Core Binding Factor A1 Research Articles

Biosafety and osteogenic properties of a new type of absorbable magnesium-based amorphous alloy

The treatment of bone defects has always been a difficult problem in orthopedic clinical practice, and research on new materials is currently a hot topic. In this study, the biological safety and bone induction ability of a new magnesium-based amorphous alloy (Mg66Zn30Ca3Sr1) and zinc–magnesium powder were verified and compared in animal experiments. Thirty Sprague–Dawley rats were randomly divided into three groups, and a bone defect was made in the lateral condyle of the femur of the left hind limb. The blank group closed the wound after establishing the bone defect model, the control group added zinc–magnesium powder and chondroitin sulfate gel, and the experimental group added magnesium–zinc–calcium–strontium alloy and chondroitin sulfate gel with the same volume. Peripheral venous blood of rats was collected at 4, 8, and 12 weeks postoperatively, and alanine aminotransferase (ALT), aspartate transferase, creatinine, blood urea nitrogen, magnesium ion, and zinc ion levels were evaluated. The expression levels of bone morphogenetic protein 2 (BMP-2), osteoprotegerin (OPG), osteopontin (OPN), core binding factor α1 (RUNX2), and alkaline phosphatase (ALP) were detected using enzyme-linked immunosorbent assay (ELISA). After the rats were euthanized, samples were taken. X-rays and micro-computed tomography (CT) images of the bone defect were obtained, and hematoxylin and eosin and Masson staining were performed. This study shows that amorphous magnesium–zinc–calcium–strontium alloy is a biodegradable metal with good biocompatibility and bone induction and has good application prospects in treating bone defects.

Requirement of Na+/H+ Exchanger NHE1 for Vasopressin-Induced Osteogenic Signaling and Calcification in Human Aortic Smooth Muscle Cells

Background/Aims: Vasopressin is a powerful stimulator of vascular calcification, augmenting osteogenic signaling in vascular smooth muscle cells (VSMCs) including upregulation of transcription factors such as core-binding factor α-1 (CBFA1), msh homeobox 2 (MSX2), and SRY-Box 9 (SOX9), as well as of tissue-nonspecific alkaline phosphatase (ALPL). Vasopressin-induced osteogenic signaling and calcification require the serum- and glucocorticoid-inducible kinase 1 (SGK1). Known effects of SGK1 include upregulation of Na⁺/H⁺ exchanger 1 (NHE1). NHE1 further participates in the regulation of reactive oxygen species (ROS). NHE1 has been shown to participate in the orchestration of bone mineralization. The present study, thus, explored whether vasopressin modifies NHE1 expression and ROS generation, as well as whether pharmacological inhibition of NHE1 disrupts vasopressin-induced osteogenic signaling and calcification in VSMCs. Methods: Human aortic smooth muscle cells (HAoSMCs) were treated with vasopressin in the absence or presence of SGK1 silencing, SGK1 inhibitor GSK-650394, and NHE1 blocker cariporide. Transcript levels were determined by using quantitative real-time polymerase chain reaction, protein abundance by Western blotting, ROS generation with 2′,7′-dichlorofluorescein diacetate fluorescence, and ALP activity and calcium content by using colorimetric assays. Results: Vasopressin significantly enhanced the NHE1 transcript and protein levels in HAoSMCs, effects significantly blunted by SGK1 inhibition with GSK-650394 or SGK1 silencing. Vasopressin increased ROS accumulation, an effect significantly blocked by the NHE1 inhibitor cariporide. Vasopressin further significantly increased osteogenic markers CBFA1, MSX2, SOX9, and ALPL transcript levels, as well as ALP activity and calcium content in HAoSMCs, all effects significantly blunted by SGK1 silencing or in the presence of GSK-650394 or cariporide. Conclusion: Vasopressin stimulates NHE1 expression and ROS generation, an effect dependent on SGK1 and required for vasopressin-induced stimulation of osteogenic signaling and calcification of VSMCs.

The role of TGF-β1/Smad3 signaling pathway and oxidative stress in the inhibition of osteoblast mineralization by copper chloride

To explore the relationship of oxidative stress and TGF-β 1/Smad3 pathway in the inhibition of osteoblast mineralization by copper chloride (CuCl2), the osteoblasts were treated with CuCl2 (0, 50 μM, 100 μM, 150 μM CuCl2 5H2O) for 24 h. We found that Cu impaired the osteoblast structure, inhibited the glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities, alkaline phosphatase (ALP) content, mRNA expression of collagen I (COL-I), osteocalcin (OCN), insulin-like growth factor I (IGF-I), bone morphogenetic protein-2 (BMP-2), transforming growth factor β1 (TGF-β1) and core-binding factor α1 (Cbfα1), promoted the reactive oxygen species (ROS) production, inactivated the TGF-β1/Smad3 pathway. It indicates that the inactivated TGF-β1/Smad3 pathway leads to osteoblast impairment by CuCl2. It will contribute to clarify the influence of CuCl2 on the osteoblast mineralization.

Osteogenic differentiation characteristics of hip joint capsule fibroblasts obtained from patients with ankylosing spondylitis.

BackgroundAutoimmune disease are fairly common and one that has an excessive degree of disability is Ankylosing spondylitis (AS). As the main cells in connective tissues, fibroblasts may play important roles in AS ossification. The conducted research aims to establish the osteogenic disparity characteristics of fibroblasts cultured in vitro, obtained via AS patients hip joint capsule, as well as investigating the pathological osteogenic molecular workings of AS.MethodsAS patients hip joint capsules were acquired and fracture patients as the control with the finite fibroblast line were established by using tissue culture method. AS fibroblast proliferation, cycle and apoptosis, expression of osteogenic marker genes, osteogenic phenotypes, and the activation degree of the bone morphogenetic protein (BMP)/Smads signalling pathway were detected by flow cytometry, western blotting and real-time fluorescent quantitative polymerase chain reaction.ResultsProliferative activity in AS fibroblasts were abnormally high, and the apoptotic rate decreased. Compared with normal fibroblasts, the mRNA expression of osteogenic marker genes, expression of osteogenic phenotypes, protein expression of core-binding factor a1 (Cbfa1), Smad1, Smad4, Smad5, phosphorylated (p) Smad1, and pSmad5 in AS fibroblasts were higher; however, the expression of Smad6 was lower. Moreover, recombinant human bone morphogenetic protein-2(rhBMP-2) stimulated Cbfa1 expression by normal and AS fibroblasts through the BMP/Smads signalling pathway.ConclusionsThe fibroblasts of hip joint capsules in patients with AS cultured in vitro have biologic characteristics of osteogenic differentiation and may be important target cells of AS ossification. The Activated BMP/Smads signalling pathway could potentially be a mechanism relating to fibroblasts differentiating into osteoblasts and an ossification mechanism for AS.

Translation in amino-acid-poor environments is limited by tRNAGln charging

An inadequate supply of amino acids leads to accumulation of uncharged tRNAs, which can bind and activate GCN2 kinase to reduce translation. Here, we show that glutamine-specific tRNAs selectively become uncharged when extracellular amino acid availability is compromised. In contrast, all other tRNAs retain charging of their cognate amino acids in a manner that is dependent upon intact lysosomal function. In addition to GCN2 activation and reduced total translation, the reduced charging of tRNAGln in amino-acid-deprived cells also leads to specific depletion of proteins containing polyglutamine tracts including core-binding factor α1, mediator subunit 12, transcriptional coactivator CBP and TATA-box binding protein. Treating amino-acid-deprived cells with exogenous glutamine or glutaminase inhibitors restores tRNAGln charging and the levels of polyglutamine-containing proteins. Together, these results demonstrate that the activation of GCN2 and the translation of polyglutamine-encoding transcripts serve as key sensors of glutamine availability in mammalian cells.

Zingerone Attenuates Pi-induced Vascular Calcification via AMPK-mediated TIMP4 Expression.

ObjectiveVascular calcification requires the differentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells. This phenomenon can be enhanced by inflammation and oxidative stress. Zingerone is one of the active ingredients present in the ginger plant that has anti-inflammatory and antioxidant effects. Other functions include anti-obesity, anti-nausea effects. However, the functions of zingerone on vascular calcification has not yet been elucidated. This study investigated the effect of zingerone on vascular calcification and its molecular mechanism.MethodsReverse transcription-polymerase chain reaction (PCR), real-time PCR and Western blot analysis was used to measure expression levels of osteogenic marker genes and to investigate whether calcification was regulated by the expression of AMP-activated protein kinase (AMPK) and tissue inhibitor of metalloproteinase 4 (TIMP4). Alizarin red S staining was used to measure calcium deposition. Studies were carried out in VSMCs.ResultsZingerone induced the expression of 2 markers of VSMCs differentiation (α-smooth muscle actin (α-SMA) and smooth muscle 22α (SM22α)) and decreased the expression of core-binding factor α-1 (CBFA1). Additionally, zingerone decreased inorganic phosphate (Pi)-induced expression of distal-less homeobox 5 and CBFA1. AMPK phosphorylation and TIMP4 expression were increased by zingerone. Importantly, zingerone protected VSMCs from calcification, and this protective effect was confirmed by increased TIMP4 via overexpression of AMPK, and inhibition of TIMP4 by Compound C. Zingerone upregulated AMPK/TIMP4 expression and recovered Pi-induced inhibition of TIMP4.ConclusionsTaken together, our results show that zingerone inhibits Pi-induced vascular calcification by regulating the AMPK/TIMP4 signaling cascade in VSMCs. These results suggest that the natural product zingerone could be useful for treating vascular and metabolic diseases.

Mutual promotions between periodontitis and vascular calcification by rat animal model.

To study the relationship between periodontitis and vascular calcification by establishing rat model of chronic periodontitis and vascular calcification. Forty male Wistar rats were divided into four groups randomly: control group, periodontitis group, vascular calcification group, and compound periodontitis and calcification group. Each group rats accepted the corresponding manages to establish the animal model. Clinical examinations and hematoxylin and eosin staining of periodontal tissue were taken to test the periodontal model; calcium assay, alkaline phosphatase activity, expression of mineral-related factors including osteopontin, alkaline phosphatase, core-binding factor-α1 and bone sialoprotein, hematoxylin and eosin staining and von Kossa staining of vascular tissue were taken to test the vascular calcification model; inflammatory factors including C-reactive protein, interleukin-1β, tumor necrosis factor-α, interleukin-6, prostaglandin E2, and serum lipid in serum were also detected at the same time. The rat model was established. Inflammation of periodontal tissue and alveolar bone resorption in compound group and periodontitis group were more obvious than those in control group and vascular calcification group (P<.05). However, the calcium assay, alkaline phosphatase activity, and mineralized deposition in vascular calcification group and compound group were higher than those in control group and periodontitis group (P<.05), and compound group were the highest (P<.05); as for serum lipid, the level of total cholesterol and low-density lipoprotein-cholesterol in compound group and vascular calcification group were higher than that in control group and periodontitis group (P<.05), and compound group was the highest (P<.05); but the level of high-density lipoprotein cholesterol was higher in control group and periodontitis group. Inflammatory factors expression in serum were higher in compound group and periodontitis group, while mineral-related factors expression were higher in compoundgroup and vascular calcification group. There are some mutual promotions between periodontitis and vascular calcification, which might be related to the increasing inflammatory factors, lipids level, and mineral-related factors.

Ginsenoside Rg3 Attenuates Aluminum-Induced Osteoporosis Through Regulation of Oxidative Stress and Bone Metabolism in Rats.

Aluminum (Al)-induced bone metabolism disorder is a primary cause of osteoporosis. Ginsenoside Rg3 (Rg3) has demonstrated therapeutic properties in the treatment of osteoporosis. The present study aimed to identify potential bone protection mechanisms of Rg3 against Al-induced osteoporosis in rats. In this study, forty healthy male Sprague-Dawley rats were randomly allocated into groups in which they were treated with AlCl3 (64mg/kg/day) and/or Rg3 (20mg/kg/day). AlCl3 was given orally to rats for 120days, and from the 91st day, treated orally with Rg3 for 30days. Rg3 attenuated AlCl3-induced accumulation of Al by decreasing the bone mineral density in the lumbar spines, femoral metaphysis, and tibia, and inhibited AlCl3-induced oxidative stress in rat bone by decreasing the levels of reactive oxygen species and malondialdehyde, while increasing glutathione peroxidase and superoxide dismutase activity. Rg3 facilitated bone formation by increasing the concentration of calcium, phosphorus, amino-terminal propeptide of type I procollagen, and carboxyl-terminal propeptide of type I procollagen, bone alkaline phosphatase activity in serum, and type I collagen, osteocalcin, and osteopontin protein expressions. Rg3 inhibited bone resorption by decreasing the content of N-terminal cross-linking telopeptide of type I collagen, C-terminal cross-linking telopeptide of type I collagen, and tartrate-resistant acid phosphatase 5b activity in serum. Rg3 promoted the mRNA expression of growth regulation factors by increasing transforming growth factor-β1, bone morphogenetic protein-2, insulin-like growth factor I, and core-binding factor α1. The results demonstrate that Rg3 can significantly attenuate Al accumulation, facilitate bone formation, inhibit bone resorption, resist oxidative stress, and promote the expression of factors that regulate growth. The results indicate that Rg3 is effective in alleviating AlCl3-induced osteoporosis.

Inhibitory effect of Ginkgo biloba extract on vascular calcification in rats with chronic kidney disease by ROS-NF-κB signaling pathway

In order to explore the mechanism of Extract Gingko biloba (EGb761) inhibiting vascular calcification in rats with chronic kidney disease (CKD) through ROS-NF-κB signaling pathway, adult male healthy SD (Sprague Dawley) rats were selected as the research objects. Animal models were constructed and randomly divided into control group, high phosphorus-induced residual renal vascular calcification model group (HPIAC group), and EGb761 intervention calcification group. Samples were collected from each group of rats, and the renal function, aortic calcification, and the expression of related proteins were detected by Western blot. The results showed that the contents of urea nitrogen, creatinine, and phosphorus in calcification model group increased significantly, with significant statistical difference (P < 0.05), but did not decrease significantly after intervention with EGb761. In the comparative analysis of calcium content in aorta of rats in each group, it was found that the calcium content of abdominal aorta in model group was significantly higher than that in control group. After the intervention of EGb761, the calcium content of abdominal aorta was significantly decreased, with significant statistical difference (P < 0.05). Western blot was used to detect the expression levels of NF-κB (nuclear factor-κB) P65, cbfα1 (core binding factor-α1), and α-SM (a-smooth muscle) actin in thoracic aorta of rats in each group. It was found that the expression levels of NF-κB p65 and cbfα1 in thoracic aorta of rats in HPIAC group increased significantly, while the expression of α-SM actin decreased significantly, with significant statistical difference (P < 0.05). After the intervention of EGb761, the change of the protein was alleviated obviously, with significant statistical difference (P < 0.05). Therefore, through this study, it was found that EGb761 can partially alleviate the calcification of chronic renal failure (CRF) caused by high phosphorus by interfering with the expression of NF-κB p65 protein in rat thoracic artery. Although there are some shortcomings in the experiment, it still provides experimental basis for the treatment of CRF in the later clinical stage.

TAZ contributes to osteogenic differentiation of periodontal ligament cells under tensile stress.

Bone remodeling during orthodontic treatment is achieved by the osteogenesis of human periodontal ligament cells (PDLCs) subjected to mechanical loadings. Transcriptional co-activator with PDZ-binding motif (TAZ) mediates bone remodeling in response to extracellular mechanical signals. This study aims to investigate the role of TAZ in osteogenesis of PDLCs under tensile strain. A uniaxial cyclic tensile stress (CTS) at 12% elongation and 6 cycles/min (5s on and 5s off) was applied to PDLCs. The osteogenic differentiation was determined by the protein and gene expressions of osteogenic markers using qRT-PCR and Western blot, respectively, and further by alkaline phosphatase (ALP) activity and Alizarin Red S staining. The interaction of TAZ with core-binding factor α1 (Cbfα1) was examined by co-immunoprecipitation. The immunofluorescence histochemistry was used to examine the nucleus aggregation of TAZ and the reorganization of actin filaments. Moreover, small interfering RNA-targeting TAZ (TAZsiRNA) was used for TAZ inhibition and Y-27632 was employed for Ras homologue-associated coiled-coil protein kinase (ROCK) signaling blockage. CTS clearly stimulated the nucleus accumulation of TAZ and its interaction with Cbfα1. CTS-induced osteogenesis in PDLCs was significantly abrogated by the infection with TAZsiRNA, as shown by the decreased stained nodules and protein expressions of Cbfα1, collagen type I, osterix, and osteocalcin, along with the inhibition of β-catenin signaling. Moreover, ROCK inhibition by Y-27632 hindered TAZ nucleus aggregation and its binding with Cbfα1, which subsequently lead to the decreased osteoblastic differentiation of PDLCs. Taken together, we propose that TAZ nucleus localization and its interaction with Cbfα1 are essential for the CTS-induced osteogenic differentiation in PDLCs. And such TAZ activation by CTS could be mediated by ROCK signaling, indicating the pivot role of ROCK-TAZ pathway for PDLCs differentiation.

Rapid Repair of Goat Large Segmental Loading Bone Defect and Functional Reconstruction by Interventional Micro-Circulation System

Objective: This study aimed to explore the efficacy of interventional micro-circulation system (IMCS), combining the stromal cell-derived factor 1 (SDF-1)/type I collagen/chitosan composite membrane into TEB in treating with the model of goat tibial LSBD. Methods: The model of goat tibial LSBD was constructed by introducing the IMCS and SDF-1 guiding membrane. Perfusion treatment of CXCR4 + -BMSCs and BMP-2 was performed on the animal model, general conditions of the goats were observed, and changes in the goat's tibia was observed at different time points through imaging techniques. Using RNA extraction and reverse transcription, cDNA was acquired. Alkaline phosphatase (ALP), core binding factor-α1 (Cbfα1) and osteocalcin (OC) content, as well as the mRNA of transformation growth factor-β1 (TGF-β1) in fixed bone tissue samples of the goat, were detected by real-time quantitative PCR (RT-qPCR). Results: The model of goat tibial LSBD repaired by IMCS was successfully constructed. The standing, walking and running functions of the goat recovered well after two months; and imaging revealed that the callus was closely connected and most of the scaffold materials of TEB were absorbed. The detection of the ALP gene in bone tissue samples failed, and the amplified product was non-specific. Furthermore, the levels of Cbfα1, OC and mRNA of TGF-β1 were significantly higher than in normal bones (P &lt; 0.05). Conclusion: The combination treatment of IMCS with slow controlled drug delivery systems and TEB functioned well in repairing goat tibial LSBD. This is a novel method for repairing bone defects.

Cbfα1 hinders autophagy by DSPP upregulation in odontoblast differentiation.

Odontoblasts forms dentin as the main hard tissue of the tooth. Autophagy is a highly conserved homeostasis involved in odontoblast differentiation. DSPP (Dentin sialophosphoprotein) and Cbfα1 (Core binding factor α1) are typical factors in odontoblasts of the dentin-pulp complex. However, the relationship between Cbfα1 and DSPP as well as the role of Cbfα1 involved in the autophagy during odontoblast differentiation are poorly understood. In this study, we found the stimulation with FGF2 in MDPC-23 cells resulted in the suppression of autophagy and promotion of cell differentiation. We demonstrated that both mRNA and protein levels of Cbfα1 and DSPP were elevated upon FGF2 stimulation coupled with the inhibition of autophagy in MDPC-23 cells. Further results revealed that overdose of Cbfα1 increased the expression of DSPP, and also suppressed autophagy in MDPC-23 cells. Using luciferase reporter assay and EMSA, we illustrated that Cbfα1 upregulated DSPP expression through binding to the specific sites 5'-TACCTCA (-3950 bp to -3944 bp) and 5'-ACCACA (-3106 bp to -3101 bp) in DSPP promoter. Furthermore, ChIP assay results revealed that FGF2 treatment enhanced this binding of Cbfα1 to DSPP promoter. Signaling pathway inhibitor screening further revealed Cbfα1 suppressed autophagy involved in Wnt I signaling. Moreover, knockdown of Cbfα1 using siRNA silencing approach reversed the FGF2-mediated suppression of autophagy in MDPC-23 cells. Thus, these findings illustrate a novel regulatory mechanism by which Cbfα1 transcriptionally enhances DSPP expression, and also provide an insight into the inhibitory action of Cbfα1 in autophagy thereby facilitating odontoblast differentiation upon FGF2 stimulation.

ADAM28 dramatically regulates the biological features of human gingival fibroblasts.

This study was to explore the effects of a disintegrin and metalloproteinase 28 (ADAM28) on the proliferation, differentiation, and apoptosis of human gingival fibroblasts (HGFs) and probable mechanism. After ADAM28 antisense oligodeoxynucleotide (AS-ODN) and sense oligodeoxynucleotide (S-ODN) were transfected into HGFs by Lipofectamine 2000, respectively, the expression discrepancies of ADAM28 among various groups were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Western-blotting. Methabenzthiazuron (MTT) and cell-cycle assays were used to test the HGFs proliferation activity. Annexin V fluorescein isothiocyanate (FITC)/propidium iodide (PI) and alkaline phosphatase (ALP) analysis were performed separately to measure apoptosis and the cytodifferentiation standard. Immunocytochemistry and Western-blotting were carried out to determine the influence of ADAM28 AS-ODN on HGFs expressing core binding factor α1 (Cbfα1), cementum protein 1 (CEMP1), osteopontin (OPN) and dentin matrix protein 1 (DMP1). The AS-ODN group displayed the lowest expression level in HGFs, meanwhile the ADAM28 S-ODN group showed the highest. Furthermore, blocking of ADAM28 could inhibit the proliferation of HGFs, enhance HGFs differentiation and induce apoptosis of HGFs. Whereas, overexpression of ADAM28 generated the opposite effects and inhibited apoptosis. ADAM28 AS-ODN was able to notably suppress the expressions of Cbfα1 and CEMP1, and ADAM28 had positive correlations with cbfα1 and CEMP1. These provided conspicuous evidence that ADAM28 may play a crucial role in root development as a potential regulator of growth, differentiation, and apoptosis of HGFs.

Friday, September 28, 2018 1:00 PM–2:30 PM abstracts: achieving lumbar interbody fusion: 192. Clinical evaluation of a new allograft cage designed for TLIF: a minimal 1-year follow-up

BACKGROUND CONTEXT Currently most cages are made of metal, carbon fiber, polyether ether ketone (PEEK), and bio-absorbable materials. But there are some drawbacks of these materials in terms of biology, biomechanics, etc., mainly in terms of the elastic modulus is too large, poor biocompatibility, and unable to absorb. Allogeneic bone is recognized bone graft material of great osteogenic effect. It has the closest physicochemical properties, structure and activity of protein-based composition of autologous bone. It in rich in a variety of cytokines required for osteogenesis: bone morphogenetic protein-2, vascular endothelial growth factor, core binding factor α1, et. PURPOSE The purpose of this study was to observe the preliminary clinical results of the new allograft cage. STUDY DESIGN/SETTING A retrospective research. PATIENT SAMPLE A total of 152 patients. OUTCOME MEASURES Visual Analogue Scale (VAS) and The Oswestry Disability Index (ODI);Relative intervertebral disc height (RIDH); fusion rate. METHODS From 2012 to 2015, a retrospective review of the first 152 consecutive cases using the new allograft cage in our department was performed. The follow-up ranged from 12 to 36 months (average 18 months). Intraoperative index, clinical outcome and complications were recorded. Radiographs evaluatedrelative intervertebral disc height (RIDH) and fusion rate. RESULTS Between September 2012 and November 2014, total 152 patients surgery using the new allograft cage in our department. A total of 133 c (87.6%) met the inclusion criteria and exclusion criteria. There are 78 males and 55 females. The average follow-up time is 15.2±5.3 months. The main diagnosis are lumbar disc herniation (68/133), Lumbar spinal stenosis (44/133), SpondyloliSthesis (21/133). There are 99 one segments fusion and 33 two segments fusion. Respectively, the average blood loss and operation time is 335.7±214.2ml and 175.2±40.9 minutes in single segments fusion, and 488.3±173.2ml, 335.7±214.2 minutes in double segments fusion. The VAS score decreased significantly from 6.89 ±2.51 preoperatively to 2.72±0.97 in 3 months postoperation. At final follow up, the VAS score is 2.96 ±1.02. There was also a significant improvement of the ODI from 38.3±15.9preoperatively to 14.6±6.3 in 3 months postoperation (P CONCLUSIONS It has the potential ability in promoting fusion rate in relative short time (approximately 6 months) postoperatively. We consider it have good clinical outcomes. However, it is just the preliminary data with a short-term follow up. Long-term follow-up is still needed proved.

Polysaccharide from Fuzi protects against Ox‑LDL‑induced calcification of human vascular smooth muscle cells by increasing autophagic activity.

Polysaccharide from Fuzi (FPS) is a water-soluble polysaccharide isolated from the traditional Chinese herbal medicine Fuzi. It has been demonstrated to protect hepatocytes against ischemia-reperfusion injury through its potent antioxidant effects, and to attenuate starvation-induced cytotoxicity in H9c2 cells by increasing autophagic activity. In the present study, Alizarin Red S staining was used to detect mineral deposition and reverse transcription-quantitative polymerase chain reaction was used to detect the core binding factor α1 and smooth muscle 22α mRNA expression. To analyze autophagic activity, western blotting was used to detect microtubule-associated protein 1A/1B light chain 3 and nucleoporin P62 expression. In addition, green fluorescent protein-LC3 dots-per-cell was observed by fluorescence microscopy. It was demonstrated that oxidized low-density lipoprotein (Ox-LDL) could increase the calcification of human vascular smooth muscle cells (VSMCs) in a concentration-dependent manner, and that FPS treatment had a significant protective effect against Ox-LDL-induced calcification of human VSMCs. Furthermore, FPS treatment alleviated the Ox-LDL-induced downregulation of autophagic activity, and the protective effect of FPS on Ox-LDL-induced calcification was attenuated by the autophagy inhibitor 3-methyladenine. In conclusion, the present study demonstrated for the first time to the best of the authors' knowledge that FPS can protect against Ox-LDL-induced vascular calcification in human VSMCs, and that this likely occurs via the activation of autophagy. This supports the hypothesis that autophagy may be an endogenous protective mechanism counteracting vascular calcification, and that FPS may be used as a potential therapeutic for vascular calcification.

MMP-2 silencing reduces the osteogenic transformation of fibroblasts by inhibiting the activation of the BMP/Smad pathway in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a common type of rheumatoid disease, which has recently been demonstrated to be associated with the expression of matrix metalloproteinase (MMP)-2. The aim of the present study was to investigate whether MMP-2 interference reduced the osteogenic differentiation of fibroblasts and to explore the mechanism involved in the differentiation. Fibroblasts from patients with AS were divided into control, mock and small interfering (si)RNA-MMP-2 groups. Cell viability was assessed using the MTT assay. mRNA and protein expression levels of MMP-2, core-binding factor a1 (Cbfa-1) and bone morphogenetic proteins/Smad-signalling molecules (BMP/Smad) were measured using reverse transcription-quantitative polymerase chain reaction and western blotting. The results indicated that cell viability and fibroblast morphology did not differ significantly between healthy volunteers and patients with AS. However, MMP-2 expression levels in AS fibroblasts were substantially higher. MMP-2 gene silencing markedly downregulated the expression of MMP-2 and Cbfa-1, and inhibitied the activation of the BMP/Smad signalling pathway consequent to the reduction in levels of BMP-2, Smad1, Smad4 and Smad1/5/8. The results showed that MMP-2 gene silencing may reduce the osteogenesis of fibroblasts in AS by inhibiting the activation of the BMP/Smad signalling pathway.

Inhibition of osteo/chondrogenic transformation of vascular smooth muscle cells by MgCl2 via calcium-sensing receptor.

The progression of vascular calcification, an active process promoted by osteo/chondrogenic transformation of vascular smooth muscle cells (VSMCs) is attenuated by activation of the calcium-sensing receptor (CASR). Recent in-vitro studies revealed that vascular calcification could be blunted by Mg, but the underlying mechanisms remained elusive. The present study explored whether the effects of MgCl2 on vascular calcification involve the CASR. Experiments were performed in primary human aortic smooth muscle cells (HAoSMCs) and in the mouse vascular calcification model of vitamin D3 overload. Phosphate-induced calcium deposition and mRNA expression of the osteogenic markers msh homeobox 2 (MSX2), CBFA1 (core-binding factor α 1), and ALPL (tissue-nonspecific alkaline phosphatase) in HAoSMCs were blunted by additional treatment with MgCl2. MgCl2 upregulated CASR mRNA expression in HAoSMCs in a dose-dependent manner. Furthermore, the inhibitory effects of MgCl2 on phosphate-induced calcium deposition and osteogenic markers mRNA expression were mimicked by the CASR agonist GdCl3 and reversed by additional treatment with the CASR antagonist NPS-2143 or by silencing of the CASR gene in HAoSMCs. MgCl2 also blunted the osteogenic transformation of VSMCs induced by hydroxyapatite particles. High-dosed cholecalciferol treatment induced vascular calcification and upregulated aortic osteogenic markers Msx2, Cbfa1 and Alpl and collagen type I (Col1a1), collagen type III (Col3a1) and fibronectin (Fbn) mRNA expression in mice, effects reduced by additional treatment with MgCl2. These effects were paralleled by increased aortic Casr mRNA expression in cholecalciferol-treated mice, which was further augmented by MgCl2. The protective effects of MgCl2 on osteo/chondrogenic transformation of VSMCs and vascular calcification involve regulation of CASR and CASR-dependent signaling.

The effects of platelet-rich fibrin extract on MC3T3-E1 cells cultured on the titanium discs

Objective: To evaluate the effect of platelet-rich fibrin extract (PRFe) on the adhesion, proliferation and differentiation of MC3T3-E1 cells cultured on the titanium discs. Methods: Samples were divided into experimental group (P) and control group (D). Group P used the α-minimal essential medium (α-MEM) containing PRFe (0.5%), while group D used only the α-MEM. Cell adhesion and cytoskeleton were observed using scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM). Methyl thiazolyl tetrazolium (MTT) assay to detect the number of the osteoblasts at 1, 3, 5, 7 d; the activity of alkaline phosphatase (ALP) to detect the differentiation of osteoblast at 1, 3, 5, 7 d; the level of osteogenetic biomarkers core-binding factorα1 (cbfα1) and osteocalcin (OCN) were quantified by quantitative real-time PCR (qRT-PCR) at 3 and 7 d. Results: SEM and LSCM showed that the adhesion and filaments of group P were higher than those of group D at each time point. MTT assay showed that the absorbance were significantly increased in group P (1 d: 0.299±0.002, 3 d: 0.517±0.004, 5 d: 0.810±0.002, 7 d: 1.203±0.011) compared with group D (1 d: 0.198±0.003, 3 d: 0.399±0.002, 5 d: 0.588±0.002, 7 d: 0.897±0.005) at each time points (P<0.05). Furthermore, the ALP activity of group P (1 d: 0.162±0.004, 3 d: 0.289±0.001, 5 d: 0.491±0.006, 7 d: 0.647±0.005) was significantly higher than that of group D (1 d: 0.121±0.003, 3 d: 0.191± 0.006, 5 d: 0.252±0.004, 7 d: 0.365±0.012), (P<0.05). Moreover, the qRT-PCR showed that the Cbfα1 and OCN gene expression in group P (Cfbα1, 3 d: 1.50±0.04, 7 d: 1.94±0.06; OCN, 3 d: 3.37±0.17, 7 d: 3.92± 0.04) were significantly higher than that in group D(Cfbα1, 3 d: 1, 7 d: 1.18±0.13; OCN, 3 d: 1, 7 d: 2.34± 0.09) (P<0.05). Conclusions: PRFe promoted the adhension, proliferation and differentiation of MC3T3-E1 cells on the titanium discs.

CML/RAGE signal induces calcification cascade in diabetes.

ObjectiveVascular calcification is a significant predictor of coronary heart disease events, stroke, and lower-limb amputation. Advanced glycation end-products (AGEs) play a key role in the development of vascular calcification. However, the role of Nε-carboxymethyl-lysine (CML), a major active ingredient of heterogeneous AGEs, in the development of atherosclerotic calcification in diabetic patients and the underlying mechanism remain unclear. Hence, the role and the mechanism of CML in the transmission pathway of diabetic calcification cascade were investigated in the present study.MethodsIn vivo and in vitro investigations were performed. In study I, 45 diabetic patients hospitalized for above-knee amputation in the Department of Orthopedics, Affiliated Hospital of Jiangsu University were recruited from February 2010 to June 2015. The patients were categorized based on the severity of anterior tibial artery stenosis, which was assessed by color Doppler ultrasound, into mild stenosis (0% < stenosis < 50%, n = 15), moderate stenosis (50 ≤ stenosis < 70%, n = 15), and severe stenosis/occlusion groups (70 ≤ stenosis ≤ 100%, n = 15). In study II, the specific mechanism of CML in the transmission pathway of the diabetic calcification cascade signal was investigated in A7r5 aortic smooth muscle cells under high-lipid, apoptosis-coexisting conditions. ELISA (for serum CML concentration of patients), ultrasound (for plaque size, calcification, blood flow filling, vascular stenosis etc.), H&E staining (for plaque morphology), vonKossa staining (for qualitative analysis of calcification), calcium content assay (for quantitative analysis of calcification), and Western blot analyses of CML, receptor for advanced glycation end products (RAGE), NADPH oxidase 4, phosphorylated p38, core-binding factor α1 (cbfα1), alkaline phosphatase (ALP) and β-actin were then performed.ResultsMorphological analysis revealed extensive calcification lesions in the intima and media of the anterior tibial artery. The extent and area of calcium deposition in the intima significantly increased with disease progression. Interestingly, spotty calcification was predominant in the atherosclerotic plaques of diabetic patients with amputation, and macrocalcification was almost invisible. Pearson correlation analysis revealed that serum CML level exhibited a significant positive correlation with calcium content in the arterial wall (R2 = 0.6141, P < 0.0001). Semi-quantitative Western blot analysis suggested that the intensity of CML/RAGE signal increased with progression of atherosclerotic calcification in diabetic patients. In subsequent in vitro study, the related pathway was blocked by anti-RAGE antibody, NADPH oxidase inhibitor DPI, p38MAPK inhibitor SB203580, and anti-cbfa1 antibody in a step-wise manner to observe changes in calcium deposition and molecular signals. Results suggested that CML may play a key role in atherosclerotic calcification mainly through the CML/RAGE- reactive oxygen species (ROS)-p38MAPK-cbfα1-ALP pathway.ConclusionSpotty calcification was predominant in the atherosclerotic plaques of amputated diabetic patients. CML/RAGE signal may induce the calcification cascade in diabetes via ROS-p38MAPK.

Bushen Qiangji Granule () medicated serum inhibits osteogenic differentiation of fibroblasts in ankylosing spondylitis by inhibiting the BMP/Smads signal pathway in vitro.

To explore the mechanism of Bushen Qiangji Granule (, BSQJ) in restraining the osteogenic differentiation of ankylosing spondylitis (AS) fifibroblasts. Hip joint capsules were obtained from AS patients (n=10) receiving total hip replacement and healthy hip joint capsules from patients with hip fracture (n=10) receiving surgery as a control. Finite fifibroblast lines were established from these tissue samples to observe the effect of BSQJ on suppressing osteogenic differentiation of fifibroblasts. The expression of osteogenic marker gene corebinding factor a1 (Cbfa1) and Smad family proteins were examined by Western blot and real-time quantitative polymerase chain reaction (qPCR). The mRNA expression level of Cbfa1 was significantly higher in AS fibroblasts than that in normal fibroblasts and the expression of pSmad1, pSmad5, Smad4 and Cbfa1 in AS fibroblasts was also higher, demonstrating the activation of the BMP/Smads signal pathway in AS fifibroblasts. BSQJ-medicated serum not only restrained the mRNA and protein expression levels of Cbfa1 and inhibited protein expression level of Smad4 but also decreased the expression quantities of pSmad1 and pSmad5. BSQJ can inhibit osteogenic differentiation of AS fifibroblasts in vitro by suppressing the activation of the BMP/Smads signal pathway. This may be the important molecular mechanism of BSQJ in regulating AS ossifification.