Inhibitory effect of alcohol on osteogenic differentiation in human bone marrow-derived mesenchymal stem cells

BACKGROUND: Alcohol-induced osteoporosis is characterized by a considerable suppression of osteogenesis. The objective of this investigation was to determine the effect of alcohol on gene expression, protein synthesis, and mineralization in human bone marrow-derived mesenchymal stem cells induced toward osteogenic differentiation in vitro. METHODS: Human bone marrow-derived mesenchymal stem cells induced toward osteogenesis were cultured in the presence or absence of 50 mM alcohol. Stem cells were characterized by using SH2 antibody to the cell-surface antigen CD105/endoglin, and their proliferation in the presence of alcohol was quantified. The expression of genes for early, middle, and late markers of the osteogenic lineage was quantified by Northern analysis, and bone matrix protein synthesis was assayed. The effect of alcohol on cell-mediated matrix mineralization in terminally differentiated cultures was determined by von Kossa staining. RESULTS: Fluorescence-activated cell sorting analysis of human mesenchymal stem cells separated with a Percoll gradient proved 99% homogeneity by using SH2 antibody to the surface antigen CD105. Dose-dependent inhibition of proliferation of these stem cells occurred at concentrations greater than 50 mM alcohol. Gene expression of osteoblast-specific factor 2/core binding factor a1 (Osf2/Cbfa1), type I collagen, alkaline phosphatase, and osteocalcin (early, middle, and late markers for osteogenesis, respectively) was analyzed with and without osteogenic induction and treatment with 50 mM alcohol. After induction, Osf2/Cbfa1 levels were unresponsive to alcohol. To determine the effect of alcohol on human mesenchymal stem cell progression along the osteogenic pathway, messenger RNA (mRNA) levels for type I collagen, alkaline phosphatase, and osteocalcin were examined after osteogenic induction. After osteogenic induction, alcohol down-regulated the gene expression of type I collagen and significantly reduced its synthesis. Alcohol did not alter mRNA expression of alkaline phosphatase, a midstage marker for osteogenesis, but significantly decreased its activity compared with osteogenic induction alone. After induction, osteocalcin remained unchanged by alcohol at both the mRNA and protein levels. Histochemistry revealed decreased alkaline phosphatase staining and fewer alkaline phosphatase-positive cells in alcohol-treated human mesenchymal stem cell cultures. von Kossa staining revealed a reduction in the number of mineralizing nodules in stem cell cultures after alcohol treatment. CONCLUSIONS: Collectively, the data suggest that alcohol alters osteogenic differentiation in human bone marrow-derived mesenchymal stem cell cultures during lineage progression and provide further insight into alcohol-induced reduced bone formation.     

The aminobisphosphonate risedronate preserves localized mineral and material properties of bone in the presence of glucocorticoids

OBJECTIVE: Glucocorticoids (GCs) alter bone strength such that patients receiving these medications have a high rate of fragility-related fractures. The purpose of this study was to assess whether concurrent treatment with GCs (prednisolone) and risedronate (an aminobisphosphonate) would prevent the reduction in bone strength induced by GCs, in a mouse model of GC-induced bone loss and in patients enrolled in a clinical study. METHODS: We evaluated mice treated with prednisolone pellets alone, GCs plus risedronate, or placebo alone and iliac crest biopsy specimens obtained from patients who were treated with GCs plus placebo or GCs plus risedronate for 1 year. We measured the mass, architecture, and physical and material properties of bone (subject to therapeutic treatments) at nanoscale to macroscopic dimensions, using synchrotron x-ray tomography, elastic modulus mapping, transmission electron microscopy, and small-angle x-ray scattering techniques. RESULTS: GC treatment reduced trabecular bone mass, microarchitecture, and the degree of bone mineralization and elastic modulus within the trabeculae. Concurrent treatment with GCs and risedronate prevented the deterioration of trabecular bone architecture, reduced the degree of mineralization, and preserved elastic modulus within the trabeculae, in both mouse and human bone. In addition, treatment with risedronate plus GCs in mice appeared to preserve bone crystal orientation, compared with treatment with GCs alone. CONCLUSION: Risedronate prevented the localized changes in mineral and material properties of bone induced by GCs, which may ultimately improve bone strength.     

Scaffold-free Three-dimensional Graft From Autologous Adipose-derived Stem Cells for Large Bone Defect Reconstruction: Clinical Proof of Concept

Long bone nonunion in the context of congenital pseudarthrosis or carcinologic resection (with intercalary bone allograft implantation) is one of the most challenging pathologies in pediatric orthopedics. Autologous cancellous bone remains the gold standard in this context of long bone nonunion reconstruction, but with several clinical limitations. We then assessed the feasibility and safety of human autologous scaffold-free osteogenic 3-dimensional (3D) graft (derived from autologous adipose-derived stem cells [ASCs]) to cure a bone nonunion in extreme clinical and pathophysiological conditions.Human ASCs (obtained from subcutaneous adipose tissue of 6 patients and expanded up to passage 4) were incubated in osteogenic media and supplemented with demineralized bone matrix to obtain the scaffold-free 3D osteogenic structure as confirmed in vitro by histomorphometry for osteogenesis and mineralization. The 3D “bone-like” structure was finally transplanted for 3 patients with bone tumor and 3 patients with bone pseudarthrosis (2 congenital, 1 acquired) to assess the clinical feasibility, safety, and efficacy. Although minor clones with structural aberrations (aneuploidies, such as tri or tetraploidies or clonal trisomy 7 in 6%–20% of cells) were detected in the undifferentiated ASCs at passage 4, the osteogenic differentiation significantly reduced these clonal anomalies. The final osteogenic product was stable, did not rupture with forceps manipulation, did not induce donor site morbidity, and was easily implanted directly into the bone defect. No acute (<3 mo) side effects, such as impaired wound healing, pain, inflammatory reaction, and infection, or long-term side effects, such as tumor development, were associated with the graft up to 4 years after transplantation.We report for the first time that autologous ASC can be fully differentiated into a 3D osteogenic-like implant without any scaffold. We demonstrated that this engineered tissue can safely promote osteogenesis in extreme conditions of bone nonunions with minor donor site morbidity and no oncological side effects.     

The modulation of placental lactogen release by opioids: a role for extracellular calcium

We previously reported that kappa opioids stimulated the release of human placental lactogen (hPL) from trophoblastic cells and that this effect was prevented by co-incubation with naloxone. We also reported that adenylate cyclase was not directly involved in this process. In order to understand the post-receptor events mediating hPL release by opioids in the human placenta, we studied the role of extracellular calcium. Human trophoblastic cells obtained by trypsin digestion were cultured for 48 h in Ham's F-10 medium supplemented with 10% fetal bovine serum (FBS), 200 U/ml penicillin, and 200 μg/ml streptomycin. ⁴⁵Ca²⁺ influx was then measured by filtration on glass-fiber filters. We observed a time- and dose-dependent stimulation of ⁴⁵Ca²⁺ influx by ethylketocyclazocine (EKC) with an EC₅₀ of 0.5 nM and a maximal stimulation of 196% over control. This effect was completely blocked by naloxone, a non-specific opioid antagonist, and by nor-binaltorphimine, a specific kappa antagonist. We also demonstrated that U-50, 488 (kappa agonist) had the same stimulatory effect as EKC (221 ± 25% of control). D-Ala²,NMe-Phe⁴,Gly-ol⁵)-enkephalin (DAGO) (mu agonist) slightly stimulated Ca²⁺ influx (128 ± 5% of control, p > 0.05) whereas D-Ser²,Leu, Thr⁶)-enkephaIin (DSLET) (delta agonist) had no effect. Pre-incubation of trophoblastic cells with pertussis toxin (PTX) did not affect the EKC-induced ⁴⁵Ca²⁺ influx, suggesting that this placental opiate effect is not coupled with PTX-sensitive G proteins. The role of extracellular calcium in the regulation of hPL release by kappa opiates was confirmed by experiments in which ionomycin (ionophore) and Bay K 8644 (calcium-channel agonist) mimicked the stimulatory effect of EKC on hPL release. Also, without extracellular Ca²⁺, the basal (control) and EKC-stimulated hPL release were significantly lower than with 1.8 mM Ca²⁺. Moreover, nifedipine (calcium-channel antagonist) inhibited both basal and EKC-stimulated hPL release. These results suggest that calcium influx is one mechanism mediating opioid-stimulated hPL release by human term trophoblastic cells.     

PI3K／Akt信号通路在骨髓间充质干细胞增殖及成骨分化调控中的作用

目的：研究PI3K/Akt信号通路抑制剂LY294002对骨髓间充质干细胞（mesenchymal stem cells,MSCs）增殖及分化的影响。方法采用贴壁法体外分离人骨髓间充质干细胞（hMSCs）,加入PI3K抑制剂LY294002（1、10μmol/L）,应用MTT法测定细胞增殖,常规成骨诱导分化培养3或7d,采用碱性磷酸酶（ALP）染色观察成骨分化水平,化学比色法测定ALP活性,茜素红染色后观察矿化钙结节数量并定量分析,Westernblot检测磷酸化Akt蛋白表达,应用Realtime-PCR检测各组细胞BMP2、Runx2、OPN及Osterix等成骨分化标记物的基因表达水平。结果从24至72h,LY294002对hMSCs增殖均产生显著抑制,随时间推延,可见抑制增殖效果增强（P＜0.05）。ALP染色和定量测定提示10μmol/L的ALP活性最强,在不同时间显著高于对照组和1μmol/L组（P＜0.05）。成骨诱导培养3和7d,1、10μmol/L组矿化量都显著高于对照组（P＜0.05）。10μmol/L组矿化量在成骨诱导7d也显著高于1μmol/L组（P＜0.05）。Westernblot检测结果证实成骨诱导可激活Akt磷酸化蛋白表达,但LY294002可抑制该蛋白磷酸化。成骨诱导分化7d,1、10μmol/L均明显促进BMP2、Runx2、OPN、Osterix4种基因mRNA表达（均P＜0.05）。结论PI3K/Akt信号通路参与hMSCs增殖和分化过程。成骨分化伴随下游Akt蛋白表达。PI3K抑制剂可抑制hMSCs增殖,但同时促进其向成骨分化和矿化。     

Estrogen deficiency does not decrease the in vitro osteogenic potential of rat adipose-derived mesenchymal stem cells

Osteoporosis due to estrogen deficiency is an increasing bone health issue worldwide: new strategies are being studied for regenerative medicine of bone pathologies in these patients. The most commonly used cells for tissue engineering therapy are the bone marrow mesenchymal stem cells (BMSCs), but they might be negatively affected by aging and estrogen deficiency. Besides the general advantages of adipose-derived mesenchymal stem cells (ADSCs) over BMSCs, ADSCs also seem to be less affected by aging than BMSCs, but in the literature, little is known about ADSCs in estrogen deficiency. The present study investigated the in vitro behavior of ADSCs, isolated from healthy (SHAM) and estrogen-deficient (OVX) rats. Phenotype, clonogenicity, viability, and osteogenic differentiation, at both cellular and molecular levels, were evaluated with or without osteogenic stimuli. Pro-inflammatory cytokines, growth factors, and adipogenic differentiation markers were also analyzed. There were no significant differences between OVX and SHAM ADSCs in some analyzed parameters. In addition, clonogenicity, osteopontin (Spp1) gene expression, alkaline phosphatase (ALP) activity at 2 weeks of culture, total collagen (COLL), osteocalcin (Bglap) gene expression and production, and matrix mineralization were significantly higher in OVX than in SHAM ADSCs. Besides the increase in some osteogenic markers, peroxisome proliferator-activated receptor gamma (Pparg) gene was also more expressed in OVX in osteogenic medium, with a concomitant estrogen receptor 1 (Esr1) gene expression decrease. These results underlined that ADSCs were not affected by estrogen deficiency in an osteogenic microenvironment.     

Role of calponin in the generation and differentiation of blood vessels - increased bone formation in mice lacking smooth muscle calponin -

Mice lacking smooth muscle calponin (also called basic calponin or calponin h1) expression displayed enhanced entopic bone formation in vivo, induced by recombinant human bone morphogenetic protein-2 (rhBMP-2) and an augmentation of the degree of osteoblastic differentiation of embryonic mesenchymal cells when they were stimulated by rhBMP-2. Calponin messenger RNA was shown to be expressed in developing and healing bone tissues. An examination of the skeletons of mutated mice showed increased postnatal bone formation characterized by an increase in the number of activated periosteal osteoblasts. These data revealed that calponin plays an important role in regulating the osteogenic differentiation of mesenchymal cells.     

High density lipoproteins stimulate placental lactogen release and adenosine 3',5'-monophosphate (cAMP) production in human trophoblast cells: evidence for cAMP as a second messenger in human placental lactogen release

Recent studies from our laboratory indicate that high density lipoproteins (HDL) and apolipoproteins-AI, -AII, and -CI stimulate the release of human placental lactogen (hPL) from human placental explants and trophoblast cells. However, the intracellular mechanisms by which HDL stimulate hPL release are unknown. Since earlier investigations from our laboratory indicated that pharmacological agents that increase intracellular cAMP levels [such as (Bu)2cAMP, isobutylmethylxanthine, and cholera toxin] also stimulate hPL release, we have performed studies to determine whether the stimulation of hPL release by HDL may be mediated by cAMP. Exposure of an enriched fraction of enzymatically dispersed hPL-producing cells to HDL (10-1500 micrograms/ml) for 0.5 h resulted in a significant dose-dependent stimulation of both cAMP production and hPL release. The half-maximal effective HDL dose for cAMP production and hPL release was 60-100 micrograms/ml. When perifused trophoblast cells were exposed to HDL (150 micrograms/ml) for 25 min, the increase in cAMP production preceded the increase in hPL release. The increase in cAMP production occurred during the first 5 min of exposure to HDL and reached a maximum (12.1 +/- 1.6-fold greater than base-line levels) 10 min after exposure. The increase in hPL release occurred about 10 min after the initial increase in cAMP production, reaching a maximum (9.7 +/- 1.4-fold greater than base-line levels) 15 min after exposure to HDL. HDL (300 micrograms/ml) also stimulated a 2.7-fold (P less than 0.001) increase in adenylate cyclase activity in placental plasma membranes, a magnitude of stimulation comparable to that caused by epinephrine (1 mM). Apolipoprotein-AII stimulated cAMP formation and hPL release in trophoblast cells with a pattern identical to that of HDL. These results, the first to demonstrate an effect of HDL on adenylate cyclase activity and cAMP production, strongly suggest a role for cAMP as a second messenger in HDL-stimulated hPL release.     

Biological effects of melatonin on osteoblast/osteoclast cocultures,bone, and quality of life: Implications of a role for MT2 melatonin receptors,MEK1/2, and MEK5 in melatonin‐mediated osteoblastogenesis

The Melatonin Osteoporosis Prevention Study (MOPS) demonstrated that nightly melatonin resulted in a time‐dependent decrease in equilibrium ratios of serum osteoclasts and osteoblasts in perimenopausal women. This study examines mechanisms related to the ratios of osteoblasts and osteoclasts using coculture models (transwell or layered) of human mesenchymal stem cell (MSC) and human peripheral blood monocytes (PBMCs). Human MSC/PBMC cocultures exposed to melatonin in osteogenic (OS+) medium for 21 days induced osteoblast differentiation and mineralization; however, only in layered cocultures did melatonin inhibit osteoclastogenesis. Melatonin effects were mediated through MT2 melatonin receptors, MEK1/2, and MEK5. In layered but not transwell cocultures, melatonin increased OPG:RANKL ratios by inhibiting RANKL, suggesting that contact with osteoclasts during osteoblastogenesis inhibits RANKL secretion. Melatonin modulated expression of ERK1/2, ERK5, β1 integrin, GLUT4, and IRβ that was dependent upon the type of coculture; however, in both cultures, melatonin increased RUNX2 and decreased PPARγ expression, indicating a role for metabolic processes that control osteogenic vs adipogenic cell fates of MSCs. Furthermore, melatonin also has osteoblast‐inducing effects on human adipose‐derived MSCs. In vivo, one‐year nightly melatonin (15 mg/L) given to neu female mice in their drinking water increased pErk1/2, pErk5, Runx2, and Opg and Rankl levels in bone consistent with melatonin's already reported bone‐enhancing effects. Finally, analysis of daily logs from the MOPS demonstrated a significant improvement in mood and perhaps sleep quality in women receiving melatonin vs placebo. The osteoblast‐inducing, bone‐enhancing effects of melatonin and improvement in quality of life suggest that melatonin is a safe and effective bone loss therapy.     

Normal matrix mineralization induced by strontium ranelate in MC3T3-E1 osteogenic cells

There is growing evidence that strontium ranelate (SR; S12911-2, PROTELOS; Institut de Recherches Internationales Servier, Courbevoie, France), a compound containing 2 atoms of stable strontium (Sr), influences bone cells and bone metabolism in vitro and in vivo. We previously reported that SR increases bone mass in rats and mice by stimulating bone formation and inhibiting bone resorption. We also showed that short-term treatment with SR enhances osteoblastic cell recruitment and function in short-term rat calvaria cultures. Because Sr incorporates into the bone matrix, it was of interest to determine whether SR may affect matrix mineralization in long-term culture. To this goal, osteogenic mouse calvaria-derived MC3T3-E1 osteoblastic cells were cultured for up to 14 days in the presence of ascorbic acid and phosphate to induce matrix formation and mineralization. Matrix formation was determined by incorporation of tritiated proline during collagen synthesis. Matrix mineralization was quantified by measuring the number and surface of mineralized nodules using a digital image analyzer. In this model, 1,25(OH)2 vitamin D (1 nmol/L) used as internal control, increased alkaline phosphatase (ALP) activity, an early osteoblast marker, on days 4, 10, and 14 of culture. Treatment with SR (1 mmol/L Sr(2+)) increased ALP activity at days 4 and 14 of culture. SR also increased collagen synthesis at days 4 and 10 of culture. In contrast, 1,25(OH)2 vitamin D (1 nmol/L) inhibited collagen synthesis at 4 to 14 days of culture. Long-term treatment with SR (0.1 to 1 mmol/L Sr(2+)) dose dependently increased Sr concentration into the calcified nodules, but did not alter matrix mineralization in long-term culture, as shown by the ratio of the surface of mineralized nodules to the number of mineralized nodules on day 14 of culture. These results show that long-term treatment with SR increases collagenous matrix formation by MC3T3-E1 osteoblasts without inducing deleterious effect on matrix mineralization.     

罗格列酮对经诱导的骨髓间充质干细胞ALP、BMP-2及TGF-β1分泌的影响

目的 体外观察罗格列酮对骨髓间充质干细胞(BMSCs)向成骨细胞分化的影响,并观察对碱性磷酸酶(ALP)、骨形成蛋白-2(BMP-2)、转化生长因子β1(TGF-β1)分泌的影响,探讨其对骨代谢的作用机制.方法 无菌条件下从大鼠长骨骨髓中分离获取BMSCs,采用全骨髓贴壁培养法对BMSCs进行纯化、传代扩增,随后在1、2、5、10 μmol/L罗格列酮干预下诱导成骨细胞分化培养21 d,进行茜素红染色观察矿化结节,并测定成骨细胞标记物ALP、BMP-2及TGF-β1的分泌.结果 1、2、5、10 μmol/L罗格列酮干预组与成骨经典组对比,成骨细胞的钙结节形成比例显著降低(P＜0.05),ALP、BMP-2、TGF-β1水平呈剂量依赖性地下降(均P＜0.05).结论 罗格列酮可剂量依赖性地抑制BMSCs向成骨细胞分化,这可能是罗格列酮致骨质疏松的重要机制.

Abstract：

Objective To observe the effects of rosiglitazone on differentiation of rat bone-marrow stromal cells (BMSCs) into osteoblasts (OB) and on secretion of alkaline phosphatase (ALP), bone morphogenetic protein2 (BMP-2), and transforming growth factor-β1 (TGF-β1) in order to investigate its mechanism of the impact on the bone metabolism.Methods BMSCs from long bone were bred by using differential time adherent culture method,and then were interfered with 1,2,5,10 μmol/L rosiglitazone to differentiate into osteoblasts in the presence of an osteogenic medium.The rate of mineralization was examined by staining mineralized nodules with Alizarin red S,and the secretion of ALP, BMP-2, and TGF-β1 was examined by enzyme linked immunosorbent assay (ELISA)after 21 d of culture.Results Compared with the classic group, the rate of mineralization was significantly decreased by 1,2,5 and 10 μmoL/L rosiglitazone (P＜0.05), the levels of ALP, BMP-2, and TGF-β1 decreased in a dose-depedent manner (P＜0.05).Conclusion Rosiglitazone dose-dependently inhibits differentiation of BMSCs into osteoblasts, which may be an important mechanism in causing osteoporosis.     

Characterization and partial purification of a serum protein which stimulates the release of human placental lactogen in vitro

The factors that regulate the release of human placental lactogen (hPL) are poorly understood. To determine whether hPL is regulated by a factor(s) in pregnancy serum, placental explants were exposed for up to 9 h to a pool of serum samples from 50 women in the third trimester. In static explant cultures, the addition of the serum (0.6-10.8 mg protein/ml) caused a dose-dependent and reversible increase in hPL release during a 6-h period. The maximum release by the explants exposed to pregnancy serum was 200-250% greater than that of control explants, and the half-maximal dose was 2-3 mg/ml. Perifusion of placental explants with 15% pregnancy serum (final concentration, 10.5 mg protein/ml) also caused a significant release in hPL within 15 min, which reached a maximum of 200-225% above control levels. Two other pools of pregnancy serum samples as well as individual samples from four pregnant women also stimulated hPL release. Although pregnancy serum significantly stimulated hPL release, there was no increase in either the release of hCG or trichloroacetic acid-precipitable 35S-labeled proteins. Serum from nonpregnant women and men, as well as bovine serum, also stimulated hPL release, but their potencies were only 20-25% that of pregnancy serum. Chicken and porcine serum (10.8 mg/ml each) caused only small (less than 10%) increases in hPL release, and purified human albumin and ovalbumin had no effect. Dialysis or ultrafiltration of pregnancy serum using membranes with mol wt exclusions of 10K daltons caused no loss of activity. Delipidation of pregnancy serum with acetone-ethanol or acid-charcoal also caused no loss of activity, but treatment with trypsin caused greater than 95% loss of activity. Purification of the stimulatory activity by successive chromatographies on Sephadex G-150, Cibacron blue, and Sephadex G-75 resulted in an approximately 800-fold increase in specific activity. Approximately 90% of the total activity eluted from Sephadex G-75 with an apparent mol wt of 31,000, the remainder eluted in the void volume. Although partially purified pregnancy serum stimulated hPL release, the active fractions did not affect the release of rat LH, FSH, or GH from rat pituitary cells or the release of PRL from human decidual explants. Incubation of placental explants in calcium-deficient medium blocked the stimulatory effect of the partially purified pregnancy serum by greater than 90%. These studies indicate that human serum contains a protein(s) that causes a specific, rapid, dose-dependent, and reversible increase in hPL release.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bone mineral content of the radius: Good correlations with physicochemical determinations in iliac crest trabecular bone of normal and osteoporotic subjects

Specific gravity, porosity index (physical parameters), hydroxyproline, calcium, magnesium, and phosphorus (chemical parameters) were determined in iliac crest trabecular bone of normal and osteoporotic subjects. These physical and chemical parameters were compared to bone mineral content (BMC) measurements by x-ray photodensitometry of the radius. BMC values correlated negatively with porosity index, specific gravity, and degree of mineralization of trabecular bone matrix, which all increase with osteoporosis. There was a negative correlation between calcium and magnesium contents per net bone volume. “Distal” scans of the radius reflected better the axial skeleton mass than “proximal” scans, and physicochemical data correlated better with bone mineral content values than with bone mineral mass (BMM) values.     

Connectivity Map-based discovery of parbendazole reveals targetable human osteogenic pathway

Osteoporosis is a common skeletal disorder characterized by low bone mass leading to increased bone fragility and fracture susceptibility. In this study, we have identified pathways that stimulate differentiation of bone forming osteoblasts from human mesenchymal stromal cells (hMSCs). Gene expression profiling was performed in hMSCs differentiated toward osteoblasts (at 6 h). Significantly regulated genes were analyzed in silico, and the Connectivity Map (CMap) was used to identify candidate bone stimulatory compounds. The signature of parbendazole matches the expression changes observed for osteogenic hMSCs. Parbendazole stimulates osteoblast differentiation as indicated by increased alkaline phosphatase activity, mineralization, and up-regulation of bone marker genes (alkaline phosphatase/ALPL, osteopontin/SPP1, and bone sialoprotein II/IBSP) in a subset of the hMSC population resistant to the apoptotic effects of parbendazole. These osteogenic effects are independent of glucocorticoids because parbendazole does not up-regulate glucocorticoid receptor (GR) target genes and is not inhibited by the GR antagonist mifepristone. Parbendazole causes profound cytoskeletal changes including degradation of microtubules and increased focal adhesions. Stabilization of microtubules by pretreatment with Taxol inhibits osteoblast differentiation. Parbendazole up-regulates bone morphogenetic protein 2 (BMP-2) gene expression and activity. Cotreatment with the BMP-2 antagonist DMH1 limits, but does not block, parbendazole-induced mineralization. Using the CMap we have identified a previously unidentified lineage-specific, bone anabolic compound, parbendazole, which induces osteogenic differentiation through a combination of cytoskeletal changes and increased BMP-2 activity.Osteoporosis is a common and devastating bone disease characterized by reduced bone mass and increased fragility and fracture risk. It has been estimated that an osteoporotic fracture occurs once every 8 s worldwide (1), and direct healthcare costs in Europe alone are at least €31.7 billion annually (2). Osteoporosis means porous bones and occurs when bone remodeling is disrupted. Bone remodeling is a balancing act between removal of old bone and formation of new bone, which is achieved by two distinct cells, the osteoclast and osteoblast, respectively. When uncoupling of these two processes takes place, bone resorption can overtake bone formation, resulting in osteoporosis. Most osteoporosis treatments, such as bisphosphonates, reduce bone resorption and result in modest increases in bone density; however, these treatments do not result in a true bone anabolic effect, so patients do not regain bone that has been lost at time of diagnosis. An ideal treatment would stimulate bone formation as well, to help repair the damage already done to the bone microarchitecture and strength; with this in mind, our goal was to search for previously unidentified molecules and/or mechanisms that stimulate human osteoblast differentiation and bone formation.The connectivity map (CMap) is a web-based tool that allows for screening of compounds against a genome-wide disease or physiological gene signature (3, 4). This screening is achieved by comparing microarray data from more than 1,300 small molecules to a user’s selected gene signature of the phenotype of interest using a pattern-matching algorithm with a high level of resolution and specificity. The screening results in a list of compounds with a highly correlating gene expression pattern to that of the phenotype of interest, which has the potential to aid in finding a novel treatment for a disease or to identify novel pathways or genes involved in a complex biological process. To date, the CMap has been successfully used to identify compounds and combination therapies that show promise in the treatment of osteoarthritic pain (5), adenocarcinoma (6), kidney disease (7), gliomas (8), and NK cell neoplasms (9).Our aim was to identify previously unidentified anabolic therapeutic targets by genomic, proteomic, and bioinformatic dissection of human mesenchymal stromal cell (hMSC)-derived osteoblasts. Therefore, we used the CMap to identify compounds with a matching gene expression profile to human mesenchymal stem cells undergoing osteogenic differentiation. By following this approach, we aimed to not only discover novel compounds that stimulate osteogenic differentiation, but also novel processes underlying this process.     

Bone marrow stromal cells from aged male rats have delayed mineralization and reduced response to mechanical stimulation through nitric oxide and ERK1/2 signaling during osteogenic differentiation

Bone marrow stromal cells (MSCs) are a source of osteoblast precursors that can be recruited during bone remodeling or injury, both important processes in aging populations. With advancing age, alterations in bone structure and mineralization are often associated with an increase in osteoporosis and fracture risk. Changes in the number of osteoprogenitor cells and their osteogenic potential may occur with advancing age; however few studies have considered the influence of mechanical conditions. Here, we investigated the ability of bone MSCs from mature and aged rats to differentiate into osteoblasts and to respond to short and long periods of mechanical stimulation through signaling by ERK1/2, nitric oxide (NO), and prostaglandin E₂ (PGE₂) during differentiation. Mineralization was delayed and reduced, but extracellular matrix production appeared less affected by increased age. Differentiating MSCs from aged animals had a decreased response to short and long periods of mechanical stimulation through ERK1/2 signaling, and to long periods of mechanical loading through NO signaling early and late during differentiation. Increases in relative PGE₂ signaling were higher in MSCs from aged animals, which could compensate for reduced ERK1/2 and NO signaling. The decreased mineralization may decrease the ability of cells from aged animals to respond to mechanical stimulation through ERK1/2 and NO signaling, with increased impairment over differentiation time. Decreasing the delay in mineralization of MSCs from aging animals might improve their ability to respond to mechanical stimulation during bone remodeling and injury, suggesting therapies for bone fragility diseases and tissue engineering treatments in elderly populations.     

Bone mineral and collagen quality in iliac crest biopsies of patients given teriparatide: new results from the fracture prevention trial

CONTEXT: Evidence suggests that both bone mineral density and bone quality should be taken into account when assessing bone strength and fracture risk. Bone quality is a multifactor entity, of which bone architecture and material properties are two important components. Matrix mineralization, hydroxyapatite characteristics, and collagen cross-link ratio are key determinants of material properties. Fourier transform infrared imaging (FTIRI) yields data on these characteristics from bone sections. OBJECTIVE: We sought to determine collagen cross-link ratios and matrix mineralization of bone from patients randomized to teriparatide [recombinant human PTH (1-34)] treatment using FTIRI. DESIGN: The Fracture Prevention Trial was randomized, double blind, and placebo-controlled. SETTING: The trial was conducted at global clinical research centers. PATIENTS: Patients consisted of postmenopausal women with osteoporosis. INTERVENTIONS: Patients were randomized to receive daily sc injections of placebo (n = 12) or 20 microg (n = 13) or 40 microg (n = 13) teriparatide. Biopsies were obtained after 12 months of treatment or at the end of treatment (range, 19-24 months for end of treatment paired biopsies). MAIN OUTCOME MEASURES: Biopsies were analyzed by FTIRI to determine the matrix mineralization (mineral to matrix), mineral crystallinity, and collagen cross-link ratio (pyridinoline/dehydrodihydroxylysinonorleucine) with a spatial resolution of approximately 6.3 microm. RESULTS: Patients administered teriparatide 20 and 40 microg/d exhibited significantly lower matrix mineralization, mineral crystallinity, and collagen cross-link ratio when compared with placebo. CONCLUSIONS: These findings indicate that the bone-forming effect of teriparatide results in bone with a molecular profile reminiscent of younger bone.     

Methotrexate (MTX) inhibits osteoblastic differentiation in vitro: possible mechanism of MTX osteopathy

OBJECTIVE: To clarify the mechanism of impaired bone formation during low dose methotrexate (MTX) therapy. METHODS: The in vitro effects of MTX on the function and differentiation of osteoblastic cells were investigated using (1) a mouse osteogenic cell line (MC3T3-E1) with the capacity to differentiate into osteoblastic or osteocytes, (2) a human osteoblastic osteosarcoma cell line (SaOS-2) with a mature osteoblastic phenotype, and (3) mouse bone marrow stromal cells containing osteoblast precursors. Osteoblast function was assessed by measuring the cellular activity of alkaline phosphatase (ALP) and the mineralization capacity of cultures. RESULTS: MTX suppressed ALP activity dose-dependently in growing MC3T3-E1 cells, but proliferation of these cells was only inhibited by a high concentration of MTX. In contrast, inhibition of ALP activity in MC3T3-E1 cells of mature osteoblastic phenotype was only observed with 10(-8) M and 10(-7) M MTX, and proliferation was not influenced. ALP activity and the proliferation of SaOS-2 cells were not inhibited by MTX, even when growing cells were treated. However, both ALP activity and formation of calcified nodules in bone marrow stromal cell cultures were significantly suppressed by MTX at concentrations between l0(-10) and 10(-7) M. CONCLUSION: These results suggest that MTX suppresses bone formation by inhibiting the differentiation of early osteoblastic cells.