Phillygenin prevents osteoclast differentiation and bone loss by targeting RhoA.

Forsythia suspensa tea is a popular traditional Chinese medicine decoction for its healthy and therapeutic benefits. However, its effects in bone metabolism were not clear. In recent study, we uncovered anti-osteoclastogenesis property of Phillygenin (Phi), a compound abundant in Forsythia suspensa leaves, and aimed to investigate the effect and mechanism of Phi on bone metabolism in vivo and in vitro. Lipopolysaccharides-induced murine calvaria osteolysis and ovariectomy-induced bone loss animal models were used to identify the bone-protective effect of Phi in vivo and micro-CT, pQCT, and TRAP staining were applied. We used CCK8, TUNEL, BrdU, and TRAP staining to evaluate the efficacy of Phi on the proliferation and formation of OCs in primary mBMMs. RNA sequence, activity-based protein profiling, molecular docking, G-LISA, and WB were used to inspect the target and underlying mechanism of Phi's actions in mBMMs. We found Phi significantly inhibited bone resorption in vivo and inhibited mBMMs osteoclastogenesis in vitro. Ras homolog gene family member A (RhoA) was identified as the direct target of Phi. It counteracted the effects of RhoA activator and acted as a RhoA inhibitor. By targeting RhoA, Phi modulated Rho-associated coiled-coil containing protein kinase 1 (ROCK1) activity and regulated its downstream NF-κB/NFATc1/c-fos pathway. Furthermore, Phi depressed the disassembling of F-actin ring through cofilin and myosin1a. Our findings provided Phi as a potential option for treating bone loss diseases by targeting RhoA and highlighted the importance of F. suspensa as a preventive approach in bone disorders.

Clinical Characteristics, Mechanism, and Outcome of Humeral Shaft Fractures Sustained during Arm Wrestling in Young Men: A Retrospective Study.

OBJECTIVE: Humeral fractures are common in arm wrestling and other sports and military activities requiring similar movements; however, the precise mechanism is poorly understood. Here, we present an overview of the characteristics, possible mechanisms, and treatment of humeral shaft fractures sustained during arm wrestling. METHODS: We reviewed 8 years (January 2013 to January 2021) of medical records and retrospectively analyzed data from 27 patients with humeral shaft fractures sustained during arm wrestling. The clinical data included sex, age, affected arm, alcohol consumption, muscle warm-up, history of competitive participation, opponents' characteristics, wrist position, and post-fracture radial nerve injuries. The fracture configurations were radiographically assessed and analyzed. Surgical management included single or dual plating. Scores on the Disability of the Arm, Shoulder, and Hand questionnaire (DASH) were evaluated preoperatively and postoperatively at the last follow-up visit. RESULTS: All fractures sustained during arm wrestling were spiral fractures of the distal third of the humerus. Of these, 11 were 12-A1 type and 16 were 12-B2 type with a wedge fragment. The two subtypes differed in the total fracture line length (12-A1: 0.18 ± 0.04; 12-B2: 0.23 ± 0.04; P < 0.001). The radial nerve injury rate was 0/11 (0%) in patients with 12-A1 type fractures and 7/16 (43.8%) in patients with 12-B2 type fractures (P = 0.011). Most patients were young men (mean age, ~25 years) with a history of competitively participating in arm wrestling for >2 years. Cold seasonal temperatures and a lack of warm-ups increased the risk of injury. All patients showed improved DASH scores at the last follow-up (12-A1:77.82 ± 5.14 to 10.25 [5.38]; 12-B2:78.91 ± 7.46 to 8.95 [3.17]; P < 0.001). No significant differences were observed among the different surgical treatments. CONCLUSIONS: Individuals who participated in arm wrestling were at risk of humeral shaft fractures (type 12-A1 or 12-B2). The 12-B2 type occurs with a wedge fragment and is frequently accompanied by radial nerve injuries. The characteristics of arm-wrestling fractures and the mechanism(s) underlying these fractures can help orthopedic surgeons understand the causes of these fractures and similar fractures sustained in traditional sports. This understanding will help surgeons choose more effective surgical treatments that will result in more desirable functional outcomes and a faster return to work.

Adipose stem cells-derived exosomes modified gelatin sponge promotes bone regeneration.

Background: Large bone defects resulting from trauma and diseases still a great challenge for the surgeons. Exosomes modified tissue engineering scaffolds are one of the promising cell-free approach for repairing the defects. Despite extensive knowledge of the variety kinds of exosomes promote tissue regeneration, little is known of the effect and mechanism for the adipose stem cells-derived exosomes (ADSCs-Exos) on bone defect repair. This study aimed to explore whether ADSCs-Exos and ADSCs-Exos modified tissue engineering scaffold promotes bone defects repair. Material/Methods: ADSCs-Exos were isolated and identified by transmission electron microscopy nanoparticle tracking analysis, and western blot. Rat bone marrow mesenchymal stem cells (BMSCs) were exposed to ADSCs-Exos. The CCK-8 assay, scratch wound assay, alkaline phosphatase activity assay, and alizarin red staining were used to evaluate the proliferation, migration, and osteogenic differentiation of BMSCs. Subsequently, a bio-scaffold, ADSCs-Exos modified gelatin sponge/polydopamine scaffold (GS-PDA-Exos), were prepared. After characterized by scanning electron microscopy and exosomes release assay, the repair effect of the GS-PDA-Exos scaffold on BMSCs and bone defects was evaluated in vitro and in vivo. Results: The diameter of ADSCs-exos is around 122.1 nm and high expressed exosome-specific markers CD9 and CD63. ADSCs-Exos promote the proliferation migration and osteogenic differentiation of BMSCs. ADSCs-Exos was combined with gelatin sponge by polydopamine (PDA)coating and released slowly. After exposed to the GS-PDA-Exos scaffold, BMSCs have more calcium nodules with osteoinductive medium and higher expression the mRNA of osteogenic related genes compared with other groups. The quantitative analysis of all micro-CT parameters showed that GS-PDA-Exos scaffold promote new bone formed in the femur defect model in vivo and confirmed by histological analysis. Conclusion: This study demonstrates the repair efficacy of ADSCs-Exos in bone defects, ADSCs-Exos modified scaffold showing a huge potential in the treatment of large bone defects.

The Safety Assessment of Irrigation Fluid Management for Shoulder Arthroscopy and Its Effect on Postoperative Efficacy.

OBJECTIVE: Fluid extravasation is a potentially dangerous complication associated with shoulder arthroscopy. Most relevant studies have involved respiratory system, while the primary purpose was to reveal the effects of the fluid extravasation on cardiovascular system and postoperative function. METHODS: The clinical data of 92 patients was retrospective analyzed, in which 84 cases with rotator cuff injury, three cases with shoulder instability, three cases with fractures of the greater tuberosity of the humerus, and two cases with frozen shoulder. All the patients were undergoing shoulder arthroscopy. The relationship between the basic information of the patients and cardiac index (CI) or pulse pressure variation (PPV) were evaluated by linear regression analysis. The change of CI or PPV at different states were evaluated by the one-way analysis of variance. The liquid retention (TR) and postoperative clinical outcomes was analyzed using linear regression. RESULTS: The preoperative CI was affected by anesthesia status and body position, while PPV was not affected. Multivariate mixed-effects model analysis of CI found that there was a statistically significant difference in groups of older than 55 years old and those with obesity (BMI > 24). After the operation, the retention of irrigation fluid significantly influenced the circumference of the deltoid (P < 0.001 (95%CI: [0.30, 1.00])), but not on the circumference of the deltoid, neck, and arm. The multivariate analysis of the American Shoulder and Elbow Surgery (ASES) scores at 3 and 6 months after surgery showed that the fluid retention volume was correlated with the ASES score at 3 months after surgery, especially when the retention volume was greater than 2 L (P = 0.001 (95%). %CI: [-12.49, -3.22]). CONCLUSION: The retention of irrigation fluid after shoulder arthroscopic surgery causes swelling of local limbs, and has an effect on peripheral blood vessels, which is mainly reflected in its influence on PPV and the postoperative function. Therefore, surgeons need to improve their surgical technique, shorten the operation time and reduce fluid retention.

Tea polyphenol/glycerol-treated double-network hydrogel with enhanced mechanical stability and anti-drying, antioxidant and antibacterial properties for accelerating wound healing.

Frequent dressing changes can result in secondary wound damage. Therefore, it is of great significance to construct a wound dressing that can be used for a long time without changing. Here, a double-network hydrogel was synthesized through hydrogen bonding interactions of tea polyphenol (TP)/glycerol with photo-crosslinked N-acryloyl glycinamide (NAGA), gelatin methacrylate (GelMA), and nanoclay hydrogel. The glycerol/water solvent slowed the diffusion of TP into the NAGA/GelMA/Laponite (NGL)hydrogel, thereby avoiding excessive crosslinking, and forming a uniform network. The hydrogel exhibited excellent water retention (84% within 28 days). Additionally, due to the hygroscopicity of glycerol, the hydrogel's mechanical strength (0.73-1.14 MPa) and tensile strain (207%-353%) increased further after 14 days in an open environment. Additionally, the hydrogel exhibited superior anti-ultraviolet and antioxidant properties, which effectively alleviated the wound site's oxidative stress and accelerated wound healing. Moreover, antibacterial activity was observed against both E. coli and S. aureus in the hydrogel wound dressing. Thus, by promoting wound closure, angiogenesis and collagen deposition, the double-network NGLG20/TG hydrogel dressing can successfully accelerate wound healing. The multifunctional double-network hydrogel, therefore, shows immense potential as an ideal candidate for wound dressings because it is long-lasting and prevents secondary damage caused by frequent dressing changes.

Bipolar Metal Flexible Electrospun Fibrous Membrane Based on Metal-Organic Framework for Gradient Healing of Tendon-to-Bone Interface Regeneration.

Metal ions play a significant role in tissue repair, with widely application in clinical treatment. However, the therapeutic effect of metal ions is always limited due to metabolization and narrow repair capability. Here, a bipolar metal flexible electrospun fibrous membrane based on a metal-organic framework (MOF), which is bioinspired by the gradient structure of the tendon-to-bone interface, with a combination of regulating osteoblasts differentiation and angiogenesis properties, is constructed successfully by a continuous electrospinning technique and matching the longitudinal space morphology for synchronous regeneration. Furthermore, the MOF, acting as carriers, can not only achieve the sustainable release of metal ions, but promote the osteogenesis and tenogenesis on the scaffold. The in vitro data show that this novel hierarchical structure can accelerate the tenogenesis, the biomineralization, and angiogenesis. Moreover, in the in vivo experiment, the flexible fibrous membrane can promote tendon and bone tissue repair, and fibrocartilage reconstruction, to realize the multiple tissue synchronous regeneration at the damaged tendon-to-bone interface. Altogether, this newly developed bipolar metal flexible electrospun fibrous membrane based on a MOF, as a new biomimetic flexible scaffold, has great potential in reconstruct the tissue damage, especially gradient tissue damage.

Circular RNA circLGMN facilitates glioblastoma progression by targeting miR-127-3p/LGMN axis.

Glioblastoma (GBM) is one of the most devastating cancers and is characterized by rapid cell proliferation and aggressive invasiveness. Legumain (LGMN), a substrate-specific protease, is associated with poor progression of GBM. Circular RNAs (circRNAs) are aberrantly expressed in various cancers and play crucial roles in tumor progression; however, the functional roles of circRNAs originating from LGMN remain largely unknown in GBM. Herein, we found that hsa_circ_0033009 (circLGMN) was the most abundantly expressed circRNA derived from LGMN. CircLGMN was upregulated in high-grade glioma (HGG), and high expression of circLGMN was associated with poor prognosis in patients with glioma. CircLGMN overexpression promoted GBM cell proliferation and enhanced cell invasion. Mechanistically, circLGMN acts as a sponge for miR-127-3p, and prevents miR-127-3p-mediated degradation of LGMN mRNA, ultimately leading to increased LGMN protein expression. Treatment with miR-127-3p mimic suppressed proliferation and reduced invasion of GBM cells overexpressing circLGMN. Moreover, circLGMN overexpression promoted GBM malignancy in vivo, while miR-127-3p overexpression alleviated this effect. Taken together, circLGMN is a novel tumor-promoting circRNA that acts by sponging miR-127-3p, which ultimately leads to LGMN upregulation. Thus, targeting the circLGMN/miR-127-3p/LGMN axis might be a promising strategy for GBM treatment. More importantly, the discovery of the self-regulatory mechanism of LGMN expression by circLGMN, will facilitate further research on LGMN.

Balloon Test Occlusion of Internal Carotid Artery in Recurrent Nasopharyngeal Carcinoma Before Endoscopic Nasopharyngectomy: A Single Center Experience.

OBJECTIVES: Endoscopic nasopharyngectomy (ENPG) is a promising way in treating recurrent nasopharyngeal carcinoma (rNPC), but sometimes may require therapeutic internal carotid artery (ICA) occlusion beforehand. Balloon test occlusion (BTO) is performed to evaluate cerebral ischemic tolerance for ICA sacrifice. However, absence of neurological deficits during BTO does not preclude occur of delayed cerebral ischemia after permanent ICA occlusion. In this study, we evaluate the utility of near-infrared spectroscopy (NIRS) regional cerebral oxygen saturation (rSO2) monitoring during ICA BTO to quantify cerebral ischemic tolerance and to identify the valid cut-off values for safe carotid artery occlusion. This study also aims to find out angiographic findings of cerebral collateral circulation to predict ICA BTO results simultaneously. MATERIAL AND METHODS: 87 BTO of ICA were performed from November 2018 to November 2020 at authors' institution. 79 angiographies of collateral flow were performed in time during BTO and classified into several Subgroups and Types according to their anatomic and collateral flow configurations. 62 of 87 cases accepted monitoring of cerebral rSO2. Categorical variables were compared by using Fisher exact tests and Mann-Whitney U tests. Receiver operating characteristic curve analysis was used to determine the most suitable cut-off value. RESULTS: The most suitable cut-off △rSO2 value for detecting BTO-positive group obtained through ROC curve analysis was 5% (sensitivity: 100%, specificity: 86%). NIRS rSO2 monitoring wasn't able to detect BTO false-negative results (p = 0.310). The anterior Circle was functionally much more important than the posterior Circle among the primary collateral pathways. The presence of secondary collateral pathways was considered as a sign of deteriorated cerebral hemodynamic condition during ICA BTO. In Types 5 and 6, reverse blood flow to the ICA during BTO protected patients from delayed cerebral ischemia after therapeutic ICA occlusion (p = 0.0357). In Subgroup IV, absence of the posterior Circle was significantly associated with BTO-positive results (p = 0.0426). CONCLUSION: Angiography of cerebral collateral circulation during ICA BTO is significantly correlated with ICA BTO results. Angiographic ICA BTO can be performed in conjunction with NIRS cerebral oximeter for its advantage of being noninvasive, real-time, cost-effective, simple for operation and most importantly for its correct prediction of most rSO2 outcomes of ICA sacrifice. However, in order to ensure a safe carotid artery occlusion, more quantitative adjunctive blood flow measurements are recommended when angiography of cerebral collateral circulation doesn't fully support rSO2 outcome among clinically ICA BTO-negative cases.

Self-healing polyurethane-elastomer with mechanical tunability for multiple biomedical applications in vivo.

The unique properties of self-healing materials hold great potential in the field of biomedical engineering. Although previous studies have focused on the design and synthesis of self-healing materials, their application in in vivo settings remains limited. Here, we design a series of biodegradable and biocompatible self-healing elastomers (SHEs) with tunable mechanical properties, and apply them to various disease models in vivo, in order to test their reparative potential in multiple tissues and at physiological conditions. We validate the effectiveness of SHEs as promising therapies for aortic aneurysm, nerve coaptation and bone immobilization in three animal models. The data presented here support the translation potential of SHEs in diverse settings, and pave the way for the development of self-healing materials in clinical contexts.

Gradient bimetallic ion-based hydrogels for tissue microstructure reconstruction of tendon-to-bone insertion.

Although gradients play an essential role in guiding the function of tissues, achieving synchronous regeneration of gradient tissue injuries remains a challenge. Here, a gradient bimetallic (Cu and Zn) ion-based hydrogel was first constructed via the one-step coordinative crosslinking of sulfhydryl groups with copper and zinc ions for the microstructure reconstruction of the tendon-to-bone insertion. In this bimetallic hydrogel system, zinc and copper ions could not only act as crosslinkers but also provide strong antibacterial effects and induce regenerative capacity in vitro. The capability of hydrogels in simultaneously promoting tenogenesis and osteogenesis was further verified in a rat rotator cuff tear model. It was found that the Cu/Zn gradient layer could induce considerable collagen and fibrocartilage arrangement and ingrowth at the tendon-to-bone interface. Overall, the gradient bimetallic ion-based hydrogel ensures accessibility and provides opportunities to regenerate inhomogeneous tissue with physiological complexity or interface tissue.

The LGMN pseudogene promotes tumor progression by acting as a miR-495-3p sponge in glioblastoma.

Pseudogenes, which are long noncoding RNAs that originate from protein-coding genes, have been suggested to play important roles in disease. Although studies have revealed high expression of legumain (LGMN) in many types of tumors, the regulation of LGMN remains largely unknown. Here, we found that a novel LGMN pseudogene (LGMNP1) was upregulated in glioblastoma (GBM) tissues and high LGMNP1 expression in GBM cells enhanced proliferation and invasion. Biochemical analysis showed that cytoplasmic LGMNP1 functionally targeted miR-495-3p in a manner involving an RNA-induced silencing complex. Dual-luciferase reporter assays demonstrated that LGMN was a target of miR-495-3p, and LGMN was upregulated and positively correlated with LGMNP1 in GBM. Moreover, miR-495-3p was downregulated and negatively correlated with LGMNP1 in GBM tissues. Notably, the tumor-promoting effects of LGMNP1 upregulation could be alleviated by miR-495-3p mimics. Furthermore, GBM cells overexpressing LGMNP1 exhibited more aggressive tumor progression and elevated LGMN expression in vivo. Thus, our data illustrate that LGMNP1 exerts its oncogenic activity, at least in part, as a competitive endogenous RNA (ceRNA) that elevates LGMN expression by sponging miR-495-3p. CeRNA-mediated miRNA sequestration might be a novel therapeutic strategy in GBM.

An Integrative Survival Analysis for Multicentric Low-Grade Glioma.

OBJECTIVE: This study aimed to perform a survival analysis of patients with multicentric low-grade gliomas (MLGGs) and to assess the influence of various prognostic factors on progression-free survival (PFS) and overall survival. METHODS: A literature search on Web of Science and PubMed was performed for literature in English published from 1963 to September 2018. Detailed information including demographics, clinical characteristics, treatments, critical events, and time to events for survival analysis were extracted from the included articles. RESULTS: A total of 36 cases from published articles were selected for analysis. Univariate analysis showed that age (<31 years or ≥31 years), grade (pure low grade/low and high grade) and glioma type (astrocytoma/oligodendroglioma) had a significant relationship with PFS. Cox regression analysis showed that tumor grade was an independent prognostic factor for PFS. No factors correlated with overall survival. CONCLUSIONS: This integrative analysis of MLGGs patients revealed that age younger than 31 years, pure MLGG, and oligodendroglioma were significantly associated with improved PFS, and pure MLGGs was an independent prognostic factors for PFS.

C1ql1/Ctrp14 and C1ql4/Ctrp11 promote angiogenesis of endothelial cells through activation of ERK1/2 signal pathway.

C1ql-like (C1QL)-1 and -4 proteins are encoded by homologous genes that are highly expressed in brain and adipose tissues. However, functional properties of C1QL proteins outside of the brain and adipocytes remain unknown. Here, we report that the globular domain of C1ql1/Ctrp14 and C1ql4/Ctrp11 proteins directly stimulate the angiogenesis of endothelial cells. In this study, soluble C1ql1/CTRP14 and C1ql4/Ctrp11 proteins, produced in prokaryote expression system, are co-cultured with human umbilical vein endothelium cells (HUVECs), which phenotype is identified with von Willebrand factor antibody. C1ql1/Ctrp14 and C1ql4/Ctrp11 promote the migration and capillary tube formation of HUVECs in a dose-dependent manner. During this process, phosphorylation of c-Raf, MEK1/2, ERK1/2, and p90RSK are activated by C1ql1/Ctrp14 and C1ql4/Ctrp11. MEK1/2 inhibitor, U0126, blocks C1ql1/Ctrp14-, and C1ql4/Ctrp11-induced capillary tube formation and cell migration. Moreover, the immunoreactivity of the receptor of C1QL1-C1QL4, brain-specific angiogenesis inhibitor 3 (BAI3), is detected in HUVECs, suggesting that BAI3 may mediate C1QL1/CTRP14- and C1QL4/CTRP11-induced angiogenesis. Meanwhile, C1ql1/Ctrp14 and C1ql4/Ctrp11 exposure also causes a stimulatory response of angiogenesis in chick yolk sac membrane. These data demonstrate that C1ql1/Ctrp14 and C1ql4/Ctrp11 stimulate the new blood vessel growth by activation of ERK1/2 signal pathway. The proangiogenic activity of C1ql1/Ctrp14 and C1ql4/Ctrp11 provides novel insights into the new opportunities for therapeutic intervention by targeting C1QLs in tumorigenesis, tissue regeneration, and recovery of ischemic heart disease.

Overexpression of Gremlin1 in Mesenchymal Stem Cells Improves Hindlimb Ischemia in Mice by Enhancing Cell Survival.

Mesenchymal stem cells (MSCs) are a promising cell resource for the treatment of ischemic diseases, partially through paracrine effects. One of the major obstacles of MSC treatment is the poor survival rate and low efficiency of transplanted stem cells due to ischemic or inflammatory environments. Gremlin1 (GREM1), a regulator of growth, differentiation and development, has been identified as a novel proangiogenic factor. However, the role and mechanism of GREM1 in MSCs remains unclear. Therefore, we assessed the putative beneficial effects of GREM1 on MSC-based therapy for hindlimb ischemia. The lentiviral vector, EF1a-GREM1, was constructed using the Multisite Gateway System and used to transduce MSCs. In vitro studies demonstrated increased survival of GREM1-MSCs exposed to H2 O2 , which is consistent with the activation of caspase-3. Conditional medium from GREM1-MSCs (GREM1-MSC-CM) increased the anti-apoptotic effects of human umbilical vein endothelial cells (HUVECs), and this effect was attenuated by treatment with the PI3K/Akt pathway inhibitor LY294002. MSCs modified with GREM1 could significantly increase blood perfusion of the ischemic hindlimb in vivo in a mouse model, which was correlated to improved MSC survival. This study demonstrates that overexpression of GREM1 in MSCs have greater therapeutic effects against ischemia compared with wild-type MSCs by enhancing the survival of MSCs and ECs, which may provide new tools for studies investigating the treatment of ischemic diseases. J. Cell. Physiol. 232: 996-1007, 2017. © 2016 Wiley Periodicals, Inc.

Exposure to Excess Phenobarbital Negatively Influences the Osteogenesis of Chick Embryos.

Phenobarbital is an antiepileptic drug that is widely used to treat epilepsy in a clinical setting. However, a long term of phenobarbital administration in pregnant women may produce side effects on embryonic skeletogenesis. In this study, we aim to investigate the mechanism by which phenobarbital treatment induces developmental defects in long bones. We first determined that phenobarbital treatment decreased chondrogenesis and inhibited the proliferation of chondrocytes in chick embryos. Phenobarbital treatment also suppressed mineralization in both in vivo and in vitro long bone models. Next, we established that phenobarbital treatment delayed blood vessel invasion in a cartilage template, and this finding was supported by the down-regulation of vascular endothelial growth factor in the hypertrophic zone following phenobarbital treatment. Phenobarbital treatment inhibited tube formation and the migration of human umbilical vein endothelial cells. In addition, it impaired angiogenesis in chick yolk sac membrane model and chorioallantoic membrane model. In summary, phenobarbital exposure led to shortened lengths of long bones during embryogenesis, which might result from inhibiting mesenchyme differentiation, chondrocyte proliferation, and delaying mineralization by impairing vascular invasion.

Dexamethasone Exposure Accelerates Endochondral Ossification of Chick Embryos Via Angiogenesis.

Dexamethasone (Dex) is widely used to treat chronic inflammatory diseases in the clinic. Increasingly, there is more attention being paid to the side effect of Dex. In this study, we investigated the involvement and mechanism of Dex exposure in accelerating mineralization during long bone formation. We first determined that Dex exposure could accelerate long bone mineralization in vivo, but there was no apparent difference between control and Dex-treated in the phalanges model in vitro. Next, we established that Dex exposure promoted angiogenesis in the chick yolk sac membrane model. In addition, it increased human umbilical vein endothelial cell proliferation and migration in culture. We found that Dex could enhance angiogenesis when phalanges were cultured on chick chorioallantoic membrane and correspondingly increased the expression of angiogenesis-related genes in the phalanges. Furthermore, we also revealed that Dex exposure reduced the number of osteoblasts and simultaneously increased the number of osteocytes in ex vivo-cultured phalanges. Runx-2 and Col10α1 expressions were up-regulated by Dex exposure, indicating that Dex exposure accelerated the terminal differentiation of osteoblasts. Lastly, we demonstrated that MC3T3-E1 cells cultured in the presence of Dex accelerated their mineralization. In summary, we have shown that the ability of Dex to initiate angiogenesis is the mechanism that allows it to accelerate mineralization during long bone formation.

Effects of 2,5-hexanedione on angiogenesis and vasculogenesis in chick embryos.

n-Hexane is widely used in industry and its metabolite, 2,5-hexanedione (2,5-HD), has been implicated as a neural toxin in the developing fetus. Using the chick embryo model, we have previously revealed the neurotoxicity of 2,5-HD during development and established that high dose of 2,5-HD was embryo lethal. In view of the close linkage in biology for neurogenesis and angiogenesis, we speculated that it was most likely caused by cardiovascular dysplasia, therefore in this study, we investigated the effects of 2,5-HD on the development of the vasculature, which involves vasculogenesis and angiogenesis. Using gastrulating chick embryos as a model, we demonstrated that the hemangioblasts (precursor of hematopoietic and endothelial cells) migrated to the area opaca where they form the blood islands. However, this process was impaired when the embryos were treated with 2,5-HD, suggesting that 2,5-HD is capable of impairing vasculogenesis. To study the effect of 2,5-HD exposure on angiogenesis, we used the chick yolk-sac membrane (YSM) and chorioallantoic membrane (CAM) models. We found that, at low (0.02M) concentration, 2,5-HD stimulated angiogenesis while at higher concentrations (>0.1M) it inhibited this process. This biphasic response of angiogenesis to 2,5-HD exposure was found to be associated with altered expression of the VEGF-R, FGF-2 and angiogenin. Moreover, we also determined that 2,5-HD exposure increased the reactive oxygen species (ROS) production. In conclusion, 2,5-HD could induce dysplasia in the developing vasculature, which in turn could cause extravascular hemolysis and the embryos to die.