Calvaria Cultures Research Articles

KDM6A Mediated Regulation of Cranial Frontal Bone Suture Fusion in Mice is Sex-Dependent.

The five flat bones of developing cranial plates are bounded by fibrous sutures, which remain open during development to accommodate for the growing brain. Kdm6A is a demethylase which removes the epigenetic repressive mark, tri-methylated lysine 27 on histone 3 (H3K27me3), from the promoters of osteogenic genes, and has previously been reported to promote osteogenesis in cranial bone cells. The present study generated a mesenchyme-specific deletion of a histone demethylase, Kdm6a, to assess the effects of Kdm6a loss, in cranial plate development and suture fusion. The results showed that the loss of Kdm6a in Prx-1+ cranial cells caused increased anterior width and length in the calvaria of both male and female mice. However, the posterior length was further decreased in female mice. Moreover, loss of Kdm6a resulted in suppression of late suture development and calvarial bone formation predominantly in female mice. In vitro assessment of calvaria cultures isolated from female Kdm6a knockout mice, found significantly suppressed calvarial osteogenic differentiation potential, associated with decreased gene expression levels of Runx2 and Alkaline Phosphatase and increased levels of the suppressive mark, H3K27me3 on the respective gene promoters. Conversely, cultured calvaria bone cultures isolated from male Kdm6a knockout mice exhibited an increased osteogenic differentiation potential. Interestingly, the milder effects on cranial suture development in Kdm6a knockout male mice, were associated with an overcompensation of the Kdm6a Y-homolog, Kdm6c and increased expression levels of Kdm6b in calvarial bone cultures. Taken together, these data demonstrate a role for Kdm6a during calvarial development and patterning, predominantly in female mice, and highlights the potential role of Kdm6 family members in patients with unexplained craniofacial deformities.

Lanthanum promoting bone formation by regulating osteogenesis, osteoclastogenesis and angiogenesis

With lanthanum (La) in agriculture, medicine, and the chemical industry, La shows accumulation in the body, especially in the bone, because of its similar structure to calcium. Moreover, La has a direct role in bone formation regulating osteoblasts and osteoclasts. Nevertheless, bone formation is complex under the regulation of osteogenesis, osteoclastogenesis, and angiogenesis in the bone microenvironment. It is difficult to comprehensively understand the regulation of bone homeostasis from a single kind of cell. Herein, some bone microenvironment-related cells and mouse calvaria culture models were used to study the regulatory effect of La-based compound-lanthanum nitrate (La(NO3)3) on bone formation. La(NO3)3 has good biological safety, and the osteogenic differentiation is significantly increased under La(NO3)3 treatment for bone marrow mesenchymal stem cells (BMMSC). In contrast, the differentiation, maturation, and bone erosion ability of osteoclasts are significantly decreased. Meanwhile, the angiogenesis ability of human umbilical vein endothelial cells (HUVEC) is significantly promoted when treated with La(NO3)3. Furthermore, bone metabolism and angiogenesis are improved under La(NO3)3 treatment in the calvaria ex vivo culture model and BMMSC-HUVEC co-culture system. These results suggest that La has the beneficial effect of promoting bone metabolism and improving bone formation in the bone microenvironment.

Elevation of the unfolded protein response increases RANKL expression.

Increased production of the osteoclastogenic cytokine RANKL is a common feature of pathologic bone loss, but the underlying cause of this increase is poorly understood. The unfolded protein response (UPR) is activated in response to accumulation of misfolded proteins in the endoplasmic reticulum (ER). Failure to resolve misfolding results in excess UPR signaling that stimulates cytokine production and cell death. We therefore investigated whether RANKL is one of the cytokines stimulated in response to elevated UPR in bone cells. Pharmacologic induction of UPR with tunicamycin (Tm)‐stimulated RANKL expression in cultures of primary osteoblastic cells and in osteoblast and osteocyte cell lines. Pharmacologic inhibition of the UPR blunted Tm‐induced RANKL production. Silencing Edem1 or Sel1l, proteins that aid in degradation of misfolded proteins, also induced UPR and increased RANKL mRNA. Moreover, Tm or hypoxia increased RANKL and bone resorption in cultures of neonatal murine calvaria. Administration of Tm to adult mice caused dilation of ER in osteoblasts and osteocytes, elevated the UPR, and increased RANKL expression and osteoclast number. These findings support the hypothesis that excessive UPR signaling stimulates the expression of RANKL by osteoblasts and osteocytes, and thereby facilitates excessive bone resorption and bone loss in pathologic conditions.

The Ex Vivo Organ Culture of Bone.

The ex vivo organ culture of bone provides many of the advantages of both the whole organism and isolated cell strategies and can deliver valuable insight into the network of processes and activities that are fundamental to bone and cartilage biology. Through maintaining the bone and/or cartilage cells in their native environment, this model system provides the investigator with a powerful experimental protocol to address specific facets of skeletal growth and development. In this chapter, we outline the basic protocols and possible readouts of organ culture models to replicate; (a) linear bone growth (murine metatarsal culture model), (b) bone and cartilage metabolism (murine femoral head culture model), (c) bone response to mechanical stimulation (bovine trabecular core culture model), and (d) bone resorption and formation (murine calvaria culture model).

Peptides from Antarctic krill (Euphausia superba ) ameliorate senile osteoporosis via activating osteogenesis related BMP2/Smads and Wnt/β-catenin pathway

The senescence-accelerated mouse strain P6 (SAMP6) was applied to investigate the effect of treatment with peptides from Antarctic Krill (AKP) on senile osteoporosis in vivo. Results showed that AKP significantly increased bone mineral density, improved bone biomechanical properties, and ameliorated trabecular bone microstructure by promoting bone anabolism in SAMP6. Neonatal murine calvaria culture system further demonstrated that AKP stimulated osteoblast-induced new osteoid and bone formation ex vivo. The mechanism revealed that AKP induced the secretion and expression of bone morphogenetic protein 2 (BMP2), which was identified as a potent osteoinductive factor, and activated BMP2-dependent Smads signaling by phosphorylation of Smad1. Wnt signaling shares the same osteogenesis related transcriptional factor Runx2 with BMP2/Smads pathway. AKP also induced the activation of Wnt/β-catenin signaling by upregulating the expression of Wnt10b and β-catenin. In conclusion, AKP ameliorated senile osteoporosis by activating BMP2/Smads and Wnt/β-catenin molecular pathways, thereby promoting bone formation. Practical applications As a novel food material, Antarctic krill possesses an enormous biomass and utilization potential. It contains the largest amount of protein among all recorded organisms worldwide and the protein biological value is better than other animal proteins. This study demonstrated that peptides hydrolyzed from Antarctic Krill exhibit antiosteoporotic activity, which may provide some theoretical basis for the high-value of Antarctic Krill protein as antiosteoporotic functional foods in future.

Coordinate regulatory osteogenesis effects of icariin, timosaponin B II and ferulic acid from traditional Chinese medicine formulas on UMR-106 osteoblastic cells and osteoblasts in neonatal rat calvaria cultures

Ethnopharmacological significanceIcariin (I), ferulic acid (F) and timosaponin B II (T) derived respectively from the leaf of Epimedium brevicornu Maxim (EBM, Berberidaceae), rhizome of Anemarrhena asphodeloides Bunge (AAB, Liliaceae) and root of Angelica sinensis (Oliv.) Diels (ASD, Umbelliferae) are included in several traditional Chinese medicine (TCM) formulas for the treatment of osteoporosis. In addition, the medicinal materials and chemical constituents in many traditional Chinese formulas have been shown to have potential synergistic, additive and antagonistic effects. Aim of studyTo explore the action mechanism and interactions between I, T and F as bone anabolic ingredients on osteoblasts, and fully understand their action mechanism and rationality of the formula design. Materials and methodsAn osteoporotic model was established in bilaterally ovariectomized mice. Bone mineral density (BMD), bone mineral content (BMC) and serum biochemical parameters including alkaline phosphatase (ALP), tartrate resistant acid phosphatase (TRAP), osteoprotegerin (OPG) and deoxypyridinoline cross-links (DPD) were measured to evaluate the effects of I, T or F alone and their combinations on osteoporotic mice. UMR-106 osteoblastic cells and primary osteoblasts in neonatal rat calvarias were used to evaluate the osteogenesis effect. The immunohistochemical method and Western-blot analysis were used to detect the expression of critical proteins in the process of proliferation and differentiation of osteoblasts. ResultsIFT combinations enhanced the therapeutic effect without increasing the adverse effects on osteoporotic mice, synergistically increased the osteoblast proliferation, ALP activity and mineralized nodule formation, and promoted the expression of bone matrix by regulating BMP and Wnt/β-catenin signaling pathways in osteoblasts. ConclusionIFT combinations reinforced the therapeutic effect on osteoporosis by modulating multi-signaling pathways and action targets.

Analysis of constituents of the eastern Nigeria mistletoe, Loranthus micranthus linn revealed presence of new classes of osteogenic compounds

Ethnopharmacological relevanceMistletoe extracts (decoctions) are used traditionally in eastern Nigeria for the management of bone pain, post menopausal syndrome and diabetes amongst several other ailments. While scientific evidence supporting its folkloric use as an antidiabetic agent has been documented, the age-long practice of its use in treatment of post menopausal syndrome has not been scientifically validated. Postmenopausal osteoporosis accounts for one of the prevalent disease conditions in aging population globally. This situation is exacerbated by the lack of osteogenic therapy. In search for plants of Nigerian origin with osteogenic potential, we evaluated eastern Nigerian mistletoe, having ethnotraditional claims of anti-diabetic, anti-hypertensive and anti-cancer activities as well as preventive effect in various post-menopausal syndromes. Materials and MethodsMethanolic extracts of mistletoe leaves harvested from three host tress – Kola acuminata (KM), Citrus spp (CM) and Garcinia kola (GKM) – were evaluated for osteoblast viability and osteogenic activities using primary rat calvaria culture. Lupeol (1) was isolated from the stem bark of Bombax ciba and its congener, dihydoxylupeol palmitate (2) in addition to three other compounds; 3-methoxy quercetin (3), 3,4,5-trimethoxy gallate (4), and friedelin (5) were isolated from the leaves of mistletoes species. Following their chemical characterization, the compounds were evaluated for osteogenic potential using validated models including alkaline phosphatase (ALP) assay, mineralization assay and expression of osteogenic genes – bone morphogenetic protein-2 (BMP2) and osteoblast transcription factor (RUNX2) – in primary calvarial cultures harvested from neonatal rats. Uterine estrogenicity of the extracts was tested in adult female Sprague Dawley rats. ResultsMethanol extracts of mistletoe from three hosts exhibited increase in ALP activity (a marker of osteoblast differentiation) at lower concentrations (0.2–0.8μg/ml) and either no or inhibitory effect at higher concentrations (1.6 and 3.2μg/ml). None of the extract had cytotoxicity to osteoblasts at the concentrations tested. Five compounds viz. 1 from Bombax ciba, and 2–5 were isolated from the mistletoe leaves. Out of these, 5 exhibited significant loss of osteoblast viability and hence it was not considered further. All four compounds exhibited stimulatory effects on osteoblast differentiation as assessed by ALP assay and determination of osteogenic gene expression. Compound 2 was relatively more potent than its precursor, compound 1 in stimulating BMP2 upregulation. KM did not show uterine estrogenicity. ConclusionMethanolic extracts from the three mistletoes species possess in vitro osteogenic activity, and from these extracts three new classes of compounds have been found to promote osteoblast differentiation in vitro. In light of these findings, we propose that mistletoe species may be developed as safer alternative(s) in the management of diseases where lack of bone formation is the pathology.

Inhibition of the canonical Wnt pathway by high glucose can be reversed by parathyroid hormone‐related protein in osteoblastic cells

Recent in vivo findings suggest that the bone sparing effect of parathyroid hormone-related protein (PTHrP) in diabetic mice might occur at least in part through targeting a suppressed Wnt/β-catenin pathway in osteoblasts. We here aimed to examine the inhibitory action of a high glucose environment on specific components of the canonical Wnt pathway, and the putative compensatory effects of PTHrP, in osteoblastic cell cultures. Mouse osteoblastic MC3T3-E1 cells and primary cultures of fetal mouse calvaria were exposed to normal (5.5 mM) or high (25 mM) D-glucose (HG), with or without PTHrP (1-36) or PTHrP (107-139) for different times. In some experiments, MC3T3-E1 cells were incubated with the Wnt pathway activators Wnt3a and LiCl, or were transfected with plasmids encoding either a mutated β-catenin that cannot be targeted for degradation or a human PTHrP (-36/+139) cDNA, or the corresponding empty plasmid, in the presence or absence of HG. The gene expression of Wnt3a and low density receptor-like proteins (LRP)-5 and 6, as well as β-catenin protein stabilization and β-catenin-dependent transcription activity were evaluated. Oxidative stress status under HG condition was also assessed. The present data demonstrate that HG can target different components of the canonical Wnt pathway, while β-catenin degradation appears to be a key event leading to inhibition of Wnt/β-catenin signaling in mouse osteoblastic cells. Both PTHrP peptides tested were able to counteract this deleterious action of HG. These in vitro findings also provide new clues to understand the underlying mechanisms whereby PTHrP can increase bone formation.

RETRACTED: Synthetic triterpenoids, CDDO-Imidazolide and CDDO-Ethyl amide, induce chondrogenesis

Novel methods for inducing chondrogenesis are critical for cartilage tissue engineering and regeneration. Here we show that the synthetic oleanane triterpenoids, CDDO-Imidazolide (CDDO-Im) and CDDO-Ethyl amide (CDDO-EA), at concentrations as low as 200 nM, induce chondrogenesis in organ cultures of newborn mouse calvaria. The cartilage phenotype was measured histologically with metachromatic toluidine blue staining for proteoglycans and by immunohistochemical staining for type II collagen. Furthermore, real-time polymerase chain reaction (PCR) analysis using mRNA from calvaria after 7-day treatment with CDDO-Im and CDDO-EA showed up-regulation of the chondrocyte markers SOX9 and type II collagen (alpha1). In addition, TGF-β; BMPs 2 and 4; Smads 3, 4, 6, and 7; and TIMPs-1 and -2 were increased. In contrast, MMP-9 was strongly down-regulated. Treatment of human bone marrow-derived mesenchymal stem cells with CDDO-Im and CDDO-EA (100 nM) induced expression of SOX9, collagen IIα1, and aggrecan, as well as BMP-2 and phospho-Smad5, confirming that the above triterpenoids induce chondrogenic differentiation. This is the first report of the use of these drugs for induction of chondrogenesis.

Abstract 1767: Targeting prostate cancer osteoblastic progression with VEGF121/rGel, a single agent targeting osteoblasts, osteoclasts, and tumor neovasculature

Abstract Purpose: A hallmark of prostate cancer (PCa) progression is the development of osteoblastic bone metastases, which respond poorly to available therapies. We previously reported that VEGF121/rGel targets osteoclast precursors and tumor neovasculature. Here we tested the hypothesis that targeting non-tumor cells expressing the receptors for VEGF121, namely VEGFR-1 and VEGFR-2, can inhibit tumor progression in a clinically relevant model of osteoblastic PCa. Experimental Design: We examined the effect of VEGF121/rGel on osteoblast precursors and several PCa cell lines in vitro; on osteoid formation in a mouse calvaria culture assay ex vivo; and on PCa osteoblastic progression in the femurs of mice injected with MDA PCa 118b, a PCa xenograft obtained from a bone metastasis in a patient with castrate-resistant PCa. Results: VEGF121/rGel was cytotoxic in vitro to osteoblast precursor cells. This cytotoxicity was specific as VEGF121/rGel internalization into osteoblasts was VEGF121 receptor driven. Furthermore, VEGF121/rGel significantly inhibited PCa-induced bone formation in a mouse calvaria culture assay. In vivo, systemic administration of VEGF121/rGel significantly inhibited the osteoblastic progression of PCa cells in the femurs of nude mice. Microcomputed tomography analysis revealed that VEGF121/rGel restored the bone volume fraction of tumor-bearing femurs to values similar to those of the contralateral (non-tumor bearing) femurs. VEGF121/rGel significantly reduced the number of tumor-associated osteoclasts but did not change the numbers of peritumoral osteoblasts. Importantly, VEGF121/rGel-treated mice had significantly less tumor burden than control mice did. Our results thus indicate that VEGF121/rGel inhibits osteoblastic tumor progression by targeting angiogenesis, osteoclastogenesis, and bone formation. Conclusions: Targeting VEGFR-1- or VEGFR-2-expressing cells is effective in controlling the osteoblastic progression of PCa in bone. These findings provide the basis for an effective multitargeted approach for metastatic PCa. We believe that VEGF121/rGel in combination with tumor cell-targeting therapies (e.g., chemotherapy) constitutes a novel strategy for advanced PCa. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1767. doi:10.1158/1538-7445.AM2011-1767

Inhibition of Prostate Cancer Osteoblastic Progression with VEGF121/rGel, a Single Agent Targeting Osteoblasts, Osteoclasts, and Tumor Neovasculature

A hallmark of prostate cancer (PCa) progression is the development of osteoblastic bone metastases, which respond poorly to available therapies. We previously reported that VEGF(121)/rGel targets osteoclast precursors and tumor neovasculature. Here we tested the hypothesis that targeting nontumor cells expressing these receptors can inhibit tumor progression in a clinically relevant model of osteoblastic PCa. Cells from MDA PCa 118b, a PCa xenograft obtained from a bone metastasis in a patient with castrate-resistant PCa, were injected into the femurs of mice. Osteoblastic progression was monitored following systemic administration of VEGF(121)/rGel. VEGF(121)/rGel was cytotoxic in vitro to osteoblast precursor cells. This cytotoxicity was specific as VEGF(121)/rGel internalization into osteoblasts was VEGF(121) receptor driven. Furthermore, VEGF(121)/rGel significantly inhibited PCa-induced bone formation in a mouse calvaria culture assay. In vivo, VEGF(121)/rGel significantly inhibited the osteoblastic progression of PCa cells in the femurs of nude mice. Microcomputed tomographic analysis revealed that VEGF(121)/rGel restored the bone volume fraction of tumor-bearing femurs to values similar to those of the contralateral (non-tumor-bearing) femurs. VEGF(121)/rGel significantly reduced the number of tumor-associated osteoclasts but did not change the numbers of peritumoral osteoblasts. Importantly, VEGF(121)/rGel-treated mice had significantly less tumor burden than control mice. Our results thus indicate that VEGF(121)/rGel inhibits osteoblastic tumor progression by targeting angiogenesis, osteoclastogenesis, and bone formation. Targeting VEGF receptor (VEGFR)-1- or VEGFR-2-expressing cells is effective in controlling the osteoblastic progression of PCa in bone. These findings provide the basis for an effective multitargeted approach for metastatic PCa.

Tissue engineering of bone an ectopic rat model

Tissue engineering is attempting to recreate the complexity of living tissues. In order to test a variety of scaffolds or cells that are constantly being developed, we describe here a model where tissue engineering of bone in a non-osseous environment at subcutaneous thoracic site of DA rats generates. In this model, cell - matix interactions can mimic the normal cascade of bone development into a well organized ossicle like structure including newly formed bone marrow, during 3-4 weeks. Histogenesis of cartilage, bone and bone marrow is closely related to changes in molecular expression of essential early transcriptional regulators of osteoblast differentiation. We tested different organic, anorganic and polymeric scaffolds and their interaction with mesenchymal stem cells present in fresh bone marrow. In another series of experiments we tested mesenchymal populations separated from cultures of calvaria and periosteum for their ability to form bone in the same rat model. It is concluded that this in vivo model is very potent in studying cell-scaffold interactions affecting the temporal and spatial tissue engineering of bone.

Marginal Zinc Deficiency in Pregnant Rats Impairs Bone Matrix Formation and Bone Mineralization in Their Neonates

Zinc (Zn) deficiency during pregnancy may result in a variety of defects in the offspring. We evaluated the influence of marginal Zn deficiency during pregnancy on neonatal bone status. Nine-week-old male Sprague-Dawley rats were divided into two groups and fed AIN-93G-based experimental diets containing 35 mg Zn/kg (Zn adequately supplied, N) or 7 mg Zn/kg (low level of Zn, L) from 14-day preconception to 20 days of gestation, that is, 1 day before normal delivery. Neonates were delivered by cesarean section. Litter size and neonate weight were not different between the two groups. However, in the L-diet-fed dam group, bone matrix formation in isolated neonatal calvaria culture was clearly impaired and was not recovered by the addition of Zn into the culture media. Additionally, serum concentration of osteocalcin, as a bone formation parameter, was lower in neonates from the L-diet-fed dam group. Impaired bone mineralization was observed with a significantly lower content of phosphorus in neonate femurs from L-diet-fed dams compared with those from N-diet-fed dams. Moreover, Zn content in the femur and calvaria of neonates from the L-diet group was lower than that of the N-diet-fed group. In the marginally Zn-deficient dams, femoral Zn content, serum concentrations of Zn, and osteocalcin were reduced when compared with control dams. We conclude that maternal Zn deficiency causes impairment of bone matrix formation and bone mineralization in neonates, implying the importance of Zn intake during pregnancy for proper bone development of offspring.

Estrogens attenuate oxidative stress and the differentiation and apoptosis of osteoblasts by DNA-binding-independent actions of the ERα

Estrogens diminish oxidative stress in bone and bone marrow, attenuate the generation of osteoblasts, and decrease the prevalence of mature osteoblast apoptosis. We have searched for the molecular mechanism of these effects using as tools a mouse model bearing an estrogen receptor α (ERα) knock-in mutation that prevents binding to DNA (ERαNERKI/−) and several osteoblast progenitor cell models expressing the wild-type ERα or the ERαNERKI/−. We report that the ability of estrogens to diminish the generation of reactive oxygen species, stimulate the activity of glutathione reductase, and decrease the phosphorylation of p66shc, as well as osteoblastogenesis and osteoblast number and apoptosis, were fully preserved in ERαNERKI/− mice, indicating that the DNA-binding function of the ERα is dispensable for all these effects. Consistent with the attenuation of osteoblastogenesis in this animal model, 17β-estradiol attenuated bone morphogenetic protein 2 (BMP-2)–induced gene transcription and osteoblast commitment and differentiation in murine and human osteoblastic cell lines. Moreover, 17β-estradiol attenuated BMP-2-induced differentiation of primary cultures of calvaria- or bone marrow–derived osteoblastic cells from ERαNERKI/− mice as effectively as in cells from wild-type littermates. The inhibitory effect of the hormone on BMP-2 signaling resulted from an ERα-mediated activation of ERKs and the phosphorylation of Smad1 at the linker region of the protein, which leads to proteasomal degradation. These results illustrate that the effects of estrogens on oxidative stress and the birth and death of osteoblasts do not require the binding of ERα to DNA response elements, but instead they result from the activation of cytoplasmic kinases. © 2010 American Society for Bone and Mineral Research

Iron overload alters iron-regulatory genes and proteins, down-regulates osteoblastic phenotype, and is associated with apoptosis in fetal rat calvaria cultures

Iron overload has been implicated in decreased bone mineral density. However, the effect of iron overload on osteoblast lineage cells remains poorly understood. The purpose of this study was to examine osteoblast differentiation, function, and apoptosis in iron-loaded cells from fetal rat calvaria. Cells were incubated with media supplemented with 0–10 μM ferrous sulfate (FeSO 4) during differentiation (days 6–20). Intracellular iron status was assessed by measuring iron content in cell layers and changes in transferrin receptor (TrfR) and ferritin gene and protein expression. Osteoblast differentiation and function were evaluated by measuring osteoblast phenotypic gene markers and capacity of cultures to form mineralized bone nodules. Apoptotic hallmarks were evaluated by microscopy. A 2.3-fold increase in media iron concentration resulted in saturable accumulation of iron in the cell layer 20-fold higher than control ( p < 0.05) by mid-differentiation (day 15, D15). Iron accumulation resulted in rapid and sustained down-regulation of TrfR gene and protein levels (within 24 h) and up-regulation of light and heavy chain ferritin protein levels at late differentiation (day 20, D20). Concurrently, osteoblast phenotype gene markers were suppressed by D15 and a decreased number of mineralized nodules at D20 were observed. Apoptotic events were observed within 24 h of iron loading. These results provide evidence that iron overload alters iron metabolism and suppresses differentiation and function of cells in the osteoblast lineage associated with increased apoptosis.

Inhibition of osteoclast formation and function by bicarbonate: Role of soluble adenylyl cyclase

High [HCO(3)(-)] inhibits and low [HCO(3)(-)] stimulates bone resorption, which mediates part of the effect of chronic acidosis or acid feeding on bone. Soluble adenylyl cyclase (sAC) is a bicarbonate sensor that can potentially mediate the effect of bicarbonate on osteoclasts. Osteoclasts were incubated in 0, 12, and 24 mM HCO(3)(-) at pH 7.4 for 7-8 days and assayed for tartrate-resistant acid phosphatase (TRAP) and vacuolar-ATPase expression, and H+ accumulation. Total number and area of TRAP (+) multinucleated osteoclasts was decreased by HCO(3)(-) in a dose-dependent manner. V-ATPase expression and H+ accumulation normalized to cell cross-sectional area or protein were not significantly changed. The HCO(3)(-) -induced inhibition of osteoclast growth and differentiation was blocked by either 2-hydroxyestradiol, an inhibitor of sAC or sAC knockdown by sAC specific siRNA. The model of HCO(3)(-) inhibiting osteoclast via sAC was further supported by the fact that the HCO(3)(-) dose-response on osteoclasts is flat when cells were saturated with 8-bromo-cAMP, a permeant cAMP analog downstream from sAC thus simulating sAC activation. To confirm our in vitro findings in intact bone, we developed a 1-week mouse calvaria culture system where osteoclasts were shown to be viable. Bone volume density (BV/TV) determined by micro-computed tomography (microCT), was higher in 24 mM HCO(3)(-) compared to 12 mM HCO(3)(-) treated calvaria. This HCO(3)(-) effect on BV/TV was blocked by 2-hydroxyestradiol. In summary, sAC mediates the inhibition of osteoclast function by HCO(3)(-), by acting as a HCO(3)(-) sensor.

Antiosteoporotic activity of phenolic compounds from Curculigo orchioides

Six phenolic compounds isolated from Curculigo orchioides, including 2,6-dimethoxy benzoic acid (1), curculigoside A (2), curculigoside B (3), curculigine A (4), curculigine D (5) and 3,3',5,5'-tetramethoxy-7,9':7',9-diepoxylignan-4,4'-di-O-beta-D-glucopyranoside (6), together with the ethanol extract of Curculigo orchioides were evaluated for their activity on osteoblasts in neonatal rat calvaria cultures and multinucleated osteoclasts derived from rat marrow cells so as to characterize the antiosteoporotic components of this plant and explore the relationship of chemical structure with antiosteoporotic activity. The proliferation of osteoblast was assayed by MTT methods. The activity of ALP (alkaline phosphatase) and TRAP (tartrate-resistant acid phosphatase) was measured by p-nitrophenyl sodium phosphate assay. The TRAP stain was used to identify osteoclast in morphology. The resorption pit area on the bone slices formed by osteoclast was measured by computer image processing. The ethanol extract exhibited stimulatory effect on both the osteoblast proliferation and the ALP activity. Six compounds all increased the osteoblast proliferation, and compounds (1), (2) and (4) also slightly increased the osteoblastic ALP activity. Compounds (1), (2), (3), (6) and the ethanol extract decreased area of bone resorption pit, osteoclastic formation and TRAP activity. These results indicated that phenolic compounds are antiosteoporotic chemical constituents from Curculigo orchioides, and their activities are related with chemical structures.

Disruption of BMP Signaling in Osteoblasts Through Type IA Receptor (BMPRIA) Increases Bone Mass

Bone morphogenetic proteins (BMPs) are known as ectopic bone inducers. The FDA approved BMPs (BMP2 and BMP7) for clinical use. However, direct effects of BMPs on endogenous bone metabolism are not yet well known. We conditionally disrupted BMP receptor type IA (BMPRIA) in osteoblasts during weanling and adult stages to show the impact of BMP signaling on endogenous bone modeling and remodeling. Cre recombination was detected in immature osteoblasts in the periosteum, osteoblasts, and osteocytes but not in chondrocytes and osteoclasts after tamoxifen administration. Bmpr1a conditional knockout mice (cKO) showed increased bone mass primarily in trabecular bone at P21 and 22 wk as determined by H&E staining. Vertebrae, tails, and ribs showed increased radiodensity at 22 wk, consistent with a significant increase in BMD. Both muCT and histomorphometry showed an increase in trabecular BV/TV and thickness of cKO adult bones, whereas osteoclast number, bone formation rate, and mineral apposition rate were decreased. Expression levels of bone formation markers (Runx2 and Bsp), resorption markers (Mmp9, Ctsk, and Tracp), and Rankl were decreased, and Opg was increased in adult bones, resulting in a reduction in the ratio of Rankl to osteoprotegerin (Opg). The reduction in osteoclastogenesis through the RANKL-OPG pathway was also observed in weanling stages and reproduced in newborn calvaria culture. These results suggest that Bmpr1a cKO increased endogenous bone mass primarily in trabecular bone with decreased osteoclastogenesis through the RANKL-OPG pathway. We conclude that BMPRIA signaling in osteoblasts affects both bone formation and resorption to reduce endogenous bone mass in vivo.

Antiosteoporotic chemical constituents from Er-Xian Decoction, a traditional Chinese herbal formula

Aim of the study Er Xian Decoction (EXD), a traditional Chinese medicine formula, has long been used for the treatment of osteoporosis and menopausal syndrome in China. The present study was designed to investigate the antiosteoporotic constituents of EXD, and evaluate their antiosteoporotic effects in ovariectomized rats. Materials and methods Osteoblasts in neonatal calvaria cultures and osteoclasts derived from rat marrow cells were used to bioactivity-guided screen the active constituents. The proliferation of osteoblast was assayed by MTT methods. The activity of ALP and TRAP was measured by p- nitrophenyl sodium phosphate assay. The antiosteoporotic effects of icariin (1), anemarsaponin B II (8) and berberine (6) were verified by using OVX rats model. The bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry using the small animal scan mode. The undecalcified longitudinal proximal tibial metaphysical (PTM) sections were cut and stained for the bone histomorphometric analysis. Results Bioactivity-guided fractionation has led to the successful isolation of antiosteoporotic constituents, i.e., icariin (1), icariside I (2), baohuoside I (3), mangiferin (4), neomangiferin (5), berberine (6), anemarsaponin B (7), anemarsaponin BII (8), anemarsaponin C (9), anemarrhenasaponin I (10), rubiadin-1-methyl ether (11) and obaculactone (12) from EXD. Further study showed that icariin (1), anemarsaponin BII (8) and berberine (6) increased the BMD in ovariectomized rats, and icariin (1) not only increased the bone formation, but also inhibited bone resorption; anemarsaponin BII (8) mainly increased bone formation and berberine (6) only inhibited the bone resorption in ovariectomized rats. Conclusion Our findings demonstrate that multiple ingredients are responsible for antiosteoporotic activity in traditional Chinese medicine formula Er-Xian decoction.

Evaluation of cell affinity on poly(L‐lactide) and poly(ε‐caprolactone) blends and on PLLA‐b‐PCL diblock copolymer surfaces

An evaluation of cell proliferation and adhesion on biocompatible film supports was performed. A series of films were compression molded from commercially available poly (L-lactide), PLLA, and poly(epsilon-caprolactone), PCL, and from their melt mixed blends (PLLA/PCL blends). These were compared with compression molded films of PLLA-b-PCL model diblock copolymers. The samples were analyzed by differential scanning calorimetry (DSC), contact angle measurements, and scanning force microscopy (SFM). Cell adhesion and proliferation were performed with monkey derived fibroblasts (VERO) and with osteoblastic cells obtained either enzymatically or from explants cultures of Sprague-Dawley rat calvaria. Migration studies were performed with bone explants of the same origin. The results obtained indicate that although all materials tested were suitable for the support of cellular growth, a PLLA-b-PCL diblock copolymer sample with 93% PLLA was significantly more efficient. This sample exhibited a unique surface morphology with long range ordered domains (of the order of 2-3 mum) of edge-on PLLA lamellae that can promote "cell contact guidance." The influence of other factors such as chemical composition, degree of crystallinity, and surface roughness did not play a major role in determining cell preference toward a specific surface for the materials employed in this work.