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1.
Braz Dent J ; 31(3): 304-309, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32667511

RESUMO

Among other factors, types of bisphosphonates and treatment regimens seem to be strongly associated with the success or failure of installation of osseointegrated implants. This study investigated the influence of two bisphosphonates, sodium alendronate (SA) and zoledronic acid (ZA), on the metabolism of osteoblasts. Human osteoblasts (Saos-2) were seeded onto machined or acid-treated titanium discs previously placed on 24-well plates in complete culture medium. After 24 h, cells were exposed to bisphosphonates at 0.5, 1 or 5 µM for 24 h, 48 h or 7 days. The effects of SA and ZA on osteoblasts were assessed based on the adhesion of these cells to the titanium surfaces by direct fluorescence, cell viability, total protein and collagen synthesis. Alkaline phosphatase activity and mineral nodule deposition by these cells were also evaluated. Data were evaluated by ANOVA and Tukey tests (α=0.05). Decreased adhesion of cells to the titanium discs was observed when exposed to both bisphosphonates; however, this lack of cell adhesion was more evident for ZA-treated cells. In addition, the exposure of osteoblasts to ZA decreased the viability, ALP activity and mineral nodule deposition, which may be related to poor osseointegration after implant installation.


Assuntos
Difosfonatos , Titânio , Fosfatase Alcalina , Adesão Celular , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos , Osteoblastos , Propriedades de Superfície , Ácido Zoledrônico
2.
Artigo em Inglês | MEDLINE | ID: mdl-32559043

RESUMO

Piezoelectric surgery utilizes ultrasonic vibrations to cut bone more precisely and less traumatically than conventional methods. The regional acceleratory phenomenon following bone injury has a demineralization phase followed by a remineralization phase. Part I of this study on rats assessed the biologic modifications following bone injuries with the piezoelectric knife at 10-Hz and 30-Hz modulation frequencies. Part II focuses on piezoelectric surgery-regulated osteoblast activity and changes occurring in the bone during the regeneration phase. The results indicate that at 30 Hz, the remineralization process starts at day 14 and continues until day 70, with osteoblast progenitor cells observed in the periodontal ligament around acellular new bone as early as day 14. These findings emphasize the potential for regeneration in the late postoperative phase and the possible use of the piezoelectric knife as an adjunct for guided bone regeneration, site development, or site preparation for dental implants.


Assuntos
Implantes Dentários , Ultrassom , Animais , Regeneração Óssea , Osso e Ossos , Osteoblastos , Ratos
3.
Life Sci ; 255: 117827, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32450170

RESUMO

AIMS: Data suggest pharmacological treatment of depression with selective serotonin reuptake inhibitors (SSRI) may impair bone health. Our group has previously modeled compromised craniofacial healing after treatment with sertraline, a commonly prescribed SSRI, and hypothesized potential culprits: alterations in bone cells, collagen, and/or inflammation. Here we interrogate bone lineage cell alterations due to sertraline treatment as a potential cause of the noted compromised bone healing. MAIN METHODS: Murine pre-osteoblast, pre-osteoclast, osteoblast, and osteoclast cells were treated with clinically relevant concentrations of the SSRI. Studies focused on serotonin pathway targets, cell viability, apoptosis, differentiation, and the osteoblast/osteoclast feedback loop. KEY FINDINGS: All cells studied express neurotransmitters (e.g. serotonin transporter, SLC6A4, SSRI target) and G-protein-coupled receptors associated with the serotonin pathway. Osteoclasts presented the greatest native expression of Slc6a4 with all cell types exhibiting decreases in Slc6a4 expression after SSRI treatment. Pre-osteoclasts exhibited alteration to their differentiation pathway after treatment. Pre-osteoblasts and osteoclasts showed reduced apoptosis after treatment but showed no significant differences in functional assays. RANKL: OPG mRNA and protein ratios were decreased in the osteoblast lineage. Osteoclast lineage cells treated with sertraline demonstrated diminished TRAP positive cells when pre-exposed to sertraline prior to RANKL-induced differentiation. SIGNIFICANCE: These data suggest osteoclasts are a likely target of bone homeostasis disruption due to sertraline treatment, most potently through the osteoblast/clast feedback loop.


Assuntos
Osso e Ossos/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Inibidores de Captação de Serotonina/toxicidade , Células 3T3 , Animais , Apoptose/efeitos dos fármacos , Osso e Ossos/citologia , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Camundongos , Osteoblastos/citologia , Osteoclastos/citologia , Ligante RANK/metabolismo , Células RAW 264.7 , RNA Mensageiro/metabolismo
4.
PLoS Genet ; 16(5): e1008586, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32463816

RESUMO

The strength of bone depends on bone quantity and quality. Osteocalcin (Ocn) is the most abundant noncollagenous protein in bone and is produced by osteoblasts. It has been previously claimed that Ocn inhibits bone formation and also functions as a hormone to regulate insulin secretion in the pancreas, testosterone synthesis in the testes, and muscle mass. We generated Ocn-deficient (Ocn-/-) mice by deleting Bglap and Bglap2. Analysis of Ocn-/-mice revealed that Ocn is not involved in the regulation of bone quantity, glucose metabolism, testosterone synthesis, or muscle mass. The orientation degree of collagen fibrils and size of biological apatite (BAp) crystallites in the c-axis were normal in the Ocn-/-bone. However, the crystallographic orientation of the BAp c-axis, which is normally parallel to collagen fibrils, was severely disrupted, resulting in reduced bone strength. These results demonstrate that Ocn is required for bone quality and strength by adjusting the alignment of BAp crystallites parallel to collagen fibrils; but it does not function as a hormone.


Assuntos
Apatitas/metabolismo , Calcificação Fisiológica/genética , Metabolismo dos Carboidratos/genética , Glucose/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Osteocalcina/fisiologia , Testosterona/biossíntese , Animais , Apatitas/química , Osso e Ossos/metabolismo , Colágeno/metabolismo , Cristalização , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Tamanho do Órgão/genética , Osteoblastos/metabolismo , Osteocalcina/genética , Osteogênese/genética , Testículo/crescimento & desenvolvimento , Testículo/metabolismo
5.
Life Sci ; 254: 117809, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32428598

RESUMO

Bone remodeling is a complex and constant process, which is maintained by well-regulated communication among various cells. Extracellular vesicles (EVs) are small vesicles, which could provide a protective environment for the transportation of various functional molecules. It has been shown that EVs could dock with distant and/or neighboring target cells, deliver cargoes to these specific cells and alter their fates. MicroRNAs (miRNAs), single-stranded non-coding RNAs with 22-26 nucleotides, could bind to mRNAs and repress the translation or stimulate the degradation of mRNAs. It is reported that EVs could serve as the mail carriers, which could cargo miRNAs to exchange information between different cells and act through a novel way to regulate signaling pathways during bone remodeling. In this review, we summarize the function of EV-miRNAs in the communication among mesenchymal stem cells (MSCs), osteoblasts, osteoclasts, osteocytes, and myoblasts during bone remodeling, as well as the key signaling molecules which are involved in this process. The roles of EV-miRNAs in sending intercellular messages in the microenvironment of bone remodeling could shed new light on the development of tissue engineering, and provide novel diagnostic markers and therapeutic targets of bone-related diseases.


Assuntos
Remodelação Óssea/fisiologia , Comunicação Celular/fisiologia , Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , Animais , Humanos , Células-Tronco Mesenquimais/metabolismo , Mioblastos/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo
6.
Int J Nanomedicine ; 15: 2011-2026, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32273699

RESUMO

Introduction: The bone regeneration of endosseous implanted biomaterials is often impaired by the host immune response, especially macrophage-related inflammation which plays an important role in the bone healing process. Thus, it is a promising strategy to design an osteo-immunomodulatory biomaterial to take advantage of the macrophage-related immune response and improve the osseointegration performance of the implant. Methods: In this study, we developed an antibacterial silver nanoparticle-loaded TiO2 nanotubes (Ag@TiO2-NTs) using an electrochemical anodization method to make the surface modification and investigated the influences of Ag@TiO2-NTs on the macrophage polarization, osteo-immune microenvironment as well as its potential molecular mechanisms in vitro and in vivo. Results: The results showed that Ag@TiO2-NTs with controlled releasing of ultra-low-dose Ag+ ions had the excellent ability to induce the macrophage polarization towards the M2 phenotype and create a suitable osteo-immune microenvironment in vitro, via inhibiting PI3K/Akt, suppressing the downstream effector GLUT1, and activating autophagy. Moreover, Ag@TiO2-NTs surface could improve bone formation, suppress inflammation, and promote osteo-immune microenvironment compared to the TiO2-NTs and polished Ti surfaces in vivo. These findings suggested that Ag@TiO2-NTs with controlled releasing of ultra-low-dose Ag+ ions could not only inhibit the inflammation process but also promote the bone healing by inducing healing-associated M2 polarization. Discussion: Using this surface modification strategy to modulate the macrophage-related immune response, rather than prevent the host response, maybe a promising strategy for implant surgeries in the future.


Assuntos
Autofagia/efeitos dos fármacos , Transportador de Glucose Tipo 1/metabolismo , Fatores Imunológicos/administração & dosagem , Macrófagos/efeitos dos fármacos , Nanopartículas Metálicas/administração & dosagem , Prata/farmacocinética , Animais , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Materiais Biocompatíveis/química , Técnicas Eletroquímicas , Transportador de Glucose Tipo 1/genética , Fatores Imunológicos/imunologia , Masculino , Nanopartículas Metálicas/química , Camundongos , Nanotubos/química , Osseointegração/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Próteses e Implantes , Células RAW 264.7 , Ratos Sprague-Dawley , Prata/química , Tíbia/diagnóstico por imagem , Tíbia/cirurgia , Titânio/química , Cicatrização/efeitos dos fármacos
7.
Int J Nanomedicine ; 15: 2095-2118, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32273705

RESUMO

Purpose: Zinc (Zn), an essential trace element in the body, has stable chemical properties, excellent osteogenic ability and moderate immunomodulatory property. In the present study, a Zn-incorporated TiO2 nanotube (TNT) was fabricated on titanium (Ti) implant material. We aimed to evaluate the influence of nano-scale topography and Zn on behaviors of murine RAW 264.7 macrophages. Moreover, the effects of Zn-incorporated TNT surface-regulated macrophages on the behaviors and osteogenic differentiation of murine MC3T3-E1 osteoblasts were also investigated. Methods: TNT coatings were firstly fabricated on a pure Ti surface using anodic oxidation, and then nano-scale Zn particles were incorporated onto TNTs by the hydrothermal method. Surface topography, chemical composition, roughness, hydrophilicity, Zn release pattern and protein adsorption ability of the Zn-incorporated TiO2 nanotube surface were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS), surface profiler, contact angle test, Zn release test and protein adsorption test. The cell behaviors and both pro-inflammatory (M1) and pro-regenerative (M2) marker gene and protein levels in macrophages cultured on Zn-incorporated TNTs surfaces with different TNT diameters were detected. The supernatants of macrophages were extracted and preserved as conditioned medium (CM). Furthermore, the behaviors and osteogenic properties of osteoblasts cultured in CM on various surfaces were evaluated. Results: The release profile of Zn on Zn-incorporated TNT surfaces revealed a controlled release pattern. Macrophages cultured on Zn-incorporated TNT surfaces displayed enhanced gene and protein expression of M2 markers, and M1 markers were moderately inhibited, compared with the LPS group (the inflammation model). When cultured in CM, osteoblasts cultured on Zn-incorporated TNTs showed strengthened cell proliferation, adhesion, osteogenesis-related gene expression, alkaline phosphatase activity and extracellular mineralization, compared with their TNT counterparts and the Ti group. Conclusion: This study suggests that the application of Zn-incorporated TNT surfaces may establish an osteogenic microenvironment and accelerate bone formation. It provided a promising strategy of Ti surface modification for a better applicable prospect.


Assuntos
Fatores Imunológicos/farmacologia , Macrófagos/efeitos dos fármacos , Nanotubos , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Zinco/farmacocinética , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Microscopia Eletrônica de Varredura , Nanotubos/química , Osteoblastos/citologia , Osteogênese/genética , Espectroscopia Fotoeletrônica , Células RAW 264.7 , Propriedades de Superfície , Titânio/química , Difração de Raios X , Zinco/química
8.
PLoS One ; 15(4): e0231276, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32275737

RESUMO

OBJECTIVES: Alloyed metallic nanoparticles of silver and copper are effective against intracellular infection. However, systemic toxicity may arise due to the non-specific delivery of the nanoparticles. In addressing the issue, this study deals with the targeting of silver-copper-boron (ACB) nanoparticles to infected osteoblasts, which could decrease systemic toxicity and form the basis of targeting specific markers expressed in bone infections. METHODS: ACB nanoparticles were synthesized and conjugated to the Cadherin-11 antibody (OBAb). The effect of targeting nanoparticles against extracellular and intracellular S. aureus was determined by enumeration of bacterial growth. The binding of the targeting nanoparticles to infected osteoblasts as well as the visualization of live/dead bacteria due to treatment was carried out using fluorescence microscopy. MTT assay was used to determine the viability of osteoblasts with different concentrations of the nanoparticles. RESULTS: The ACB nanoparticles conjugated to OBAb (ACB-OBAb) were effective against extracellular S. aureus. The ACB-OBAb nanoparticles showed a 1.32 log reduction of intracellular S. aureus at a concentration of 1mg/L. The ACB-OBAb nanoparticles were able to bind to the infected osteoblast and showed toxicity to osteoblasts at levels ≥20mg/L. Also, the percentage of silver, copper, and boron in the nanoparticles determined the effectiveness of their antibacterial activity. CONCLUSION: The ACB-OBAb nanoparticles were able to target the osteoblasts and demonstrated significant antibacterial activity against intracellular S. aureus. Targeting shows promise as a strategy to target specific markers expressed on infected osteoblasts for efficient nanoparticle delivery, and further animal studies are recommended to test its efficacy in vivo.


Assuntos
Ligas/farmacologia , Osso e Ossos/citologia , Boro/farmacologia , Cobre/farmacologia , Espaço Intracelular/microbiologia , Nanopartículas Metálicas/química , Prata/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/farmacologia , Caderinas/imunologia , Linhagem Celular , Endocitose/efeitos dos fármacos , Humanos , Espaço Intracelular/efeitos dos fármacos , Nanopartículas Metálicas/ultraestrutura , Testes de Sensibilidade Microbiana , Osteoblastos/efeitos dos fármacos , Osteoblastos/microbiologia
9.
Gene ; 748: 144668, 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32334025

RESUMO

KMN-159 is the lead compound from a series of novel difluorolactam prostanoid EP4 receptor agonists aimed at inducing local bone formation while avoiding the inherent side effects of systemic EP4 activation. KMN-159 is a potent, selective small molecule possessing pharmacokinetic properties amenable to local administration. Unfractionated rat bone marrow cells (BMCs) were treated once at plating with escalating doses of KMN-159 (1 pM to 10 µM). The resulting elevated alkaline phosphatase (ALP) levels measured 9 days post-dose are consistent with increased osteoblastic differentiation and exposure to KMN-159 at low nanomolar concentrations for as little as 30 min was sufficient to induce complete osteoblast differentiation of the BMCs from both sexes and regardless of age. ALP induction was blocked by an EP4 receptor antagonist but not by EP1 or EP2 receptor antagonists and was not induced by EP2 or EP3 receptor agonists. Addition of BMCs to plates coated with KMN-159 24 days earlier resulted in ALP activation, highlighting the chemical stability of the compound. The expression of phenotype markers such as ALP, type I collagen, and osteocalcin was significantly elevated throughout the osteoblastic differentiation timecourse initiated by KMN-159 stimulation. An increased number of tartrate-resistant acid phosphatase-positive cells was observed KMN-159 or PGE2 treated BMCs but only in the presence of exogenous receptor activator of nuclear factor kappa-Β ligand (RANKL). No change in the number of adipocytes was observed. KMN-159 also increased bone healing in a rat calvarial defect model with a healing rate equivalent to recombinant human bone morphogenetic protein-2. Our studies show that KMN-159 is able to stimulate osteoblastic differentiation with a very short time of exposure, supporting its potential as a therapeutic candidate for augmenting bone mass.


Assuntos
Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Ácidos Heptanoicos/farmacologia , Osteoblastos/efeitos dos fármacos , Pirrolidinas/farmacologia , Receptores de Prostaglandina E Subtipo EP4/agonistas , Fosfatase Alcalina/metabolismo , Animais , Ativação Enzimática , Feminino , Células HEK293 , Humanos , Osteoblastos/citologia , Osteoblastos/enzimologia , Ratos , Ratos Sprague-Dawley
10.
Cell Prolif ; 53(5): e12814, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32346990

RESUMO

OBJECTIVES: Increasing evidences suggest that inducing mesenchymal stem cells to differentiate into osteoblasts has been as an especially important component in the prevention and therapy for degenerative bone disease. Here, we identify a novel lncRNA, linc02349, which increases significantly during osteogenic differentiation. MATERIALS AND METHODS: Human umbilical cord-derived stem cells (hUC-MSCs) and dental pulp mesenchymal stem cells were used. Overexpression and knockdown of linc02349 in cell lines were generated using lentiviral-mediated gene delivery method. Bioinformatics prediction, Ago2-RIP assay and dual-luciferase reporter system were employed to examine miRNA which interacts with linc02349. The RNA FISH assay was performed to identify the subcelluar location of linc02349. Alizarin Red S staining, ALP staining and qPCR were applied to identify the osteogenic differentiation. The potential linc02349-regulated genes, miR-25-3p and miR-33b-5p, were explored by ChIP, RIP and Western blotting assays. Micro-CT was used to measure the osteogenic content in bone formation assay in vivo. RESULTS: Linc02349 overexpression improves osteogenic differentiation by in vitro and in vivo analysis. Mechanistically, linc02349 acts as a molecular sponge for miR-25-3p and miR-33b-5p to control expression abundance of SMAD5 and Wnt10b, respectively, which eventually activated Dlx5/OSX pathway and hence promoted osteogenic differentiation. In addition, we revealed that STAT3 interacts with linc02349 promoter region and positively regulates the linc02349 transcriptional activity. CONCLUSION: These findings identify that linc02349 modulates the osteogenic differentiation through acting as a sponge RNA of miR-25-3p and miR-33b-5p and regulating SMAD5 and Wnt10b, and proposed a new interaction between STAT3 and linc02349, which could be a potential target in the process the osteogenesis of hUC-MSCs for future clinical application.


Assuntos
Células-Tronco Mesenquimais/patologia , MicroRNAs/genética , Osteogênese/genética , RNA Longo não Codificante/genética , Cordão Umbilical/patologia , Diferenciação Celular/genética , Células Cultivadas , Células HEK293 , Humanos , Osteoblastos/patologia , Regiões Promotoras Genéticas/genética , Fator de Transcrição STAT3/genética , Transcrição Genética/genética
11.
Life Sci ; 253: 117636, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32251631

RESUMO

BMAL1 is a core component of the circadian clock loop, which directs the sophisticated circadian expression of clock-controlled genes. Skeletal Bone development is a complex biological process involving intramembranous ossification, endochondral ossification and bone remodeling, as well as specific cells, such as mesenchymal cells, osteoblasts, osteoclasts, chondrocytes, etc. Growing evidences suggest that BMAL1 is indispensable for hard tissue development, including bone, cartilage and teeth. Loss of BMAL1 in animals can inhibit bone and cartilage development, and result in abnormal bone mass. In mesenchymal cells, BMAL1 defect inhibits osteoblastic and chondrocytic differentiation. Inactivation of BMAL1 also can promote the differentiation and formation of osteoclasts and increase bone resorption. Specifically, preclinical data demonstrate that the abnormity of BMAL1 expression is associated with skeletal disorders such as skeletal mandibular hypoplasia, osteoarthritis, osteoporosis, etc. In this review, we systemically describe the impact of BMAL1 in skeletal development and homeostasis, and devote to searching new therapy strategies for bone disorders.


Assuntos
Fatores de Transcrição ARNTL/metabolismo , Desenvolvimento Ósseo/efeitos dos fármacos , Fatores de Transcrição ARNTL/genética , Animais , Densidade Óssea/efeitos dos fármacos , Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Cartilagem/metabolismo , Diferenciação Celular , Condrócitos/metabolismo , Condrogênese/efeitos dos fármacos , Relógios Circadianos/genética , Regulação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese , Dente/metabolismo
12.
Life Sci ; 253: 117660, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32294474

RESUMO

AIMS: Osteoporosis has been known to generally result from an imbalance between bone formation and resorption. Osteogenesis is the process of differentiation of mesenchymal stem cells (MSCs) into osteoblasts. Sirtuin6 (SIRT6) has been reported to mediate osteogenic differentiation (OD) in rat bone MSCs (rBMSCs). The present study aimed to assess the influence of microRNA miR-186 on the proliferation and OD potential of rBMSCs. MAIN METHODS: OD was performed and evaluated through Alizarin red S staining, alkaline phosphatase (ALP) activity, and specific marker expression. KEY FINDINGS: miR-186 downregulation was observed during OD. rBMSCs with miR-186 overexpression were generated via transfection. Compared with vehicle negative controls, miR-186 upregulation significantly repressed rBMSCs' OD, as evidenced by a reduced ALP activity and decreased mRNA levels of osteogenic markers [osteocalcin, Runx2, BSP, and ALP]. Furthermore, bioinformatic prediction and dual-luciferase reporter assay demonstrated that miR-186 targeted SIRT6 3'-UTR for silencing. SIRT6 overexpression reversed the inhibitory effect of miR-186 on the OD of rBMSCs. Additionally, further examination showed that the activation of nuclear factor-kappa B (NFκB) pathway was involved in the miR-186/SIRT6 signal axis, and phorbol 12-myristate 13-acetate, a NFκB activator, also inhibited the OD of rBMSCs. SIGNIFICANCE: The present study results may demonstrate a novel mechanism of rBMSCs OD via miR-186-SIRT6 interaction.


Assuntos
MicroRNAs/metabolismo , Osteoblastos/metabolismo , Osteogênese/genética , Sirtuínas/genética , Fosfatase Alcalina/metabolismo , Animais , Sequência de Bases , Regeneração Óssea/genética , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Diferenciação Celular/genética , Proliferação de Células/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica , Lentivirus/genética , Células-Tronco Mesenquimais/citologia , NF-kappa B/metabolismo , Osteocalcina/metabolismo , RNA Mensageiro/metabolismo , Ratos , Transdução de Sinais , Sirtuínas/metabolismo , Transfecção
13.
J Biol Regul Homeost Agents ; 34(1 Suppl. 2): 1-5, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32270661

RESUMO

Collagenated heretologous cortico-cancelleus bone mix (CHCCBM) is largely employed in maxillary and dental surgery for regeneration procedures, and is similar to human bone from chemical and physical point of view and promotes osteogenesis. In order to get more inside how this biomaterial induces osteoblast gene expression to promote bone formation, the mRNA levels of bone related genes were compared in human osteoblasts and dental pulp stem cells, using real time RT-PCR. The obtained results demonstrated that CHCCBM enhance stem cells differentiation and deposition of matrix by the activation of osteoblast related genes SP7, FOSL1 and SPP1.


Assuntos
Polpa Dentária/citologia , Osteoblastos/citologia , Osteogênese , Células-Tronco/citologia , Diferenciação Celular , Células Cultivadas , Humanos , Osteopontina/genética , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/genética , Fator de Transcrição Sp7/genética
14.
Gene ; 749: 144703, 2020 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-32339623

RESUMO

The repair of segmental bone defects and bone fractures is a clinical challenge involving high risk and postsurgical morbidity. Bone injury and partial bone tumor resection via traditional bone grafting result in high complications. Growth factors have been proposed as alternatives to promote bone repair and formation and circumvent these limitations. In this study, we classified different lengths of mechano growth factor (MGF) E peptides in different species and analyzed their effects on MC3T3-E1 cell proliferation, cell cycle, alkaline phosphatase (ALP) activity, differentiation-related factor expression, and cell mineralization. A rabbit bone injury model was constructed, and the repair function of MGF E peptide was verified by injecting the candidate MGF E peptide. We analyzed 52 different MGF-E peptides and classified them into the following four categories: T-MGF-25E, M-MGF-25E, T-MGF-19E, and M-MGF-19E. These peptides were synthesized for further study. T-MGF-19E peptide obviously promoted cell proliferation by regulating cell cycle after MGF E peptide treatment at 72 h. T-MGF-25E and T-MGF-19E peptide significantly promoted the differentiation of osteoblasts on day 14, and M-MGF-25E peptide promoted cell differentiation on day 7. T-MGF-19E, T-MGF-25E, and M-MGF-19E significantly promoted osteoblast mineralization, with T-MGF19E showing the most significant effect. These results implied that T-MGF19E peptide could remarkably promote MC3T3-E1 cell proliferation, differentiation, and mineralization. The rabbit bone defect model showed that the low-dose T-MGF-19E peptide significantly promoted bone injury healing, suggesting its promoting effect on the healing of bone injury.


Assuntos
Osso e Ossos/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Osteogênese/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Osso e Ossos/fisiologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/genética
15.
BMC Bioinformatics ; 21(1): 114, 2020 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-32183690

RESUMO

BACKGROUND: Mechanotransduction in bone cells plays a pivotal role in osteoblast differentiation and bone remodelling. Mechanotransduction provides the link between modulation of the extracellular matrix by mechanical load and intracellular activity. By controlling the balance between the intracellular and extracellular domains, mechanotransduction determines the optimum functionality of skeletal dynamics. Failure of this relationship was suggested to contribute to bone-related diseases such as osteoporosis. RESULTS: A hybrid mechanical and agent-based model (Mech-ABM), simulating mechanotransduction in a single osteoblast under external mechanical perturbations, was utilised to simulate and examine modulation of the activation dynamics of molecules within mechanotransduction on the cellular response to mechanical stimulation. The number of molecules and their fluctuations have been analysed in terms of recurrences of critical events. A numerical approach has been developed to invert subordination processes and to extract the direction processes from the molecular signals in order to derive the distribution of recurring events. These predict that there are large fluctuations enclosing information hidden in the noise which is beyond the dynamic variations of molecular baselines. Moreover, studying the system under different mechanical load regimes and altered dynamics of feedback loops, illustrate that the waiting time distributions of each molecule are a signature of the system's state. CONCLUSIONS: The behaviours of the molecular waiting times change with the changing of mechanical load regimes and altered dynamics of feedback loops, presenting the same variation of patterns for similar interacting molecules and identifying specific alterations for key molecules in mechanotransduction. This methodology could be used to provide a new tool to identify potent molecular candidates to modulate mechanotransduction, hence accelerate drug discovery towards therapeutic targets for bone mass upregulation.


Assuntos
Mecanotransdução Celular , Osteoblastos/metabolismo , Matriz Extracelular , Humanos , Proteínas/genética , Proteínas/metabolismo , Regulação para Cima
16.
Int J Nanomedicine ; 15: 1643-1659, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32210558

RESUMO

Purpose: Aseptic loosening is a major complication after total joint replacement. Reactive oxygen species generated by local tissue cells and liberated from implant surfaces have been suggested to cause implant failures. Surface modification of titanium (Ti)-based implants with proanthocyanidins (PAC) is a promising approach for the development of anti-oxidant defense mechanism to supplement the mechanical functions of Ti implants. In this study, a controlled PAC release system was fabricated on the surface of Ti substrates using the layer-by-layer (LBL) assembly. Materials and Methods: Polyethyleneimine (PEI) base layer was fabricated to enable layer-by-layer (LBL) deposition of hyaluronic acid/chitosan (HA/CS) multi-layers without or with the PAC. Surface topography and wettability of the fabricated HA/CS-PAC substrates were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), Fourier-transform infrared spectroscopy (FTIR) and contact angle measurement. PAC release profiles were investigated using drug release assays. MC3T3-E1 pre-osteoblast cells were used to assess the osteo-inductive effects of HA/CS-PAC substrates under conditions H2O2-induced oxidative stress in vitro. A rat model of femoral intramedullary implantation evaluated the osseo-integration and osteo-inductive potential of the HA/CS-PAC coated Ti implants in vivo. Results: SEM, AFM, FTIR and contact angle measurements verified the successful fabrication of Ti surfaces with multi-layered HA/CS-PAC coating. Drug release assays revealed controlled and sustained release of PAC over 14 days. In vitro, cell-based assays showed high tolerability and enhanced the osteogenic potential of MC3T3-E1 cells on HA/CS-PAC substrates when under conditions of H2O2-induced oxidative stress. In vivo evaluation of femoral bone 14 days after femoral intramedullary implantation confirmed the enhanced osteo-inductive potential of the HA/CS-PAC coated Ti implants. Conclusion: Multi-layering of HA/CS-PAC coating onto Ti-based surfaces by the LBL deposition significantly enhances implant osseo-integration and promotes osteogenesis under conditions of oxidative stress. This study provides new insights for future applications in the field of joint arthroplasty.


Assuntos
Antioxidantes/farmacologia , Osteogênese/efeitos dos fármacos , Proantocianidinas/farmacologia , Próteses e Implantes , Titânio/farmacologia , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Quitosana/química , Liberação Controlada de Fármacos , Feminino , Ácido Hialurônico/química , Peróxido de Hidrogênio/farmacologia , Espaço Intracelular/metabolismo , Camundongos , Osseointegração/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Polietilenoimina/química , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Molhabilidade , Microtomografia por Raio-X
17.
J Oral Sci ; 62(2): 212-216, 2020 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-32161234

RESUMO

The morphogenesis of long bones is a multistep process that generates a variety of genetically defined forms. The tarsometatarsal (TMT) long bone morphology in birds develops through lateral fusion of three initially independent periosteal bone cylinders (BCs). Previous studies have clarified the histological details and chronology of the changes occurring during development. The present study investigated the temporospatial distribution of osteogenic and osteoclastic cells in the embryonic chicken using histochemistry for alkaline phosphatase and tartrate-resistant acid phosphatase, with particular reference to the radial growth of BCs and their subsequent fusion process. Osteogenic cells were localized preferentially in the periosteum of radially growing BCs, leaving open cancellous spaces in the BC wall. Osteoclasts observed later than embryonic day 10 were localized preferentially in the endosteal surface, and therefore the radial growth of BCs resulting from osteoblast activity was accompanied by endosteal resorption by osteoclasts, with progressive enlargement of the bone marrow spaces. During BC fusion, trabecular bridges were formed by periosteal osteogenic cells, with removal of the bone septum by endosteal osteoclasts. These findings suggest that fusion of BCs in the embryonic chicken is mediated by cellular events constituting ordinary long bone development, and not through a defined mechanism specific for fusion.


Assuntos
Galinhas , Osteoclastos , Animais , Osso e Ossos , Embrião de Galinha , Osteoblastos , Osteogênese
18.
Med Sci Monit ; 26: e919309, 2020 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-32146478

RESUMO

BACKGROUND Osteoblast differentiation is a critical process to maintain the stability of the bone homeostasis. Zingerone, 4-(4-hydroxy-3-methoxyphenyl)-2-butanone (ZG), isolated from ginger, performs a wide range of biological functions in human diseases. The objective of this paper was to clarify the role of ZG in human bone mesenchymal stem cells (hBMSCs) and associated mechanisms of ZG promoting osteoblast differentiation. MATERIAL AND METHODS The cytotoxicity of ZG was detected by MTT assay. The expression levels of miR-200c-3p, smad7, and osteoblast differentiation markers (alkaline phosphatase [ALP], osteocalcin [OC], osterix [OSX] and runt-related transcription factor 2 [RUNX2]) were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of smad7, ALP, OC, OSX, and RUNX2 were quantified by western blot analysis. The target mRNAs were predicted by bioinformatics tools TargetScan. The interaction between miR-200c-3p and smad7 was verified by luciferase reporter assay and RIP assay. RESULTS ZG was nontoxic to hBMSCs, and it accelerated osteoblast differentiation by inducing the expression of ALP, OC, OSX, and RUNX2. MiR-200c-3p was upregulated, but smad7 was downregulated in hBMSCs treated with ZG at different concentrations at different periods. Besides, miR-200c-3p positively regulated the expression of ALP, OC, OSX, and RUNX2 in ZG-induced hBMSCs. Moreover, miR-200c-3p targeted smad7 and strengthened the expression of ALP, OC, OSX, and RUNX2 in ZG-induced hBMSCs by downregulating smad7. CONCLUSIONS ZG contributed to osteoblast differentiation via miR-200c-3p/smad7 regulatory axis by promoting the expression of ALP, OC, OSX, and RUNX2 in hBMSCs.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Guaiacol/análogos & derivados , Células-Tronco Mesenquimais/efeitos dos fármacos , MicroRNAs/genética , Osteoblastos/citologia , Osteogênese/efeitos dos fármacos , Proteína Smad7/genética , Fosfatase Alcalina/metabolismo , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Guaiacol/farmacologia , Humanos , Células-Tronco Mesenquimais/citologia , Osteoblastos/efeitos dos fármacos , Osteocalcina/metabolismo , Fator de Transcrição Sp7/metabolismo
19.
Int J Nanomedicine ; 15: 1595-1610, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32210554

RESUMO

Introduction: The development of the field of biomaterials engineering is rapid. Various bioactive coatings are created to improve the biocompatibility of substrates used for bone regeneration, which includes formulation of thin zirconia coatings with pro-osteogenic properties. The aim of this study was to assess the biological properties of ZrO2 thin films grown by Atomic Layer Deposition (ALD) technology (ZrO2 ALD). Methodology: The cytocompatibility of the obtained layers was analysed using the mice pre-osteoblastic cell line (MC3T3) characterized by decreased expression of microRNA 21-5p (miR-21-5p) in order to evaluate the potential pro-osteogenic properties of the coatings. The in vitro experiments were designed to determine the effect of ZrO2 ALD coatings on cell morphology (confocal microscope), proliferative activity (cell cycle analysis) and metabolism, reflected by mitochondrial membrane potential (cytometric-based measurement). Additionally, the influence of layers on the expression of genes associated with cell survival and osteogenesis was studied using RT-qPCR. The following genes were investigated: B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), p53 and p21, as well as osteogenic markers, i.e. collagen type 1 (Coll-1), osteopontin (Opn), osteocalcin (Ocl) and runt-related transcription factor 2 (Runx-2). The levels of microRNA (miRNA/miR) involved in the regulation of osteogenic genes were determined, including miR-7, miR-21, miR-124 and miR-223. Results: The analysis revealed that the obtained coatings are cytocompatible and may increase the metabolism of pre-osteoblast, which was correlated with increased mitochondrial membrane potential and extensive development of the mitochondrial network. The obtained coatings affected the viability and proliferative status of cells, reducing the population of actively dividing cells. However, in cultures propagated on ZrO2 ALD coatings, the up-regulation of genes essential for bone metabolism was noted. Discussion: The data obtained indicate that ZrO2 coatings created using the ALD method may have pro-osteogenic properties and may improve the metabolism of bone precursor cells. Given the above, further development of ZrO2 ALD layers is essential in terms of their potential clinical application in bone regenerative medicine.


Assuntos
Materiais Biocompatíveis/química , Osteoblastos/citologia , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Zircônio/farmacologia , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Regulação da Expressão Gênica , Teste de Materiais , Camundongos , MicroRNAs/genética , Nanotecnologia/métodos , Osteoblastos/efeitos dos fármacos , Osteoblastos/ultraestrutura , Regulação para Cima , Zircônio/química
20.
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
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