiation of rat calvarial osteoblast-like cells (ROB cells) were evaluated by MTT method， measuring the activity of alkaline phosphatase (ALP) and Oil red O measurement. Both of La³⁺ and Gd³⁺ inhibited the proliferation of ROB cells at all concentrations (1×10^-5, 1×10^-6, 1×10^-7, 1×10^-8, 1×10^-9 mol?L^-1). La³⁺ at concentration of 1×10^-5 mol?L^-1 significantly increased the alkaline phosphatase activity of ROB cells up to 3 folds (P<0.01). However, the effects reversed to inhibit at other concentrations. Gd³⁺ played a negative role in the alkaline phosphatase activity. La³⁺ inhibited the adipogenic transdifferentiation of ROB cells at all concentrations in a dose-dependent way. However, Gd³⁺ promoted the adipogenic transdifferentiation of ROB cells at 1×10^-8 and 1×10^-9 mol?L^-1. These findings suggested that the effects of rare earth elements on the proliferation, differentiation and adipogenic transdifferentiation of ROB cells were dependent on their concentrations and species     

Mineralized Composite Nanofibrous Mats for Bone Tissue Engineering

Composite nanofibrous mats consisting of poly( L-lactideco-ε-caprolactone)( PLCL) and collagen type I( COL) were fabricated by electrospinning,and ten times simulated body fluid(10SBF) were employed to mineralize nanofibrous mats. Ballshaped hydroxyapatite( HA) was deposited on the surface of nanofibrous mats in 1. 5 h at room temperature. Human fetal osteoblasts( hFob) were seeded to investigate their proliferation and differentiation on mineralized composite nanofibrous mats. The results showed that hFob grew well on mineralized composite nanofibrous mats and alkaline phosphatase( ALP) activity of hFob on mineralized composite nanofibrous mats at 14 d was much higher than that on untreated nanofibrous mats. Moreover,the expression of osteocalcin of cells on mineralized composite nanofibrous mats was also much higher than those on untreated nanofibrous mats at 7 d and 14 d. This mineralized composite nanofibrous mats may have a great potential for bone tissue engineering.     

Optimization of dual effects of Mg– 1Ca alloys on the behavior of chondrocytes and osteoblasts in vitro

Mg ions can enhance the proliferation and redifferentiation of chondrocytes and the osteogenic differentiation of osteoblasts at specific concentrations, respectively. However, degradation of Mg alloys at varying degradation rates could lead to complex changes in the surrounding tissue environment, such as changes in the dynamic concentration of Mg ions and subsequent p H value. Considering the above mentioned factors, the comprehensive effects of Mg alloys on chondrocytes and osteoblasts behaviors have not yet been optimized. In this study, we evaluated the effects of Mg–1Ca microspheres on cell behavior with an aim to optimize conditions favorable for both cell types. Cells were cultured with Mg–1Ca microspheres prepared using the following concentrations: 250 μg/ml, 500 μg/ml and 1000 μg/ml. At specific time points,cytotoxicity, expression of specific genes and extracellular matrix deposition by cells(Alizarin Red Staining of osteoblasts and Alcian blue staining for chondrocytes) were evaluated. The experimental results revealed that Mg–1Ca microspheres prepared at a concentration of 250 μg/ml were optimum for both cell types, where chondrocytes were found to be in hypertrophy state while osteoblasts in close proximity to the microspheres showed osteogenetic differentiation. Interestingly, a slight change in osteoblasts behavior was observed nearer to and at a relative distance away from Mg–1Ca microspheres, an important observation for administering the application of microspheres as potential scaffolds.     

Physiological responses of osteoblasts to cyclic stretching and the change of intracellular calcium concentration

The development of bone tissues is regulated by mechanical stimulation.Cyclic stretching was applied to the osteoblasts that were delivered from rat calvarie,The results showed that stretching at 500με increased the cell proliferation while loading at 1000με and 1500με inhabited cell growth ,Loading also increased the adhesive force between cells and substrate as well as spreading areas of osteobalsts.Furthermore,the fluorescence probe Fluo-3/AM was used to investigate the effect of stretching stimulation on the intracellular calcium concentration of osteoblasts.The intracellular calcium concentration of osteoblasts that were stretched at 500 με for 5 min was 92.9% higher than the control ,After being treated with the panax ontoginseng saponins,the streteched osteoblasts still expressed 28.6% higher intracellular calcium concentration than that of the control ,which proved that both the influx of extracellualr calcium and the release of intracellular calcium store were involved in the increase of intracellular calcium concentration when osteoblasts responded to the cyclic stretching And the influx of extracellular calcium through transmembrance channel played a main role.     

Induction of embryonic stem cells to hematopoietic cellsin vitro

In order to get hematopoietic cells from embryonic stem (ES) cells and to study development mechanisms of hematopoietic cells, the method of inducing embryonic stem cells to hematopoietic cells was explored by differenciating mouse ES cells and human embryonic cells in three stages. The differentiated cells were identified by flow cytometry, immunohistochemistry and Wright’s staining. The results showed that embryoid bodies (EBs) could form when ES cells were cultured in the medium with 2-mercaptoethanol (2-ME). However, cytokines, such as stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), interleukin-6 (IL-6), erythropoietin (EPO) and granular colony stimulating factor (G-CSF), were not helpful for forming EBs. SCF, TPO and embryonic cell conditional medium were useful for the differentiation of mouse EBs to hematopoietic progenitors. Eighty-six percent of these cells were CD34⁺ after 6-d culture. Hematopoietic progenitors differentiated to B lymphocytes when they were cocultured with primary bone marrow stroma cells in the DMEM medium with SCF and IL-6. 14 d later, most of the cells were CD34^－CD38⁺. Wright’s staining and immunohistochemistry showed that 80% of these cells were plasma-like morphologically and immunoglubolin positive. The study of hematopoietic cells from human embryonic cells showed that human embryonic cell differentiation was very similar to that of mouse ES cells. They could form EBs in the first stage and the CD34 positive cells account for about 48.5% in the second stage.     

Effect of E. coli coli on Anti-disease Activities of Scailops: Argopecten irradians

The effect of acute E. coli challenge on the anti-disease activity of scallops Argopecten irra-dians is examined. The treatments of scallop from which hemolymph samples were taken for study included (1) control scallops, (2) sham-injected scallops, (3) PSW-injected scallops and (4) E. coli-injected scallops. From the beginning, the anti-disease activities of scallops are deter -mined at 12 hr and 24 hr.The concentrations of circulating hemocytes, the total serum protein concentrations and the activities of alkaline phosphatase, acid phosphatase and superoxide dismutase in the scallops Argopecten irradians are determined.Injection with E. coli results in a significant elevation in the concentration of circulating hemocytes and in the alkaline phosphatase activity and a significant decline in the total serum protein concentration and in the superoxide dismutase activity at 24 hr postchallenge. It shows that metabolism of bay scallop is expedited to adopt the challenge.     

Effects of Quenching Temperature and Time on Pore Diameter of Poly（3-hydroxybutyrate-co-3-hydroxyhexanoate） Porous Scaffolds and MC3T3-E1 Osteoblast Response to the Scaffolds

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) scaffolds were prepared by thermally inducing phase separation (TIPS) for bone reconstruction. Scanning electron microscopy and porosity measurements were used to analyze the structure and properties of the scaffolds. The pore diameter of the scaffolds could be easily controlled by changing the quenching temperature and time. The biocompatibility was assessed by examining the proliferation and morphology of MC 3T3-E1 osteoprogenitor cells seeded on the scaffolds. Cultures grown in the presence of a source of phosphate ions showed the formation of a mineralized extracellular matrix. The results indicate that PHBHHx scaffolds prepared using TIPS are a promising candidate for bone reconstruction.     

Calcium carbonate nanoparticles promote osteogenesis compared to adipogenesis in human bone-marrow mesenchymal stem cells

Rhombohedron-like and fusiform calcium carbonate nanoparticles were fabricated using a new method. Their geometry was controlled by varying the mixing speed and ratio of ethanol versus water in reaction system. The calcium carbonate nanoparticles(CCNPs) have slight effect on viability of human bone-marrow mesenchymal stem cells(hBMSCs) with dose-dependent and shape-dependent, but they can significantly promote osteogenic differentiation of hBMSCs in vitro by 10–37% increase of alkaline phosphatase(ALP) activity, 9–36% growth of collagen secretion and 1.13–1.83 folds upregulation of osteogenesis-related genes, even at lower dose ranges(5–20 μg/ml). The efficacity of promoting osteogenesis depends on the shape and dose of CCNPs. Furthermore,adipogenesis was inhibited by less accumulation of lipid droplets, lower triglyceride(TG) secretion and downregulation of adipogenesis-related genes. These findings improve the understanding of effects CCNPs on hBMSCs fate towards osteoblasts or adipocytes and have meaningful impact for combining use of CCNPs and hBMSCs in tissue engineering and regenerative medicine fields.     

Inhibition of bromophenols against PTP1B and anti-hyperglycemic effect of Rhodomela confervoides extract in diabetic rats

Protein tyrosine phosphatase 1B (PTP1B) plays an important role as a negative regulator in insulin signaling pathways. PTP1B is an effective target for the treatment of type 2 diabetes mellitus. Four bromophenol derivatives from red algae Rhodomela confervoides, 2,2′,3,3′-tetrabromo-4,4′,5,5′-tetra- hydroxydiphenyl methane (1), 3-bormo-4,5-bis(2,3-dibromo-4,5-dihydroxybenzyl) pyrocatechol (2), bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (3) and 2,2′,3-tribromo-3′,4,4′,5-tetrahydroxy-6′-ethyloxy- methyldiphenylmethane (4) showed significant inhibitory activity against PTP1B (IC50 were 2.4, 1.7, 1.5 and 0.84 μmol/L, respectively) as potential therapeutical agents for the treatment of type 2 diabetes mellitus. The anti-hyperglycemic effects of the ethanol extracts from R. confervoides on streptozoto- cin-induced diabetes (STZ-diabetes) in male Wistar rats fed with high fat diet were investigated. The STZ-diabetic rats treated with medium-dose and high-dose alga extracts showed remarkable reductions in fasting blood glucose (FBG) as compared with the STZ-diabetic control. The results indicate that the in vivo anti-hyperglycemic activity of the R. confervoides extracts can be partially attributed to the in- hibitory actions against PTP1B of the bromophenol derivatives and that may be of clinical importance in improving the management of type 2 diabetes mellitus.     

Arbuscular mycorrhizal formation of crucifer leaf mustard induced by flavonoids apigenin and daidzein

Flavonoids from legume root secretion may probably act as signal molecules for expression of Rhizobial “nod” nodulation genes and AM fungal symbiotic gene. Leaf mustard is a non-mycorrhizal plant; it does not contain flavonoids and other signal molecules. AM fungi could not infect the roots of leaf mustard and form a symbiont in nature,when it was treated with flavonoids (apigenin or daidzein).The results of trypan blue staining showed that two kinds of AM fungi (G intraradices and G mosseae) successfully infected the roots of non-mycorrhizal plant leaf mustard. AM fungi grew towards and colonized the roots of leaf mustard,producing young spores and completing the course of life.AM fungi are the only one kind of fungi with ALP activity.The result of ALP staining has also proved that AM fungi infected successfully the roots of leaf mustard. AM fungi (G intraradices and G mosseae) that existed in the roots of non-mycorrhizal plant leaf mustard were probed by nested PCR and special molecular probes. The above-mentioned proof chains have fully proved that flavonoids induced AM fungi (G intraradices and G mosseae) to infect non-mycorrhizal plant and establish symbiotic relationship.     

Maturation of Osteoblast-Like Saos-2 on Hydroxyapatite Ceramics

Hydroxyapatite ceramics (.HA) has been proved to be excellent in biocompatibility and bioactivity.However, limited information is available concerning how HA ceramics affects the maturation of es-tcoblasts in molecular biological level /n v/tro. This study examines the mRNA expression and protein production of hone-related genes in estcoblast-like cell line(Saos-2) cultured on HA disks. Saos-2 cells are seeded onto the substrates and cultured for 18 days. Harvested cells are tested for the cell growth rate, expression of mRNAs for esteocalcin and alkaline phosphatase, and protein production of bone sialo-protein and esteocalcin. MTS assay shows that cell proliferates well on HA ceramic substrate. After 9d,bone sialoprotein and esteocalcin protein production in SAPS-2 increases more on HA surfaces than on control material. As bone sialoprotein and esteocalcin are the genes to be highly expressed at the late stage of estcoblast differentiation, this study reveals that after long time culture in HA, HA can induce Saos-2 maturation. The behavior of Saos-2 on HA surfaces revealed in this study provides valuable infor-mation for the understanding of the biocompatibility and bioactivity of HA ceramics.     

Partial Purification and Properties of an Acid Phosphatase from Pearl Oyster Pinctada Fucata

IntroductionAcid phosphatase ( ACP,EC 3.1 .3.2 ) andalkaline phosphatase ( ALP,EC 3.1 .3.1 ) havebeen found to exist widely in the biosphere frombacteria to plants,animals and humans[18] .Thetwo enzymes catalyze the hydrolysis of variousphosphate containing compounds and act astransphosphorylases at acid- and alkaline- p Hvalue.In our lab,an alkaline phosphatase fromthe pearl oyster Pinctada fucata has been purifiedand some of its kinetics has been studied[9] .　Acid phosphatases actas ma…     

The Effect of Cyclic Stretching on Matrix Production, Mineralization and Differentiation of Osteoblasts

A four-point bending apparatus is used to investigate the effects of stretching on collagen synthesis, mineralization and differentiation of osteoblasts. Cells are stretched at 1500με for 24 hours. The responses of osteoblasts to mechanical signal of physiological stretching are evaluatedfrom three aspects: collagen production, extracellular inorganic calcium secretion and ALP activity. The results show that osteoblasts decrease the collagen synthesis, calcium secretion and ALP activity compared to the control cells (65.82 %, 73.51%, 48.10 96 respectively ), confirming that cyclic stretching at 1500με inhabits the ohvsiological activity of osteoblasts.     

Phase modification and dielectric properties of a cullet-paper ash-kaolin clay-based ceramic

Novel ceramics from waste material made of (x) paper ash-(80-x) cullet-20 kaolin clay (10wt% ≤ x ≤ 30wt%) were successfully synthesized using a conventional solid-state reaction technique. Energy-dispersive X-ray analysis confirmed the presence of Si, Ca, Al, and Fe in the waste material for preparing these ceramics. The influence of the cullet content on the phase structures and the dielectric properties of these ceramics were systematically investigated. The impedance spectra were verified in the range from 1 Hz to 10 MHz at room temperature. The phase of the ceramics was found to primarily consist of wollastonite (CaSiO₃), along with minor phases of γ-dicalcium silicate (Ca₂SiO₄) and quartz (SiO₂). The sample with a cullet content of 55wt% possessed the optimum wollastonite structure and exhibited good dielectric properties. An increase of the cullet content beyond 55wt% resulted in a structural change from wollastonite to dicalcium silicate, a decrease in dielectric constant, and an increase in dielectric loss. All experimental results suggested that these novel ceramics from waste are applicable for electronic devices.>     

Preparation and oxidation characteristics of ZrC-ZrB2 composite powders with different proportions

ZrC and ZrB2 are both typical ultra-high temperature ceramics,which can be used in the hyperthermal environment.In this study,a method for preparing ultrafine ZrC-ZrB2 composite powder was provided,by using the raw materials of nano ZrO2,carbon black,B4C,and metallic Ca.It is worth pointing out that ZrC-ZrB2 composite powder with any proportion of ZrC to ZrB2 could be synthesized by this method.Firstly,a mixture of ZrC and C was prepared by carbothermal reduction of ZrO2.By adjusting the addition amount of B4C,ZrC was boronized by B4C to generate ZrC-ZrB2 composite powder with different compositions.Using this method,five composite powders with different molar ratios of ZrC and ZrB2(100ZrC,75ZrC-25ZrB2,50ZrC-50ZrB2,25ZrC-75ZrB2,and 100ZrB2)were prepared.When the temperature of boronization and decarburization process was 1473 K,the particle size of product was only tens of nanometres.Finally,the oxidation charac-teristics of different composite powders were investigated through oxidation experiments.The oxidation resistance of ZrC-ZrB2 composite powder continued to increase as the content of ZrB2 increased.