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1.
Am J Hum Genet ; 108(1): 115-133, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33308444

RESUMO

Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a member of a small family of multifunctional cell surface-anchored glycoproteins functioning as co-receptors for a variety of growth factors. Here we report that bi-allelic inactivating variants in SCUBE3 have pleiotropic consequences on development and cause a previously unrecognized syndromic disorder. Eighteen affected individuals from nine unrelated families showed a consistent phenotype characterized by reduced growth, skeletal features, distinctive craniofacial appearance, and dental anomalies. In vitro functional validation studies demonstrated a variable impact of disease-causing variants on transcript processing, protein secretion and function, and their dysregulating effect on bone morphogenetic protein (BMP) signaling. We show that SCUBE3 acts as a BMP2/BMP4 co-receptor, recruits the BMP receptor complexes into raft microdomains, and positively modulates signaling possibly by augmenting the specific interactions between BMPs and BMP type I receptors. Scube3-/- mice showed craniofacial and dental defects, reduced body size, and defective endochondral bone growth due to impaired BMP-mediated chondrogenesis and osteogenesis, recapitulating the human disorder. Our findings identify a human disease caused by defective function of a member of the SCUBE family, and link SCUBE3 to processes controlling growth, morphogenesis, and bone and teeth development through modulation of BMP signaling.


Assuntos
Osso e Ossos/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Deficiências do Desenvolvimento/metabolismo , Osteogênese/fisiologia , Transdução de Sinais/fisiologia , Animais , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células HEK293 , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células MCF-7 , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL
2.
Int J Mol Sci ; 21(24)2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-33339165

RESUMO

Styrax Japonica Sieb. et Zucc. has been used as traditional medicine in inflammatory diseases, and isolated compounds have shown pharmacological activities. Pinoresinol glucoside (PIN) belonging to lignins was isolated from the stem bark of S. Japonica. This study aimed to investigate the biological function and mechanisms of PIN on cell migration, osteoblast differentiation, and matrix mineralization. Herein, we investigated the effects of PIN in MC3T3-E1 pre-osteoblasts, which are widely used for studying osteoblast behavior in in vitro cell systems. At concentrations ranging from 0.1 to 100 µM, PIN had no cell toxicity in pre-osteoblasts. Pre-osteoblasts induced osteoblast differentiation, and the treatment of PIN (10 and 30 µM) promoted the cell migration rate in a dose-dependent manner. At concentrations of 10 and 30 µM, PIN elevated early osteoblast differentiation in a dose-dependent manner, as indicated by increases in alkaline phosphatase (ALP) staining and activity. Subsequently, PIN also increased the formation of mineralized nodules in a dose-dependent manner, as indicated by alizarin red S (ARS) staining, demonstrating positive effects of PIN on late osteoblast differentiation. In addition, PIN induced the mRNA level of BMP2, ALP, and osteocalcin (OCN). PIN also upregulated the protein level of BMP2 and increased canonical BMP2 signaling molecules, the phosphorylation of Smad1/5/8, and the protein level of Runt-related transcription factor 2 (RUNX2). Furthermore, PIN activated non-canonical BMP2 signaling molecules, activated MAP kinases, and increased ß-catenin signaling. The findings of this study indicate that PIN has biological roles in osteoblast differentiation and matrix mineralization, and suggest that PIN might have anabolic effects in bone diseases such as osteoporosis and periodontitis.


Assuntos
Calcificação Fisiológica , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Glicosídeos/farmacologia , Lignanas/farmacologia , Osteoblastos/efeitos dos fármacos , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Linhagem Celular , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Styrax/química
3.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 34(9): 1170-1176, 2020 Sep 15.
Artigo em Chinês | MEDLINE | ID: mdl-32929912

RESUMO

Objective: To investigate the effects of three-dimensional (3D) printed Ti6Al4V-4Cu alloy on inflammation and osteogenic gene expression in mouse bone marrow mesenchymal stem cells (BMSCs) and mouse mononuclear macrophage line RAW264.7. Methods: Ti6Al4V and Ti6Al4V-4Cu alloys were prepared by selective laser melting, and the extracts of the two materials were prepared according to the biological evaluation standard of medical devices. The effects of two kinds of extracts on the proliferation of mouse BMSCs and mouse RAW264.7 cells were detected by cell counting kit 8 method. After co-cultured with mouse BMSCs for 3 days, the expression of osteogenesis- related genes [collagen type Ⅰ (Col-Ⅰ), alkaline phosphatase (ALP), Runx family transcription factor 2 (Runx-2), osteoprotegerin (OPG), and osteopontin (OPN)] were detected by real-time fluorescence quantitative PCR. After co-cultured with mouse RAW264.7 cells for 1 day, the expressions of inflammation-related genes [interleukin 4 (IL-4) and nitric oxide synthase 2 (iNOS)] were detected by real-time fluorescence quantitative PCR, and the supernatants of the two groups were collected to detect the secretion of vascular endothelial growth factor a (VEGF-a) and bone morphogenetic protein 2 (BMP-2) by ELISA. The osteogenic conditioned medium were prepared with the supernatants of the two groups and co-cultured with BMSCs for 3 days. The expressions of osteogenesis-related genes (Col-Ⅰ, ALP, Runx-2, OPG, and OPN) were detected by real-time fluorescence quantitative PCR. Results: Compared with Ti6Al4V alloy extract, Ti6Al4V-4Cu alloy extract had no obvious effect on the proliferation of BMSCs and RAW264.7 cells, but it could promote the expression of OPG mRNA in BMSCs, reduce the expression of iNOS mRNA in RAW264.7 cells, and promote the expression of IL-4 mRNA. It could also promote the secretions of VEGF-a and BMP-2 in RAW264.7 cells. Ti6Al4V-4Cu osteogenic conditioned medium could promote the expressions of Col-Ⅰ, ALP, Runx-2, OPG, and OPN mRNAs in BMSCs. The differences were all significant ( P<0.05). Conclusion: 3D printed Ti6Al4V-4Cu alloy can promote RAW264.7 cells to secret VEGF-a and BMP-2 by releasing copper ions, thus promoting osteogenesis through bone immune regulation, which lays a theoretical foundation for the application of metal prosthesis.


Assuntos
Ligas , Osteogênese , Animais , Células da Medula Óssea , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular , Células Cultivadas , Regulação da Expressão Gênica , Camundongos , Titânio , Fator A de Crescimento do Endotélio Vascular/metabolismo
4.
PLoS One ; 15(8): e0237479, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790806

RESUMO

OBJECTIVE: As native cartilage consists of different phenotypical zones, this study aims to fabricate different types of neocartilage constructs from collagen hydrogels and human mesenchymal stromal cells (MSCs) genetically modified to express different chondrogenic factors. DESIGN: Human MSCs derived from bone-marrow of osteoarthritis (OA) hips were genetically modified using adenoviral vectors encoding sex-determining region Y-type high-mobility-group-box (SOX) 9, transforming growth factor beta (TGFB) 1 or bone morphogenetic protein (BMP) 2 cDNA, placed in type I collagen hydrogels and maintained in serum-free chondrogenic media for three weeks. Control constructs contained unmodified MSCs or MSCs expressing GFP. The respective constructs were analyzed histologically, immunohistochemically, biochemically, and by qRT-PCR for chondrogenesis and hypertrophy. RESULTS: Chondrogenesis in MSCs was consistently and strongly induced in collagen I hydrogels by the transgenes SOX9, TGFB1 and BMP2 as evidenced by positive staining for proteoglycans, chondroitin-4-sulfate (CS4) and collagen (COL) type II, increased levels of glycosaminoglycan (GAG) synthesis, and expression of mRNAs associated with chondrogenesis. The control groups were entirely non-chondrogenic. The levels of hypertrophy, as judged by expression of alkaline phosphatase (ALP) and COL X on both the protein and mRNA levels revealed different stages of hypertrophy within the chondrogenic groups (BMP2>TGFB1>SOX9). CONCLUSIONS: Different types of neocartilage with varying levels of hypertrophy could be generated from human MSCs in collagen hydrogels by transfer of genes encoding the chondrogenic factors SOX9, TGFB1 and BMP2. This technology may be harnessed for regeneration of specific zones of native cartilage upon damage.


Assuntos
Proteína Morfogenética Óssea 2/genética , Hidrogéis/química , Fatores de Transcrição SOX9/genética , Fator de Crescimento Transformador beta1/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Cartilagem/citologia , Cartilagem/metabolismo , Cartilagem/patologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Condrogênese/genética , Colágeno Tipo I/química , Colágeno Tipo X/genética , Meios de Cultura Livres de Soro/química , Glicosaminoglicanos/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , RNA Mensageiro/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
5.
Life Sci ; 257: 118044, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32622944

RESUMO

AIMS: High-dose glucocorticoid (GC) administration causes osteoporosis. Many previous studies from our group and other groups have shown that melatonin participates in the regulation of osteoblast proliferation and differentiation, especially low concentrations of melatonin, which enhance osteoblast osteogenesis. However, the role of melatonin in glucocorticoid-induced osteoblast differentiation remains unknown. MATERIALS AND METHODS: An examination of the expression of osteoblast differentiation markers (ALP, OCN, COLL-1), as well as alkaline phosphatase staining and alkaline phosphatase enzymatic activity assay to measure osteoblast differentiation and quantifying Alizarin red S staining to measure mineralization, were performed to determine the effects of dexamethasone (Dex) and melatonin on the differentiation of MC3T3-E1 cells. We used immunofluorescence staining to detect the expression of Runx2 in melatonin-treated MC3T3-E1 cells. The expression of mRNA was determined by qRT-PCR, and protein levels were measured by western blotting. KEY FINDINGS: In the present study, we found that 100 µM Dex significantly reduced osteoblast differentiation and mineralization in MC3T3-E1 cells and that 1 µM melatonin attenuated these inhibitory effects. We found that only inhibition of PI3K/AKT (MK2206) and BMP/Smad (LDN193189) signalling abolished melatonin-induced differentiation and mineralization. Meanwhile, MK2206 decreased the expression of P-AKT and P-Smad1/5/9 and LDN193189 decreased the expression of P-Smad1/5/9 but had no obvious effect on P-AKT expression in melatonin-treated and Dex-induced MC3T3-E1 cells. SIGNIFICANCE: These findings suggest that melatonin rescues Dex-induced inhibition of osteoblast differentiation in MC3T3-E1 cells via the PI3K/AKT and BMP/Smad signalling pathways and that PI3K/AKT signalling may be the upstream signal of BMP/Smad signalling.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Melatonina/metabolismo , Osteoblastos/metabolismo , Animais , Biomineralização/efeitos dos fármacos , Proteína Morfogenética Óssea 2/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Linhagem Celular , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/efeitos adversos , Glucocorticoides/farmacologia , Melatonina/farmacologia , Camundongos , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo
6.
Ecotoxicol Environ Saf ; 203: 110930, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32684523

RESUMO

Benzo[a]pyrene(BaP), a polycyclic aromatic hydrocarbons (PAH) of environmental pollutants, is one of the main ingredients in cigarettes and an agonist of the aryl hydrocarbon receptor (AhR). Mesenchymal stem cells (MSCs) including C3H10T1/2 and MEF cells, adult multipotent stem cells, can be differentiated toward osteoblasts during the induction of osteogenic induction factor-bone morphogenetic protein 2(BMP2). Accumulating evidence suggests that BaP decreases bone development in mammals, but the further mechanisms of BaP on BMP2-induced bone formation involved are unknown. Here, we researched the role of BaP on BMP2-induced osteoblast differentiation and bone formation. We showed that BaP significantly suppressed early and late osteogenic differentiation, and downregulated the runt-related transcription factor 2(Runx2), osteocalcin(OCN) and osteopontin (OPN) during the induction of BMP2 in MSCs. Consistent with in vitro results, administration of BaP inhibited BMP2-induced subcutaneous ectopic osteogenesis in vivo. Interestingly, blocking AhR reversed the inhibition of BaP on BMP2-induced osteogenic differentiation, which suggested that AhR played an important role in this process. Moreover, BaP significantly decreased BMP2-induced Smad1/5/8 phosphorylation. Furthermore, BaP significantly reduced bone morphogenetic protein receptor 2(BMPRII) expression and excessively activated Hey1. Thus, our data demonstrate the role of BaP in BMP2-induced bone formation and suggest that impaired BMP/Smad pathways through AhR regulating BMPRII and Hey1 may be an underlying mechanism for BaP inhibiting BMP2-induced osteogenic differentiation.


Assuntos
Benzo(a)pireno/toxicidade , Proteína Morfogenética Óssea 2/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Benzo(a)pireno/metabolismo , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Células HCT116 , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Nus , Osteoblastos/metabolismo
7.
Gene ; 754: 144848, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32522697

RESUMO

The TGF-beta superfamily is widely involved in cell events such as cell division and differentiation, while bone morphogenetic proteins (BMPs) belong to one of the subgroups. Their functions in crustacean spermatogenesis are still unknown. In this study, we first identified the bone morphogenetic protein 2 (bmp2) from Eriocheir sinensis (E. sinensis) testis. The es-BMP2 shows high expression in E. sinensis testis. We found that es-BMP2 is expressed in spermatids. The successfully knockdown of es-BMP2 through in vivo RNAi are used for functional analysis. Compared with the control group, the proportion of abnormal nuclear cup morphology in mature spermatozoa increased significantly after es-bmp2 RNAi, suggesting that es-BMP2 plays an important role in mature sperm morphogenesis. Immunofluorescence results confirm this finding. In order to study the specific mechanism of es-BMP2 involved in spermiogenesis, we tested kinesin-14 KIFC1, which functions in the nucleus formation of spermatozoa in E. sinensis. The results showed that knockdown of es-BMP2 caused a significant decrease of es-KIFC1 expression. We further performed es-bmp2 knockdown in vitro in primary cultured testis cells. es-KIFC1 expression was significantly reduced after es-bmp2 RNAi. The above results indicate that es-BMP2 participates in maintaining the spermiogenesis of E. sinensis by regulating es-KIFC1 expression.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Células Germinativas/citologia , Cinesina/metabolismo , Espermatogênese , Testículo/citologia , Animais , Proteína Morfogenética Óssea 2/genética , Braquiúros , Regulação da Expressão Gênica , Células Germinativas/metabolismo , Cinesina/genética , Masculino , Testículo/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(27): 15620-15631, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32576689

RESUMO

Repulsive guidance molecules (RGMs) are cell surface proteins that regulate the development and homeostasis of many tissues and organs, including the nervous, skeletal, and immune systems. They control fundamental biological processes, such as migration and differentiation by direct interaction with the Neogenin (NEO1) receptor and function as coreceptors for the bone morphogenetic protein (BMP)/growth differentiation factor (GDF) family. We determined crystal structures of all three human RGM family members in complex with GDF5, as well as the ternary NEO1-RGMB-GDF5 assembly. Surprisingly, we show that all three RGMs inhibit GDF5 signaling, which is in stark contrast to RGM-mediated enhancement of signaling observed for other BMPs, like BMP2. Despite their opposite effect on GDF5 signaling, RGMs occupy the BMP type 1 receptor binding site similar to the observed interactions in RGM-BMP2 complexes. In the NEO1-RGMB-GDF5 complex, RGMB physically bridges NEO1 and GDF5, suggesting cross-talk between the GDF5 and NEO1 signaling pathways. Our crystal structures, combined with structure-guided mutagenesis of RGMs and BMP ligands, binding studies, and cellular assays suggest that RGMs inhibit GDF5 signaling by competing with GDF5 type 1 receptors. While our crystal structure analysis and in vitro binding data initially pointed towards a simple competition mechanism between RGMs and type 1 receptors as a possible basis for RGM-mediated GDF5 inhibition, further experiments utilizing BMP2-mimicking GDF5 variants clearly indicate a more complex mechanism that explains how RGMs can act as a functionality-changing switch for two structurally and biochemically similar signaling molecules.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas Ligadas por GPI/metabolismo , Fator 5 de Diferenciação de Crescimento/metabolismo , Proteína da Hemocromatose/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 2/ultraestrutura , Moléculas de Adesão Celular Neuronais/ultraestrutura , Cristalografia por Raios X , Proteínas Ligadas por GPI/ultraestrutura , Fator 5 de Diferenciação de Crescimento/ultraestrutura , Proteína da Hemocromatose/ultraestrutura , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Proteínas do Tecido Nervoso/ultraestrutura , Domínios Proteicos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Transdução de Sinais
9.
Nat Commun ; 11(1): 3025, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32541821

RESUMO

Approximately 10% of fractures will not heal without intervention. Current treatments can be marginally effective, costly, and some have adverse effects. A safe and manufacturable mimic of anabolic bone is the primary goal of bone engineering, but achieving this is challenging. Mesenchymal stem cells (MSCs), are excellent candidates for engineering bone, but lack reproducibility due to donor source and culture methodology. The need for a bioactive attachment substrate also hinders progress. Herein, we describe a highly osteogenic MSC line generated from induced pluripotent stem cells that generates high yields of an osteogenic cell-matrix (ihOCM) in vitro. In mice, the intrinsic osteogenic activity of ihOCM surpasses bone morphogenic protein 2 (BMP2) driving healing of calvarial defects in 4 weeks by a mechanism mediated in part by collagen VI and XII. We propose that ihOCM may represent an effective replacement for autograft and BMP products used commonly in bone tissue engineering.


Assuntos
Osteogênese , Células-Tronco Pluripotentes/citologia , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Proliferação de Células , Células Cultivadas , Colágeno Tipo VI/genética , Colágeno Tipo VI/metabolismo , Colágeno Tipo XII/genética , Colágeno Tipo XII/metabolismo , Anormalidades Craniofaciais/fisiopatologia , Anormalidades Craniofaciais/terapia , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/transplante , Engenharia Tecidual
10.
Phytomedicine ; 71: 153225, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32464299

RESUMO

BACKGROUND: Impaired bone formation is one of the reasons behind osteoporosis. Alterations in the patterns of mesenchymal stromal cell differentiation towards adipocytes instead of osteoblasts contribute to osteoporosis progression. Natural anti-osteoporotic agents are effective and safe alternatives for osteoporosis treatment. PURPOSE: In this context, 3,5-dicaffeoyl­epi-quinic acid (DCEQA) which is a derivative of chlorogenic acid with reported bioactivities was studied for its osteogenic differentiation enhancing potential in vitro. METHODS: Anti-osteoporotic effects of DCEQA were investigated in human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) which were induced to differentiate into osteoblasts or adipocytes with or without DCEQA treatment. Changes in the osteogenic and adipogenic markers such as ALP activity and lipid accumulation, respectively, were observed along with differentiation-specific activation of mitogen activated protein kinase (MAPK) pathways. RESULTS: At 10 µM concentration, DCEQA increased the proliferation of bone marrow-derived human mesenchymal stromal cells (hBM-MSCs) during osteoblast differentiation. The expression of osteogenic markers ALP, osteocalcin, Runx2, BMP2 and Wnt 10a was upregulated by DCEQA treatment. The ALP activity and extracellular mineralization were also increased. DCEQA elevated the phosphorylation levels of p38 and JNK MAPKs as well as the activation of ß-catenin and Smad1/5. DCEQA suppressed the lipid accumulation and downregulated expression of adipogenic markers PPARγ, C/EBPα and SREBP1c in adipo-induced hBM-MSCs. DCEQA also decreased the phosphorylation of p38 and ERK MAPKs and stimulated the activation of AMPK in hBM-MSC adipocytes. CONCLUSION: DCEQA was suggested to enhance osteoblast differentiation via stimulating Wnt/BMP signaling. The adipocyte differentiation inhibitory effect of DCEQA was suggested to arise from its ability to increase AMPK phosphorylation. Overall, DCEQA was shown to possess osteogenesis enhancing and adipogenesis inhibitory properties which might facilitate its use against osteoporotic conditions.


Assuntos
Adipócitos/citologia , Atriplex/química , Ácido Clorogênico/análogos & derivados , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/efeitos dos fármacos , Células da Medula Óssea , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Ácido Clorogênico/farmacologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo
11.
Int J Nanomedicine ; 15: 2633-2646, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32368045

RESUMO

Objective: The aim of this study is to fabricate functional scaffolds to gene delivery bone morphogenetic protein-2 (BMP-2) plasmid for bone formation in bone tissue engineering. Methods: Dendriplexes (DPs) of generation 4 polyamidoamin (G4-PAMAM)/BMP-2 plasmid were prepared through microfluidic (MF) platform. The physiochemical properties and toxicity of DPs were evaluated by DLS, AFM, FESEM and MTT assay. In order to create a suitable environment for stem cell growth and differentiation, poly-l-lactic acid (PLLA) and poly-l-lactic acid/poly (ethylene oxide) (PLLA/PEO) scaffolds containing hydroxyapatite nanoparticles (HA) and DPs were fabricated by the electrospinning method. The osteogenic potency of the scaffolds on human adipose tissue-derived mesenchymal stem cells (hASCs) was investigated. Results: The results revealed that tuning the physical properties of DPs by adjusting flow parameters in microfluidic platform can easily improve the cell viability compared to conventional bulk mixing method. Also, the result showed that the presence of HA and DPs in PLLA/PEO scaffold enhanced alkaline phosphatase (ALP) activity and increased the amount of deposited Ca, as well as, related to osteogenesis gen markers. Conclusion: This study indicated that on using the MF platform in preparation of DPs and loading them along with HA in PLLA/PEO scaffold, the osteogenic differentiation of hASCs could be tuned.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Osso e Ossos/fisiologia , Durapatita/química , Microfluídica , Nanofibras/química , Poliaminas/química , Engenharia Tecidual/métodos , Tecidos Suporte/química , Fosfatase Alcalina/metabolismo , Cálcio/metabolismo , Adesão Celular , Morte Celular , Diferenciação Celular , Proliferação de Células , Forma Celular , DNA/metabolismo , Dendrímeros/química , Humanos , Células-Tronco Mesenquimais/metabolismo , Nanopartículas/química , Tamanho da Partícula , Plasmídeos/metabolismo , Poliésteres/química , Resistência à Tração
12.
BMC Complement Med Ther ; 20(1): 123, 2020 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321506

RESUMO

BACKGROUND: Circaea mollis Sieb. & Zucc. has been used as a traditional herbal medicine in Hani Ethnopharmacy and possesses anti-arthritic activities. This study aimed to investigate the effect of Circaea mollis Siebold & Zucc on postmenopausal osteoporosis. METHODS: For in vitro study, MCF7 breast cancer cells and MC3T3-E1 pre-osteoblast cells were utilized to estimate estrogenic and osteogenic activity. Osteoblastic markers were measured by western blot and real-time PCR. For in vivo study, female mature C57BL/6 mice were ovariectomized and oral administrated with 10 mg/kg and 40 mg/kg of EECM respectively. RESULTS: EtOH extract of Circaea mollis Siebold & Zucc. (EECM) increased alkaline phosphatase activity and osteoblast marker levels at day 7 during differentiation of mouse preosteoblasts. EECM reduced osteoclast differentiation and bone resorption in an osteoblast-osteoclast primary co-culture system. In ovariectomized mice, EECM prevented the decrease in bone mineral density and recovered OSX and Runx2 via BMP2/4, Smad1/5/9 and p38. CONCLUSIONS: The results suggest that EECM may be effective in preventing bone loss, offering a promising alternative for the nutritional management of postmenopausal osteoporosis.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteoblastos/metabolismo , Osteoporose Pós-Menopausa/tratamento farmacológico , Extratos Vegetais/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL
13.
Sci Rep ; 10(1): 4979, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32188900

RESUMO

It has been demonstrated that development of three-dimensional printing technology has supported the researchers and surgeons to apply the bone tissue engineering to the oromandibular reconstruction. In this study, poly caprolactone/beta tricalcium phosphate (PCL/ß-TCP) scaffolds were fabricated by multi-head deposition system. The feasibility of the three-dimensionally (3D) -printed PCL/ß-TCP scaffolds for mandibular reconstruction was examined on critical-sized defect of canine mandible. The scaffold contained the heterogeneous pore sizes for more effective bone ingrowth and additional wing structures for more stable fixation. They were implanted into the mandibular critical-sized defect of which periosteum was bicortically resected. With eight 1-year-old male beagle dogs, experimental groups were divided into 4 groups (n = 4 defects per group, respectively). (a) no further treatment (control), (b) PCL/ß-TCP scaffold alone (PCL/TCP), (c) PCL/ß-TCP scaffold with recombinant human bone morphogenetic protein-2 (rhBMP-2) (PCL/TCP/BMP2) and (d) PCL/ß-TCP scaffold with autogenous bone particles (PCL/TCP/ABP). In micro-computed tomography, PCL/TCP/BMP2 and PCL/TCP/ ABP groups showed significant higher bone volume in comparison to Control and PCL/TCP groups (P < 0.05). In histomorphometric analysis, a trend towards more bone formation was observed in PCL/TCP/BMP2 and PCL/TCP/ABP groups, but the results lacked statistical significance (P = 0.052). Within the limitations of the present study, 3D-printed PCL/ß-TCP scaffolds showed acceptable potential for oromandibular reconstruction.


Assuntos
Fosfatos de Cálcio/química , Mandíbula/citologia , Mandíbula/cirurgia , Reconstrução Mandibular/métodos , Poliésteres/química , Impressão Tridimensional/instrumentação , Engenharia Tecidual , Animais , Proteína Morfogenética Óssea 2/metabolismo , Regeneração Óssea , Cães , Masculino , Mandíbula/metabolismo , Proteínas Recombinantes/metabolismo , Tecidos Suporte , Fator de Crescimento Transformador beta/metabolismo
14.
Biomed Res Int ; 2020: 9467683, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32149147

RESUMO

Yishen Bugu Ye (YSBGY), a traditional Chinese medicine comprising 12 types of medicinal herbs, is often prescribed in China to increase bone strength. In this study, the antiosteoporotic effects of YSBGY were investigated in C57BL/6 mice afflicted with dexamethasone- (Dex-) induced osteoporosis (OP). The results showed that YSBGY reduced the interstitial edema in the liver and kidney of mice with Dex-induced OP. It also increased the number of trabecular bone elements and chondrocytes in the femur, promoted cortical bone thickness and trabecular bone density, and modulated the OP-related indexes in the femur and tibia of OP mice. It also increased the serum concentrations of type I collagen, osteocalcin, osteopontin, bone morphogenetic protein-2, bone morphogenetic protein receptor type 2, C-terminal telopeptide of type I collagen, and runt-related transcription factor-2 and reduced those of tartrate-resistant acid phosphatase 5 and nuclear factor of activated T cells in these mice, suggesting that it improved osteoblast differentiation and suppressed osteoclast differentiation. The anti-inflammatory effect of YSBGY was confirmed by the increase in the serum concentrations of interleukin- (IL-) 33 and the decrease in concentrations of IL-1, IL-7, and tumor necrosis factor-α in OP mice. Furthermore, YSBGY enhanced the serum concentrations of superoxide dismutase and catalase in these mice, indicating that it also exerted antioxidative effects. This is the first study to confirm the antiosteoporotic effects of YSBGY in mice with Dex-induced OP, and it showed that these effects may be related to the YSBGY-induced modulation of the osteoblast/osteoclast balance and serum concentrations of inflammatory factors. These results provide experimental evidence supporting the use of YSBGY for supporting bone formation in the clinical setting.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Animais , Densidade Óssea , Proteína Morfogenética Óssea 2/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Osso Esponjoso , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osso Cortical/metabolismo , Osso Cortical/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteocalcina/metabolismo , Osteoclastos/metabolismo , Osteopontina/metabolismo , Osteoporose/diagnóstico por imagem , Osteoporose/patologia , Peptídeos , Superóxido Dismutase/sangue , Fosfatase Ácida Resistente a Tartarato/metabolismo
15.
PLoS One ; 15(3): e0230201, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32143215

RESUMO

Medial vascular calcification (MVC) is a highly prevalent disease associated with a high risk of severe, potentially lethal, complications. While animal studies may not systematically be circumvented, in vitro systems have been proven useful to study disease physiopathology. In the context of MVC, the absence of a clinically relevant standardized in vitro method prevents the appropriate comparison and overall interpretation of results originating from different experiments. The aim of our study is to establish in vitro models mimicking in vivo vascular calcification and to select the best methods to unravel the mechanisms involved in MVC. Human aortic smooth muscle cells and rat aortic rings were cultured in different conditions. The influence of fetal calf serum (FCS), alkaline phosphatase, phosphate and calcium concentrations in the medium were evaluated. We identified culture conditions, including the herein reported Aorta Calcifying Medium (ACM), which allowed a reproducible and specific medial calcification of aortic explants. Studying cells and aortic explants cultured, the involvement of bone morphogenetic protein 2 (BMP2) pathway, fibrosis and apoptosis processes in in vitro MVC were demonstrated. Expression of osteoblastic markers was also observed suggesting the occurrence of transdifferentiation of smooth muscle cells to osteoblasts in our models. The use of these models will help researchers in the field of vascular calcification to achieve reproducible results and allow result comparison in a more consistent way.


Assuntos
Miócitos de Músculo Liso/patologia , Calcificação Vascular/patologia , Fosfatase Alcalina/metabolismo , Animais , Aorta/metabolismo , Aorta/patologia , Apoptose/fisiologia , Proteína Morfogenética Óssea 2/metabolismo , Cálcio/metabolismo , Transdiferenciação Celular/fisiologia , Células Cultivadas , Humanos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Osteoblastos/metabolismo , Fosfatos/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia , Calcificação Vascular/metabolismo
16.
Nat Commun ; 11(1): 1365, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170076

RESUMO

Nanoclays have generated interest in biomaterial design for their ability to enhance the mechanics of polymeric materials and impart biological function. As well as their utility as physical cross-linkers, clays have been explored for sustained localization of biomolecules to promote in vivo tissue regeneration. To date, both biomolecule-clay and polymer-clay nanocomposite strategies have utilised the negatively charged clay particle surface. As such, biomolecule-clay and polymer-clay interactions are set in competition, potentially limiting the functional enhancements achieved. Here, we apply specific bisphosphonate interactions with the positively charged clay particle edge to develop self-assembling hydrogels and functionalized clay nanoparticles with preserved surface exchange capacity. Low concentrations of nanoclay are applied to cross-link hyaluronic acid polymers derivatised with a pendant bisphosphonate to generate hydrogels with enhanced mechanical properties and preserved protein binding able to sustain, for over six weeks in vivo, the localized activity of the clinically licensed growth factor BMP-2.


Assuntos
Difosfonatos/metabolismo , Hidrogéis/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Nanocompostos/química , Nanopartículas/química , Animais , Proteína Morfogenética Óssea 2/metabolismo , Argila , Sistemas de Liberação de Medicamentos , Feminino , Teste de Materiais , Camundongos , Polímeros/química , Ligação Proteica , Silicatos
17.
Vascular ; 28(4): 465-474, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32089109

RESUMO

OBJECTIVE: Calcification serves as a surrogate for atherosclerosis-associated vascular diseases, and coronary artery calcification is mediated by multiple pathogenic factors. Estrogen is a known factor that protects the arterial wall against atherosclerosis, but its role in the coronary artery calcification development remains largely unclear. This study tested the hypothesis that estrogen inhibits coronary artery calcification via the hypoxia-induced factor-1α pathway. METHODS: Eight-week-old healthy female Sprague-Dawley rats were castrated, and vitamin D3 was administered orally to establish. Hypoxia-induced factor-1 inhibitor was administered to test its effect on vascular calcification and expression of bone morphogenetic protein 2 and runt-related transcription factor-2. Vascular smooth muscle cell calcification was induced with CaCl2 in rat aortic smooth muscle cells in the presence or absence of E2(17ß-estradiol) and bone morphogenetic protein 2 siRNA intervention. RESULTS: The estrogen levels in ovariectomized rats were significantly decreased, as determined by ELISA. Expression of hypoxia-induced factor-1α mRNA and protein was significantly increased in vascular cells with calcification as compared to those without calcification (p < 0.01). E2 treatment decreased the calcium concentration in vascular cell calcification and cell calcium nodules in vitro (p < 0.05). E2 also lowered the levels of hypoxia-induced factor-1α mRNA and protein (p < 0.01). Oral administration of the hypoxia-induced factor-1α inhibitor dimethyloxetane in castrated rats alleviated vascular calcification and expression of osteogenesis-related transcription factors, bone morphogenetic protein 2 and RUNX2 (p < 0.01). Finally, bone morphogenetic protein 2 siRNA treatment decreased the levels of p-Smad1/5/8 in A7r5 calcification cells (p < 0.01). CONCLUSION: Estrogen deficiency enhances vascular calcification. Treatment with estrogen reduces the expression of hypoxia-induced factor-1α as well as vascular calcification in rats. The estrogen effects occur in a fashion dependent on hypoxia-induced factor-1α regulation of bone morphogenetic protein-2 and downstream Smad1/5/8.


Assuntos
Doenças da Aorta/prevenção & controle , Estradiol/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Calcificação Vascular/prevenção & controle , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Modelos Animais de Doenças , Feminino , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Ovariectomia , Fosforilação , Ratos Sprague-Dawley , Transdução de Sinais , Proteínas Smad Reguladas por Receptor/metabolismo , Calcificação Vascular/genética , Calcificação Vascular/metabolismo , Calcificação Vascular/patologia
18.
Lasers Med Sci ; 35(7): 1519-1529, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32026163

RESUMO

The aim of this study was to evaluate the osseointegration of implants placed in areas grafted with different osteoconductive bone substitutes irradiated with infrared low-level laser therapy (LLLT). Fifty-six rats were randomly allocated into 4 groups: DBB, bone defects filled with deproteinized bovine bone graft (DBB); HA/TCP, bone defects filled with biphasic ceramic made of hydroxyapatite and ß-tricalcium phosphate (HA/TCP); DBB-L, bone defects filled with DBB and treated by LLLT; HA/TCP-L, bone defects filled with HA/TCP and treated by LLLT. Bone defects were performed in the tibia of each animal and filled with the different biomaterials. The grafted areas were treated with LLLT (λ 808 nm, 100 mW, ϕ ∼ 0.60 mm) in 7 sessions with 48 h between the irradiations. After the 60-day period, the implants were placed, and the animals were euthanized after 15 and 45 days. The osseointegration and bone repair in the grafted area were evaluated by biomechanical, microtomographic and histometric analyses, and the expression of some bone biomarkers was evaluated by immunohistochemistry analysis. LLLT induced higher degree of osseointegration, which was associated with the greater expression of BMP2 and OCN. LLLT performed in areas grafted with osteoconductive bone substitutes prior to implant placement improves osseointegration.


Assuntos
Regeneração Óssea/efeitos dos fármacos , Regeneração Óssea/efeitos da radiação , Substitutos Ósseos/farmacologia , Terapia com Luz de Baixa Intensidade , Osseointegração/efeitos dos fármacos , Osseointegração/efeitos da radiação , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Fenômenos Biomecânicos/efeitos da radiação , Proteína Morfogenética Óssea 2/metabolismo , Bovinos , Hidroxiapatitas/farmacologia , Processamento de Imagem Assistida por Computador , Masculino , Ratos
19.
Environ Toxicol Pharmacol ; 75: 103331, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32004919

RESUMO

Skeletal fluorosis is a chronic metabolic bone disease caused by excessive exposed to fluoride. Recent studies have shown that fluoride causes abnormal bone metabolism through disrupting the expression of Bone Morphogenetic Proteins (BMPs). However, the relationship between fluoride and BMPs is not fully understood, and the mechanism of fluoride on BMPs expression is still unclear. This study investigated the dose-time effects of fluoride on BMP-2 and BMP-7 levels and DNA methylation status of the promoter regions of these two genes in peripheral blood of rats. Eighty Wistar male rats were randomly divided into four groups and treated for 1 month and 3 months with distilled water (control), 25 mg/L, 50 mg/L or 100 mg/L of sodium fluoride (NaF). Rats exposed to fluoride had higher protein expression of BMP-2 and BMP-7 in plasma at 1 month and 3 months. An increase in BMP-2 expression was also observed with an increase of fluoride exposure time. Significant hypomethylation was observed in 2 CpG sites (CpGs) of BMP-2 and 1 CpG site of BMP-7 promoter regions in the fluoride treatment groups. It concludes that fluoride has a dose-response effect on BMP-2 in fluorosis rats, and fluoride-induced hypomethylation of specific CpGs may play an essential role in the regulation of BMP-2 and BMP-7 expression in rats.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 7/metabolismo , Fluoretos/toxicidade , Animais , Metilação de DNA , Masculino , Regiões Promotoras Genéticas/efeitos dos fármacos , Ratos , Ratos Wistar
20.
Med Sci Monit ; 26: e918541, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31911574

RESUMO

BACKGROUND Osteoporosis is an osteolytic disease resulted from imbalance in bone homeostasis. Studies indicated that N-myc downstream-regulated gene 2 (NDRG2) could affect the osteoclast differentiation. However, the effect of NDRG2 on osteoblastic differentiation and calcification remains unknown. Hence, we aimed to analyze the effect of NDRG2 on the proliferation and differentiation of osteoblasts. MATERIAL AND METHODS The differentiation of bone morphogenetic protein 2 (BMP2) induced MC3T3-E1 cells was observed by the microscope. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analysis detected the expression of BMP2, NDRG2, runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), osterix (OSX), and osteocalcin (OCN). Alkaline phosphatase (ALP) activity assay was detecting the ALP activity and alizarin red staining assay was analyzing intracellular calcium salt deposition. The cell transfection was also verified by RT-qPCR analysis. RESULTS The results demonstrated that BMP2 promoted the osteoblastic differentiation with the increasing expression of Runx2, OPG, OSX, and OCN. NDRG2 expression was upregulated during osteogenic differentiation. NDRG2 overexpression promoted the expression of Runx2, OPG, OSX, and OCN, and increased the ALP activity while NDRG2 inhibition reversed the changes. NDRG2 overexpression increased the intracellular calcium salt deposition and NDRG2 inhibition reversed the changes. The role of NDRG2 in osteoblastic differentiation and calcification was played through the JAK3/STAT3 signal pathway. CONCLUSIONS The presented data indicated that NDRG2 promoted BMP2-induced osteoblastic differentiation and calcification by activating the JAK3/STAT3 signal pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Janus Quinase 3/metabolismo , Osteoblastos/metabolismo , Fator de Transcrição STAT3/metabolismo , Células 3T3 , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Animais , Calcificação Fisiológica/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Camundongos , Osteocalcina/metabolismo , Osteogênese/fisiologia , Transdução de Sinais , Fator de Transcrição Sp7/metabolismo
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