OBJECTIVE

The purpose of this study was to undertake thorough genetic analysis of the bone morphogenetic protein type 2 receptor (BMPR2) gene in patients with pulmonary arterial hypertension.

METHODS

We conducted a systematic analysis for larger gene rearrangements together with conventional mutation analysis in 152 pulmonary arterial hypertension patients including 43 patients diagnosed as having idiopathic pulmonary arterial hypertension and 10 diagnosed as having familial pulmonary arterial hypertension.

RESULTS

Analysis of the BMPR2 gene revealed each of the four kinds of nonsense and frameshift mutations, one missense mutation, one splice-site mutation, and two types of exonic deletion. For cases in which exons 1-3 were deleted, the 5' and 3' break points were located in the AluY repeat sequences in the 5' side of the adjacent NOP58 gene and in the AluY repeat sequences in intron 3, suggesting an AluY-mediated nonallelic homologous recombination as the mechanism responsible for the deletion. For the case in which exon 10 was deleted, nonhomologous recombination took place between the AluSx site in intron 9 and a unique sequence in intron 10.

CONCLUSIONS

Exonic deletions of BMPR2 account for at least part of BMPR2 mutations associated with heritable pulmonary arterial hypertension in Japan, as previously reported in other populations. One of our cases was mediated via Alu-mediated nonallelic homologous recombination and another was mediated via nonhomologous recombination.

Substantial evidence has indicated that osteoblastic differentiation may be regulated by mechanical loads or <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2). BMP-2-induced in vivo osteogenesis can be significantly enhanced in the presence of mechanical stimuli, revealing the therapeutic potential of the combined application of BMP-2 and mechanical loads in clinical <em>bone</em> diseases (e.g., <em>bone</em> fractures and osteoporosis); however, the underlying mechanisms remain elusive. In this study, we found that cyclic stretch or BMP-2 alone increased the expression of osteoblastic differentiation markers, including alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2), as shown by RT-qPCR, western blot analysis and ALP activity test. Furthermore, our results revealed that cyclic mechanical stretch with <em>10</em>% elongation at 0.1 Hz significantly enhanced the BMP-2-induced upregulation of ALP and Runx2 expression in osteoblast-like MC3T3-E1 cells. Cyclic stretch also inhibited the BMP-2-induced upregulation of Hes-related family bHLH transcription factor with YRPW motif 1 (Hey1, measured by RT-qPCR and immunofluorescence staining), a potent negative regulator of osteogenesis. Moreover, the transient transfection of a Hey1 expression plasmid (pcDNA3.1-Hey1) significantly reversed the effects of cyclic stretch on the BMP-2-induced upregulation of differentiation markers in the MC3T3-E1 cells. This revealed the importance of Hey1 in modulating BMP-2-induced osteoblastic differentiation in response to cyclic stretch. Taken together, our results demonstrated that cyclic stretch enhanced the BMP-2‑induced osteoblastic differentiation through the inhibition of Hey1. The present study broadens our fundamental knowledge of osteoblastic mechanotransduction and also sheds new insight into the mechanisms through which the combined application of BMP-2 and mechanical load promotes osteogenesis.

Membrane-bound transferrin-like <em>protein</em> (MTf) is expressed in parallel with the expression of cartilage-characteristic genes during differentiation of chondrocytes, and the MTf level is much higher in cartilage than in other tissues. To investigate the role of MTf in cartilage, we examined the effects of growth factors on MTf expression in mouse prechondrogenic ATDC5 cells and the effect of MTf overexpression on differentiation of ATDC5 and mouse pluripotent mesenchymal C3H<em>10</em>T1/2 cells. In ATDC5 cultures, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 and transforming growth factor-beta as well as insulin induced MTf mRNA expression when these peptides induced chondrogenic differentiation. Forced expression of rabbit MTf in ATDC5 cells induced aggrecan, type II collagen, matrilin-1, type X collagen mRNAs, and cell-shape changes from fibroblastic cells to spherical chondrocytes. Accordingly, the synthesis and accumulation of proteoglycans were higher in MTf-expressing cultures than in control cultures. These effects of MTf overexpression correlated with the MTf <em>protein</em> level on the cell surface and decreased in the presence of anti-MTf antibody. However, the aggrecan mRNA level in the ATDC5 cells overexpressing MTf was lower than that in wild type ATDC5 cells exposed to <em>10</em> microg/ml insulin. MTf overexpression in C3H<em>10</em>T1/2 cells also induced aggrecan and/or type II collagen mRNA but not the spherical phenotype. These findings suggest that the expression of MTf on the cell surface facilitates the differentiation of prechondrogenic cells, although MTf overexpression alone seems to be insufficient to commit pluripotent mesenchymal cells to the chondrocyte lineage.

Cell fate of lower Müllerian duct epithelium (MDE), to become uterine or vaginal epithelium, is determined by the absence or presence of ΔNp63 expression, respectively. Previously, we showed that SMAD4 and runt-related transcription factor 1 (RUNX1) were independently required for MDE to express ΔNp63. Here, we report that vaginal mesenchyme directs vaginal epithelial cell fate in MDE through paracrine activation of fibroblast growth factor (FGF) receptor-MAPK pathway. In the developing reproductive tract, FGF7 and FGF<em>10</em> were enriched in vaginal mesenchyme, whereas FGF receptor 2IIIb was expressed in epithelia of both the uterus and vagina. When Fgfr2 was inactivated, vaginal MDE underwent uterine cell fate, and this differentiation defect was corrected by activation of MEK-ERK pathway. In vitro, FGF<em>10</em> in combination with <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 and activin A (ActA) was sufficient to induce ΔNp63 in MDE, and ActA was essential for induction of RUNX1 through SMAD-independent pathways. Accordingly, inhibition of type 1 receptors for activin in neonatal mice induced uterine differentiation in vaginal epithelium by down-regulating RUNX1, whereas conditional deletion of Smad2 and Smad3 had no effect on vaginal epithelial differentiation. In conclusion, vaginal epithelial cell fate in MDE is induced by FGF7/<em>10</em>-MAPK, <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4-SMAD, and ActA-RUNX1 pathway activities, and the disruption in any one of these pathways results in conversion from vaginal to uterine epithelial cell fate.

The mechanism of metastasis of osteosarcoma cells to other <em>bones</em> has not yet fully been clarified. The purpose of the present study was to examine whether various factors involve the formation of osteosarcoma metastatic foci in other <em>bones</em>. Immunohistochemically, CD31 expression in osteosarcoma with no <em>bone</em> metastasis and osteosarcoma with <em>bone</em> metastasis was noted in <em>10</em> and 75% of cases, respectively. Met/hepatocyte growth factor (HGF) receptor expression in osteosarcoma with no <em>bone</em> metastasis and osteosarcoma with <em>bone</em> metastasis was noted in 90 and 25% of cases, respectively. <em>Bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) expression in osteosarcoma with no <em>bone</em> metastasis and osteosarcoma with <em>bone</em> metastasis was noted in 20 and 75% of cases, respectively. Metastasis of osteosarcoma cells to other <em>bones</em> was significantly correlated with expression of BMP and CD31 and with no expression of Met/HGF receptor <em>protein</em> in osteosarcoma cells. In contrast, expression of insulin-like growth factor receptor in osteosarcoma cells did not correlate significantly with <em>bone</em> metastasis. These results suggest that formation of metastatic foci of osteosarcoma cells in other <em>bones</em> is regulated by CD31, which is associated with migration between endothelial cells, by BMP, which can induce and activate various mesenchymal cells affecting <em>bone</em> formation, and by escape of effect by HGF, which promotes differentiation of osteosarcoma cells.

Microporous scaffolds designed to improve bony repair have had limited success; therefore, we sought to evaluate whether time-released porous scaffolds with or without recombinant <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (rhBMP-2) could enhance stem cell osteoinduction. Custom-made 15/85 hydroxyapatite/β-tricalcium phosphate scaffolds were left empty (E) or filled with rhBMP-2 (E+), calcium sulfate (CS), or CS and rhBMP-2 (CS+). All scaffolds were placed in media and weighed daily. Conditioned supernatant was analyzed for rhBMP-2 and then used to feed human adipose-derived mesenchymal stem cells (ASCs). Adipose-derived mesenchymal stem cell ALP activity, OSTERIX expression, and <em>bone</em> nodule formation were determined. E scaffolds retained 97% (SD, 2%) of the initial weight, whereas CS scaffolds had a near-linear 30% (SD, 3%) decrease over 60 days. E+ scaffolds released 155 (SD, 5) ng of rhBMP-2 (77%) by day 2. In contrast, CS+ scaffolds released only 30 (SD, 2) ng (<em>10</em>%) by day 2, and the remaining rhBMP-2 was released over 20 days. Conditioned media from E+ scaffolds stimulated the highest ALP activity and OSTERIX expression in ACSs on day 2. However, after day 6, media from CS+ scaffolds stimulated the highest ALP activity and OSTERIX expression in ASCs. Adipose-derived mesenchymal stem cells exposed to day 8 CS+-conditioned media produced significantly more <em>bone</em> nodules (<em>10</em>.1 [SD, 1.7] nodules per high-power field) than all other scaffolds. Interestingly, day 8 conditioned media from CS scaffolds simulated significantly more <em>bone</em> nodules than either E or E+ scaffold (P < 0.05 for both). Time-released hydroxyapatite/β-tricalcium phosphate porosity provides sustained growth factor release, enhances ASC osteoinduction, and may result in better in vivo <em>bone</em> formation.

Aging is associated with decreased neurogenesis in the hippocampus and diminished hippocampus-dependent cognitive functions. Expression of <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 (BMP4) increases with age by more than <em>10</em>-fold in the mouse dentate gyrus while levels of the BMP inhibitor, noggin, decrease. This results in a profound 30-fold increase in phosphorylated-SMAD1/5/8, the effector of canonical BMP signaling. Just as observed in mice, a profound increase in expression of BMP4 is observed in the dentate gyrus of humans with no known cognitive abnormalities. Inhibition of BMP signaling either by overexpression of noggin or transgenic manipulation not only increases neurogenesis in aging mice, but remarkably, is associated with a rescue of cognitive deficits to levels comparable to young mice. Additive benefits are observed when combining inhibition of BMP signaling and environmental enrichment. These findings indicate that increased BMP signaling contributes significantly to impairments in neurogenesis and to cognitive decline associated with aging, and identify this pathway as a potential druggable target for reversing age-related changes in cognition.

<AbstractText>The number of Mesenchymal Stem/Stromal Cells (MSCs) in the human <em>bone</em> marrow (BM) is small compared to other cell types. BM aspirate concentration (BMAC) may be used to increase numbers of MSCs, but the composition of MSC subpopulations and growth factors after processing are unknown. The purpose of this study was to assess the enrichment of stem/progenitor cells and growth factors in BM aspirate by two different commercial concentration devices versus standard BM aspiration.</AbstractText><p><div><b>METHODS</b></div>120 mL of BM was aspirated from the iliac crest of <em>10</em> male donors. Each sample was processed simultaneously by either Emcyte GenesisCS^® (Emcyte) or Harvest SmartPReP2 BMAC (Harvest) devices and compared to untreated BM aspirate. Samples were analyzed with multicolor flow cytometry for cellular viability and expression of stem/progenitor cells markers. Stem/progenitor cell content was verified by quantification of colony forming unit-fibroblasts (CFU-F). Platelet, red blood cell and total nucleated cell (TNC) content were determined using an automated hematology analyzer. Growth factors contents were analyzed with <em>protein</em> quantification assays. Statistical analyses were performed by ANOVA analysis of variance followed by Tukey's multiple comparison test or Wilcoxon matched-pairs signed rank test with p < 0.05 for significance.</p><AbstractText>Cell viability after processing was approximately 90% in all groups. Compared to control, both devices significantly enriched TNCs and platelets, as well as the CD45-CD73+ and CD45-CD73+CD90+ cell populations. Further, Harvest significantly concentrated CD45-CD<em>10</em>+, CD45-CD29+, CD45-CD90+, CD45-CD<em>10</em>5+, CD45-CD119+ cells, and CD45dimCD90+CD271+ MSCs, whereas Emcyte significantly enriched CD45dimCD44+CD271+ MSCs. BM concentration also increased the numbers of CFU-F, platelet-derived growth factor, vascular endothelial growth factor, macrophage colony-stimulating factor, interleukin-1b, VCAM-1 and total <em>protein</em>. Neither system concentrated red blood cells, hematopoietic stem cells or <em>bone</em> <em>morphogenetic</em> <em>proteins</em>.</AbstractText><AbstractText>This data could contribute to the development of BMAC quality control assays as both BMAC systems concentrated platelets, growth factors and non-hematopoietic stem cell subpopulations with distinct phenotypes without loss of cell viability when compared to unprocessed BM.</AbstractText>

<em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) and their receptors are expressed in ovarian theca cells (TC) and granulosa cells (GC) and BMPs have been implicated in the regulation of several aspects of follicle development including thecal androgen production and granulosal oestrogen production. Their potential involvement in luteal function has received less attention. In this study, we first compared relative abundance of mRNA transcripts for BMPs, activin-betaA and BMP/activin receptors in bovine corpus luteum (CL) and follicular theca and granulosa layers before undertaking functional in vitro experiments to test the effect of selected ligands (BMP6 and activin A) on luteinizing bovine TC and GC. Relative to beta-actin transcript abundance, CL tissue contained more BMP4 and -6 mRNA than granulosa, more BMP2 mRNA than theca but much less activin-betaA mRNA than both granulosa and theca. Transcripts for all seven BMP/activin receptors were readily detected in each tissue and two transcripts (BMPRII, ActRIIA) were more abundant in CL than either theca or granulosa, consistent with tissue responsiveness. In vitro luteinization of TC and GC from antral follicles (4-6 mm) was achieved by culturing with 5% serum for 6 days. Treatment with BMP6 (0, 2, <em>10</em>, and 50 ng/ml) and activin A (0, 2, <em>10</em> and 50 ng/ml) under these conditions dose-dependently suppressed forskolin-induced progesterone (P4) secretion from both cell types without affecting cell number. BMP6 reduced forskolin-stimulated upregulation of STAR mRNA and raised 'basal' CYP17A1 mRNA level in theca-lutein cells without affecting expression of CYP11A1 or hydroxy-Delta-5-steroid dehydrogenase, 3 beta- and steroid Delta-isomerase 1 (HSD3B1). In granulosa-lutein cells, STAR transcript abundance was not affected by BMP6, whereas forskolin-induced expression of CYP11A1, HSD3B1, CYP19A1 and oxytocin transcripts was reduced. In both cell types, follistatin attenuated the suppressive effect of activin A and BMP6 on forskolin-induced P4 secretion but had no effect alone. Granulosa-lutein cells secreted low levels of endogenous activin A ( approximately 1 ng/ml); BMP6 reduced this, while raising follistatin secretion thus decreasing activin A:follistatin ratio. Collectively, these findings support inhibitory roles for BMP/activin signalling in luteinization and steroidogenesis in both TC and GC.

BACKGROUND

Non-weight-bearing decreases the femoral head deformity but increases bone resorption without increasing bone formation in an experimental animal model of Legg-Calvé-Perthes disease. We sought to determine if local administration of bone morphogenetic protein (BMP)-2 with or without bisphosphonate can increase the bone formation during the non-weight-bearing treatment in the large animal model of Legg-Calvé-Perthes disease.

METHODS

Eighteen piglets were surgically induced with femoral head ischemia. Immediately following the surgery, all animals received an above-the-knee amputation to enforce local non-weight-bearing (NWB). One to two weeks later, six animals received local BMP-2 to the necrotic head (BMP group), six received local BMP-2 and ibandronate (BMP+IB group), and the remaining six received no treatment (NWB group). All animals were killed at eight weeks after the induction of ischemia. Radiographic, microcomputed tomography (micro-CT), and histomorphometric assessments were performed.

RESULTS

Radiographic assessment showed that the femoral heads in the NWB, BMP, and BMP+IB groups had a decrease of 20%, 14%, and 10%, respectively, in their mean epiphyseal quotient in comparison with the normal control group. Micro-CT analyses showed significantly higher femoral head bone volume in the BMP+IB group than in the BMP group (p = 0.02) and the NWB group (p < 0.001). BMP+IB and BMP groups had a significantly higher trabecular number (p < 0.01) and lower trabecular separation (p < 0.02) than the NWB group. In addition, the osteoclast number per bone surface was significantly lower in the BMP+IB group compared with the NWB group. Calcein labeling showed significantly higher bone formation in the BMP and BMP+IB groups than in the NWB group (p < 0.05). Heterotopic ossification was found in the capsule of four hips in the BMP+IB group but not in the BMP group.

CONCLUSIONS

Administration of BMP-2 with bisphosphonate best decreased bone resorption and increased new bone formation during non-weight-bearing treatment of ischemic osteonecrosis in a pig model, but heterotopic ossification is a concern.

CONCLUSIONS

This preclinical study provides new evidence that BMP-2 with bisphosphonate can effectively prevent the extreme bone loss associated with the non-weight-bearing treatment and increase new bone formation in the femoral head in this animal model of ischemic osteonecrosis.

The aim of this study was to measure the temporal expression of osteogenic genes during the process of <em>bone</em> healing in low-intensity pulsed ultrasound (LIPUS) treated <em>bone</em> defects by means of histopathologic and real-time polymerase chain reaction (PCR) analysis. Animals were randomly distributed into two groups (n = 30): control group (<em>bone</em> defect without treatment) and LIPUS treated (<em>bone</em> defect treated with LIPUS). On days 7, 13 and 25 postinjury, <em>10</em> rats per group were sacrificed. Rats were treated with a 30 mW/cm(2) LIPUS. The results pointed out intense new <em>bone</em> formation surrounded by highly vascularized connective tissue presenting a slight osteogenic activity, with primary <em>bone</em> deposition was observed in the group exposed to LIPUS in the intermediary (13 days) and late stages of repair (25 days) in the treated animals. In addition, quantitative real-time polymerase chain reaction (RT-qPCR) showed an upregulation of <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 (BMP4), osteocalcin and Runx2 genes 7 days after the surgery. In the intermediary period, there was no increase in the expression. The expression of alkaline phosphatase, BMP4 and Runx2 was significantly increased at the last period. Our results indicate that LIPUS therapy improves <em>bone</em> repair in rats and upregulated osteogenic genes, mainly at the late stages of recovery.

Ageing is the main risk factor for osteoarthritis (OA). We investigated if expression of transforming growth factor β (TGFβ)-family components, a family which is crucial for the maintenance of healthy articular cartilage, is altered during ageing in cartilage. Moreover, we investigated the functional significance of selected age-related changes.

Age-related changes in expression of TGFβ-family members were analysed by quantitative PCR in healthy articular cartilage obtained from 42 cows (age: ¾-<em>10</em> years). To obtain functional insight of selected changes, cartilage explants were stimulated with TGFβ1 or <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) 9, and TGFβ1 and BMP response genes were measured.

Age-related cartilage thinning and loss of collagen type 2a1 expression (∼256-fold) was observed, validating our data set for studying ageing in cartilage. Expression of the TGFβ-family type I receptors; bAlk2, bAlk3, bAlk4 and bAlk5 dropped significantly with advancing age, whereas bAlk1 expression did not. Of the type II receptors, expression of bBmpr2 decreased significantly. Type III receptor expression was unaffected by ageing. Expression of the ligands bTgfb1 and bGdf5 also decreased with age. In explants, an age-related decrease in TGFβ1-response was observed for the pSmad3-dependent gene bSerpine1 (P = 0.016). In contrast, ageing did not affect BMP9 signalling, an Alk1 ligand, as measured by expression of the pSmad1/5 dependent gene bId1.

Ageing negatively affects both the TGFβ-ALK5 and BMP-BMPR signalling routes, and aged chondrocytes display a lowered pSmad3-dependent response to TGFβ1. Because pSmad3 signalling is essential for cartilage homeostasis, we propose that this change contributes to OA development.

OBJECTIVE

To detect expression of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) in oocytes, and their receptor type 2 receptor for BMPs (BMPR2) in cumulus cells in women with polycystic ovary syndrome (PCOS) undergoing in vitro fertilization (IVF), and determine if BMPR2, BMP15, and GDF9 expression correlate with hyperandrogenism in FF of PCOS patients.

METHODS

Prospective case-control study. Eighteen MII-oocytes and their respective cumulus cells were obtained from 18 patients with PCOS, and 48 MII-oocytes and cumulus cells (CCs) from 35 controls, both subjected to controlled ovarian hyperstimulation (COH), and follicular fluid (FF) was collected from small (10-14 mm) and large (>18 mm) follicles. RNeasy Micro Kit (Qiagen) was used for RNA extraction and gene expression was quantified in each oocyte individually and in microdissected cumulus cells from cumulus-oocyte complexes retrieved from preovulatory follicles using qRT-PCR. Chemiluminescence and RIA assays were used for hormone assays.

RESULTS

BMP15 and GDF9 expression per oocyte was higher among women with PCOS than the control group. A positive correlation was found between BMPR2 transcripts and hyperandrogenism in FF of PCOS patients. Progesterone values in FF were lower in the PCOS group.

CONCLUSIONS

We inferred that BMP15 and GDF9 transcript levels increase in mature PCOS oocytes after COH, and might inhibit the progesterone secretion by follicular cells in PCOS follicles, preventing premature luteinization in cumulus cells. BMPR2 expression in PCOS cumulus cells might be regulated by androgens.

The endodermal pouches are a series of reiterated structures that segment the pharyngeal arches and help pattern the vertebrate face. Multiple pathways regulate the complex process of endodermal development, including the <em>Bone</em> <em>morphogenetic</em> <em>protein</em> (Bmp) pathway. However, the role of Bmp signaling in pouch morphogenesis is poorly understood. Using genetic and chemical inhibitor approaches, we show that pouch morphogenesis requires Bmp signaling from <em>10</em>-18 h post-fertilization, immediately following gastrulation. Blocking Bmp signaling during this window results in morphological defects to the pouches and craniofacial skeleton. Using genetic chimeras we show that Bmp signals directly to the endoderm for proper morphogenesis. Time-lapse imaging and analysis of reporter transgenics show that Bmp signaling is necessary for pouch outpocketing via the Fibroblast growth factor (Fgf) pathway. Double loss-of-function analyses demonstrate that Bmp and Fgf signaling interact synergistically in craniofacial development. Collectively, our analyses shed light on the tissue and signaling interactions that regulate development of the vertebrate face.

The purpose of this investigation was to study the effect of <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP), transforming growth factor beta-induced gene h3 (betaig-h3), and chitosan on early bony consolidation in distraction osteogenesis in a dog model. Sixteen dogs were used for this study. The lateral surface of the mandibular body was exposed in the subperiosteal plane and the vertical osteotomy on the mandibular body was extended downward. An external distraction device was applied to the mandibular body, and the mandibular distraction was started 5 days after the operation at a rate of 2 mm/day up to a <em>10</em>-mm distraction after 5 days. The experimental group was then divided into a control group, a BMP group, a betaig-h3 group, and a chitosan group, depending on the type of implantation material used in the distracted area. On the same day after completing the distraction, BMP, betaig-h3, or chitosan was implanted into the distracted area. No material was implanted into the distracted area in the control group. After implanting the materials, the distraction device was left in place for 7 weeks to allow for bony consolidation. Four dogs were allocated to each group. Two dogs in each group, a total of eight dogs, were killed 4 weeks after completing the distraction and the other eight dogs were killed after 7 weeks. Serial radiographs were obtained every week after completing the distraction. New <em>bone</em> was generated in the distracted zone in all groups. In the BMP group, the formation of active woven <em>bone</em> was observed throughout the distracted zone, and the new <em>bone</em> appeared to be nearly normal cortical <em>bone</em> 7 weeks after implantation. In the betaig-h3 and chitosan groups, the development of new <em>bone</em> was observed in the distracted zone after 7 weeks; however, the amount was less than that in the BMP group. In the control group, the new <em>bone</em> was observed at the edges of the distracted zone. These findings suggest that BMP seems to be very effective in early bony consolidation in distraction osteogenesis.

Oxymatrine (OMT) is able to effectively protect against hepatic fibrosis because of its anti-inflammatory property, while the underlying mechanism remains incompletely understood. In this study, forty rats were randomly divided into five groups: control group, model group (carbon tetrachloride, CCl4) and three OMT treatment groups (30, 60, 120mg/kg). After CCl4 alone, the fibrosis score was 20.2±0.8, and the level of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hydroxyproline content, and collagen I expression was elevated, but OMT blunted these parameters. Treatment with OMT prevented CCl4-induced increases in expression of pro-inflammatory and pro-fibrotic cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α, meanwhile OMT promoted the expression of anti-inflammatory and anti-fibrotic factors such as interleukin (IL)-<em>10</em> and <em>bone</em> <em>morphogenetic</em> <em>protein</em> and activin membrane-bound inhibitor (Bambi). Moreover, lipopolysaccharides (LPS) and high mobility group box-1 (HMGB1), which activates Toll-like receptor 4 (TLR4) and modulate hepatic fibrogenesis through hepatic stellate cells (HSCs) or Kupffer cells, were significantly decreased by OMT treatment. These results were further supported by in vitro data. First, OMT suppressed the expression of TLR4 and its downstream pro-inflammatory cytokines, lowered the level of HMGB1, TGF-β1 in macrophages. Then, OMT promoted Bambi expression and thereby inhibited activation of HSCs mediated by transforming growth factor (TGF)-β1. In conclusion, this study showed that OMT could effectively attenuate the CCl4-induced hepatic fibrosis, and this effect may be due to modulation of TLR4-dependent inflammatory and TGF-β1 signaling pathways.

BACKGROUND

Current theory on normal cranial suture fusion entrusts the dura with the regulatory role. Studies suggest that the dura responds to stress with changes in gene expression. Noggin (bone morphogenetic protein inhibitor) expression is decreased in normal (rat and mouse) cranial suture fusion, but its role in craniosynostosis and the response to stress has not been studied.

METHODS

Posterior frontal (fusing) and sagittal (patent) rat cranial sutures were held static, oscillated, or distracted for 10 days in an organ culture microdistraction device beginning at 5 days of age (n = 30 sutures, or 10 sutures per group). The percentage of fusion equaled the score received for bony closure. Noggin, Runx2, and alkaline phosphatase expression was localized by immunohistochemistry for all groups.

RESULTS

Both the posterior frontal and sagittal sutures demonstrated a significant (p < 0.05) increase in fusion percentage with oscillation relative to the static control. Noggin was not expressed in the fusing posterior frontal suture but was expressed in the normally patent sagittal suture. Conversely, Runx2 was expressed in the posterior frontal suture but not in the sagittal suture. However, when a mechanical stress was applied, both the posterior frontal and sagittal sutures expressed Runx2 but not Noggin, as in the static fusing suture.

CONCLUSIONS

The application of mechanical stress to cranial sutures results in fusion of both the posterior frontal suture and the normally patent sagittal suture. Runx2 is expressed but Noggin is not expressed. Thus, mechanical stress influences sutural fusion and may play a role in craniosynostosis.

<em>Bone</em>-stimulatory therapeutics include <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (e.g. BMP2), parathyroid hormone, and antibody-based suppression of WNT antagonists. Inhibition of the epigenetic enzyme Enhancer of Zeste Homolog 2 (EZH2) is both <em>bone</em>-anabolic and osteo-protective. EZH2 inhibition stimulates key components of <em>bone</em>-stimulatory signaling pathways, including the BMP2 signaling cascade. Because of high costs and adverse effects associated with BMP2 use, here we investigated whether BMP2 dosing can be reduced by co-treatment with EZH2 inhibitors. Co-administration of BMP2 with the EZH2 inhibitor GSK126 enhanced differentiation of murine (MC3T3) osteoblasts, reflected by increased alkaline phosphatase activity, alizarin red staining, and expression of <em>bone</em>-related marker genes (e.g. Bglap and Phospho1). Strikingly, co-treatment with BMP2 (<em>10</em> ng/ml) and GSK126 (5 μM) was synergistic and was as effective as 50 ng/ml BMP2 at inducing MC3T3 osteoblastogenesis. Similarly, the BMP2-GSK126 co-treatment stimulated osteogenic differentiation of human <em>bone</em> marrow-derived mesenchymal stem/stromal cells, reflected by induction of key osteogenic markers (e.g. Osterix/SP7 and IBSP). A combination of BMP2 (300 ng local) and GSK126 (5 μg local and 5 days of 50 mg/kg systemic) yielded more consistent <em>bone</em> healing than single treatments with either compound in a mouse calvarial critical-sized defect model according to results from μCT, histomorphometry, and surgical grading of qualitative X-rays. We conclude that EZH2 inhibition facilitates BMP2-mediated induction of osteogenic differentiation of progenitor cells and maturation of committed osteoblasts. We propose that epigenetic priming, coupled with <em>bone</em> anabolic agents, enhances osteogenesis and could be leveraged in therapeutic strategies to improve <em>bone</em> mass.

Osteoporosis is the second most-prevalent epidemiologic disease in the aging population worldwide. Cross-sectional and retrospective evidence indicates that tea consumption can mitigate <em>bone</em> loss and reduce risk of osteoporotic fractures. Tea polyphenols enhance osteoblastogenesis and suppress osteoclastogenesis in vitro. Previously, we showed that (-)-epigallocatechin-3-gallate (EGCG), one of the green tea polyphenols, increased osteogenic differentiation of murine <em>bone</em> marrow mesenchymal stem cells (BMSCs) by increasing the mRNA expression of osteogenesis-related genes, alkaline phosphatase activity and, eventually, mineralization. We also found that EGCG could mitigate <em>bone</em> loss and improve <em>bone</em> microarchitecture in ovariectomy-induced osteopenic rats, as well as enhancing <em>bone</em> defect healing partially via <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP2). The present study investigated the effects of EGCG in human BMSCs. We found that EGCG, at concentrations of both 1 and <em>10</em> µmol/L, can increase mRNA expression of BMP2, Runx2, alkaline phosphatase (ALP), osteonectin and osteocalcin 48 h after treatment. EGCG increased ALP activity both 7 and 14 days after treatment. Furthermore, EGCG can also enhance mineralization two weeks after treatment. EGCG without antioxidants also can enhance mineralization. In conclusion, EGCG can increase mRNA expression of BMP2 and subsequent osteogenic-related genes including Runx2, ALP, osteonectin and osteocalcin. EGCG further increased ALP activity and mineralization. Loss of antioxidant activity can still enhance mineralization of human BMSCs (hBMSCs).

Hereditary hemorrhagic telangiectasia (HHT) is a potentially life-threatening genetic vascular disorder caused by loss-of-function mutations in the genes encoding activin receptor-like kinase 1 (ALK1), endoglin, Smad4, and <em>bone</em> <em>morphogenetic</em> <em>protein</em> 9 (BMP9). Injections of mouse neonates with BMP9/<em>10</em> blocking antibodies lead to HHT-like vascular defects in the postnatal retinal angiogenesis model. Mothers and their newborns share the same immunity through the transfer of maternal antibodies during lactation. Here, we investigated whether the transmammary delivery route could improve the ease and consistency of administering anti-BMP9/<em>10</em> antibodies in the postnatal retinal angiogenesis model. We found that anti-BMP9/<em>10</em> antibodies, when intraperitoneally injected into lactating dams, are efficiently transferred into the blood circulation of lactationally-exposed neonatal pups. Strikingly, pups receiving anti-BMP9/<em>10</em> antibodies via lactation displayed consistent and robust vascular pathology in the retina, which included hypervascularization and defects in arteriovenous specification, as well as the presence of multiple and massive arteriovenous malformations. Furthermore, RNA-Seq analyses of neonatal retinas identified an increase in the key pro-angiogenic factor, angiopoietin-2, as the most significant change in gene expression triggered by the transmammary delivery of anti-BMP9/<em>10</em> antibodies. Transmammary-delivered BMP9/<em>10</em> immunoblocking in the mouse neonatal retina is therefore a practical, noninvasive, reliable, and robust model of HHT vascular pathology.

1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] induces osteoclast formation via induction of receptor activator of NF-κB ligand (RANKL, also called TNF-related activation-induced cytokine: TRANCE) in osteoblasts. In cocultures of mouse <em>bone</em> marrow cells and osteoblasts, 1,25(OH)(2)D(3) induced osteoclast formation in a dose-dependent manner, with maximum osteoclast formation observed at concentrations greater than <em>10</em>(-9) M of 1,25(OH)(2)D(3). In the presence of <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP-2), the maximum formation of osteoclasts was seen with lower concentrations of 1,25(OH)(2)D(3) (greater than <em>10</em>(-11) M), suggesting that BMP-2 enhances osteoclast formation induced by 1,25(OH)(2)D(3). In addition, the expressions of RANKL mRNA and <em>proteins</em> were induced by 1,25(OH)(2)D(3) in osteoblasts, and further upregulated by BMP-2. In mouse <em>bone</em> marrow cell cultures without 1,25(OH)(2)D(3), BMP-2 did not enhance osteoclast differentiation induced by recombinant RANKL and macrophage colony-stimulating factor (M-CSF), indicating that BMP-2 does not target osteoclast precursors. Furthermore, BMP-2 up-regulated the expression level of vitamin D receptor (VDR) in osteoblasts. These results suggest that BMP-2 regulates mouse osteoclast differentiation via upregulation of RANKL in osteoblasts induced by 1,25(OH)(2)D(3).

METHODS

Basic science rodent model of bone morphogenetic protein-2 (BMP-2) soft-tissue inflammation.

OBJECTIVE

This study investigated the anti-inflammatory effect of human dose equivalent (HDE) dexamethasone (DM) for treatment of BMP-2-related soft-tissue inflammation in a rodent model and suggests an appropriate dose for utilization in the clinical practice of spine surgeons.

BACKGROUND

BMP-2 is frequently used in spinal surgery to augment fusion. Yet, side effects of soft-tissue inflammation have been observed. DM decreases proinflammatory cytokine production and cellular immune response. However, the anti-inflammatory effects of HDE DM in a rodent model of BMP-2-associated soft-tissue inflammation have not been reported.

METHODS

Five, 10, and 15 mg of HDE DM were administered 3 times perioperatively to rodent cohorts receiving BMP-2 paraspinal implants and compared against BMP-2 only positive controls and phosphate buffer negative controls (n = 6 subjects per group). Histopathology, magnetic resonance imaging, and gross dissection were used as measures of cellular, edematous, and exudative inflammatory response. Serial killings were made on day 2 and day 7 postoperatively.

RESULTS

Magnetic resonance imaging volume rendering demonstrated inflammatory edema decreased by 49% from 605.4 mm to 304.03 mm in subjects treated with 5, 10, or 15 mg of HDE DM (P < 0.05). Histopathological analysis demonstrated inflammatory cross-sectional area decreased 28.8% from 1.84 mm to 1.31 mm in subjects treated with 5, 10 or 15 mg of HDE DM (P < 0.05). Immune cellular infiltration depth decreased 38.5% from 0.26 mm to 0.16 in subjects treated with 15 mg of HDE DM (P < 0.05). Gross anatomical inflammatory exudates were prevented in 100% of subjects treated with 10 or 15 mg of HDE DM (P < 0.05).

CONCLUSIONS

Low-dose DM administration is effective in controlling the cellular inflammation and edema resulting from BMP-2. Ten or 15 mg of DM might be considered by spine surgeons for controlling the inflammation and edema seen in spine surgery with BMP-2.

OBJECTIVE

To determine the possibility of synergistically enhancing orthodontic tooth movement (OTM) and bone formation in vivo by administering bone morphogenetic protein type 2 (BMP-2) on the tension side or in combination with corticotomy on the pressure side.

METHODS

The sample consisted of 56 Wistar rats that were subjected to experimental OTM for 32 days using a split-mouth design. The sample was divided into 4 groups: a control group, a corticotomy group, a BMP-2 group, and a corticotomy plus BMP-2 group. An OTM force of 10 cN was applied to each group. BMP-2 18 μL was administered locally on the tension side alone or in conjunction with corticotomy and then compared with the controls using fluorescence-based tartrate-resistant acid phosphatase staining for osteoclast counts, histologic bone resorption, and clinical OTM results.

RESULTS

Corticotomy surgery increased the OTM rate (P < .05) by more than 20%. The injection of BMP-2 alone on the tension side did not induce significant changes in the degree of OTM compared with the vehicle-treated or control group (P>> .05). When BMP-2 was combined with corticotomy on the tension and pressure sides (corticotomy plus BMP-2 group), respectively, nonsignificant OTM rates were observed (P>> .05) compared with the controls; however, decreased osteoclast counts, bone resorption, and clinical results were observed in the corticotomy plus BMP-2 group.

CONCLUSIONS

In contrast to reports published to date, the present preliminary study suggests that there are limits to OTM acceleration by bone formation on the tension side and agrees with the idea that there is a single continuous periodontal compartment in OTM, rather than a pressure side and a tension side.

BACKGROUND

This study aimed to investigate new bone formation using recombinant human bone morphogenetic protein 2 (rhBMP-2) and locally applied bisphosphonate in rat calvarial defects.

METHODS

Thirty-six rats were studied. Two circular 5 mm diameter bony defect were formed in the calvaria using a trephine bur. The bony defect were grafted with Bio-Oss® only (group 1, n = 9), Bio-Oss® wetted with rhBMP-2 (group 2, n = 9), Bio-Oss® wetted with rhBMP-2 and 1 mM alendronate (group 3, n = 9) and Bio-Oss® wetted with rhBMP-2 and 10 mM alendronate (group 4, n = 9). In each group, three animals were euthanized at 2, 4 and 8 weeks after surgery, respectively. The specimens were then analyzed by histology, histomorphometry and immunohistochemistry analysis.

RESULTS

There were significant decrease of bone formation area (p < 0.05) between group 4 and group 2, 3. Group 3 showed increase of new bone formation compared to group 2. In immunohistochemistry, collagen type I and osteoprotegerin (OPG) didn't show any difference. However, receptor activator of nuclear factor κB ligand (RANKL) decreased with time dependent except group 4.

CONCLUSIONS

Low concentration bisphosphonate and rhBMP-2 have synergic effect on bone regeneration and this is result from the decreased activity of RANKL of osteoblast.