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1.
Int J Mol Sci ; 22(13)2021 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-34206740

RESUMO

N-Glycosylations are an important post-translational modification of proteins that can significantly impact cell function. Terminal sialic acid in hybrid or complex N-glycans has been shown to be relevant in various types of cancer, but its role in non-malignant cells remains poorly understood. We have previously shown that the motility of human bone marrow derived mesenchymal stromal cells (MSCs) can be modified by altering N-glycoforms. The goal of this study was to determine the role of sialylated N-glycans in MSCs. Here, we show that IFN-gamma or exposure to culture media low in fetal bovine serum (FBS) increases sialylated N-glycans, while PDGF-BB reduces them. These stimuli alter mRNA levels of sialyltransferases such as ST3Gal1, ST6Gal1, or ST3Gal4, suggesting that sialylation of N-glycans is regulated by transcriptional control of sialyltransferases. We next show that 2,4,7,8,9-pentaacetyl-3Fax-Neu5Ac-CO2Me (3F-Neu5Ac) effectively inhibits sialylations in MSCs. Supplementation with 3F-Neu5Ac increases adhesion and migration of MSCs, as assessed by both videomicroscopy and wound/scratch assays. Interestingly, pre-treatment with 3F-Neu5Ac also increases the survival of MSCs in an in vitro ischemia model. We also show that pre-treatment or continuous treatment with 3F-Neu5Ac inhibits both osteogenic and adipogenic differentiation of MSCs. Finally, secretion of key trophic factors by MSCs is variably affected upon exposure to 3F-Neu5Ac. Altogether, our experiments suggest that sialylation of N-glycans is tightly regulated in response to environmental cues and that glycoengineering MSCs to reduce sialylated N-glycans could be beneficial to increase both cell migration and survival, which may positively impact the therapeutic potential of the cells.


Assuntos
Movimento Celular , Células-Tronco Mesenquimais/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/metabolismo , Sialiltransferases/metabolismo , Adipócitos/metabolismo , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Interferon gama/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Osteoblastos/citologia , Sialiltransferases/antagonistas & inibidores
2.
Int J Mol Sci ; 22(11)2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073480

RESUMO

Mesenchymal stem cells (MSCs) can differentiate into osteoblasts, and therapeutic targeting of these cells is considered both for malignant and non-malignant diseases. We analyzed global proteomic profiles for osteoblasts derived from ten and MSCs from six healthy individuals, and we quantified 5465 proteins for the osteoblasts and 5420 proteins for the MSCs. There was a large overlap in the profiles for the two cell types; 156 proteins were quantified only in osteoblasts and 111 proteins only for the MSCs. The osteoblast-specific proteins included several extracellular matrix proteins and a network including 27 proteins that influence intracellular signaling (Wnt/Notch/Bone morphogenic protein pathways) and bone mineralization. The osteoblasts and MSCs showed only minor age- and sex-dependent proteomic differences. Finally, the osteoblast and MSC proteomic profiles were altered by ex vivo culture in serum-free media. We conclude that although the proteomic profiles of osteoblasts and MSCs show many similarities, we identified several osteoblast-specific extracellular matrix proteins and an osteoblast-specific intracellular signaling network. Therapeutic targeting of these proteins will possibly have minor effects on MSCs. Furthermore, the use of ex vivo cultured osteoblasts/MSCs in clinical medicine will require careful standardization of the ex vivo handling of the cells.


Assuntos
Células da Medula Óssea/metabolismo , Regulação da Expressão Gênica , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Proteômica , Via de Sinalização Wnt , Idoso , Células da Medula Óssea/citologia , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Pessoa de Meia-Idade , Osteoblastos/citologia
3.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071605

RESUMO

Gut microbiota has emerged as an important regulator of bone homeostasis. In particular, the modulation of innate immunity and bone homeostasis is mediated through the interaction between microbe-associated molecular patterns (MAMPs) and the host pattern recognition receptors including Toll-like receptors and nucleotide-binding oligomerization domains. Pathogenic bacteria such as Porphyromonas gingivalis and Staphylococcus aureus tend to induce bone destruction and cause various inflammatory bone diseases including periodontal diseases, osteomyelitis, and septic arthritis. On the other hand, probiotic bacteria such as Lactobacillus and Bifidobacterium species can prevent bone loss. In addition, bacterial metabolites and various secretory molecules such as short chain fatty acids and cyclic nucleotides can also affect bone homeostasis. This review focuses on the regulation of osteoclast and osteoblast by MAMPs including cell wall components and secretory microbial molecules under in vitro and in vivo conditions. MAMPs could be used as potential molecular targets for treating bone-related diseases such as osteoporosis and periodontal diseases.


Assuntos
Diferenciação Celular/fisiologia , Microbioma Gastrointestinal/fisiologia , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteócitos/metabolismo , Animais , Homeostase/fisiologia , Humanos , Osteoblastos/citologia , Osteoclastos/citologia , Osteócitos/citologia , Receptores de Reconhecimento de Padrão/metabolismo , Receptores Toll-Like/metabolismo
4.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070855

RESUMO

Lens epithelium-derived growth factor splice variant of 75 kDa (LEDGF/p75) plays an important role in cancer, but its DNA-damage repair (DDR)-related implications are still not completely understood. Different LEDGF model cell lines were generated: a complete knock-out of LEDGF (KO) and re-expression of LEDGF/p75 or LEDGF/p52 using CRISPR/Cas9 technology. Their proliferation and migration capacity as well as their chemosensitivity were determined, which was followed by investigation of the DDR signaling pathways by Western blot and immunofluorescence. LEDGF-deficient cells exhibited a decreased proliferation and migration as well as an increased sensitivity toward etoposide. Moreover, LEDGF-depleted cells showed a significant reduction in the recruitment of downstream DDR-related proteins such as replication protein A 32 kDa subunit (RPA32) after exposure to etoposide. The re-expression of LEDGF/p75 rescued all knock-out effects. Surprisingly, untreated LEDGF KO cells showed an increased amount of DNA fragmentation combined with an increased formation of γH2AX and BRCA1. In contrast, the protein levels of ubiquitin-conjugating enzyme UBC13 and nuclear proteasome activator PA28γ were substantially reduced upon LEDGF KO. This study provides for the first time an insight that LEDGF is not only involved in the recruitment of CtIP but has also an effect on the ubiquitin-dependent regulation of DDR signaling molecules and highlights the role of LEDGF/p75 in homology-directed DNA repair.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , DNA/genética , Regulação da Expressão Gênica , Reparo de DNA por Recombinação , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Antineoplásicos Fitogênicos/farmacologia , Autoantígenos/genética , Autoantígenos/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , DNA/metabolismo , Dano ao DNA , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Etoposídeo/farmacologia , Técnicas de Inativação de Genes , Histonas/genética , Histonas/metabolismo , Humanos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Transdução de Sinais , Fatores de Transcrição/deficiência , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
5.
Int J Mol Sci ; 22(9)2021 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066458

RESUMO

Paeonia suffruticosa has been extensively used as a traditional medicine with various beneficial effects; paeonolide (PALI) was isolated from its dried roots. This study aimed to investigate the novel effects and mechanisms of PALI in pre-osteoblasts. Here, cell viability was evaluated using an MTT assay. Early and late osteoblast differentiation was examined by analyzing the activity of alkaline phosphatase (ALP) and by staining it with Alizarin red S (ARS). Cell migration was assessed using wound healing and Boyden chamber assays. Western blot and immunofluorescence analyses were used to examine the intracellular signaling pathways and differentiation proteins. PALI (0.1, 1, 10, 30, and 100 µM) showed no cytotoxic or proliferative effects in pre-osteoblasts. In the absence of cytotoxicity, PALI (1, 10, and 30 µM) promoted wound healing and transmigration during osteoblast differentiation. ALP staining demonstrated that PALI (1, 10, and 30 µM) promoted early osteoblast differentiation in a dose-dependent manner, and ARS staining showed an enhanced mineralized nodule formation, a key indicator of late osteoblast differentiation. Additionally, low concentrations of PALI (1 and 10 µM) increased the bone morphogenetic protein (BMP)-Smad1/5/8 and Wnt-ß-catenin pathways in osteoblast differentiation. Particularly, PALI (1 and 10 µM) increased the phosphorylation of ERK1/2 compared with BMP2 treatment, an FDA-approved drug for bone diseases. Furthermore, PALI-mediated early and late osteoblast differentiation was abolished in the presence of the ERK1/2 inhibitor U0126. PALI-induced RUNX2 (Cbfa1) expression and nuclear localization were also attenuated by blocking the ERK1/2 pathway during osteoblast differentiation. We suggest that PALI has biologically novel activities, such as enhanced osteoblast differentiation and bone mineralization mainly through the intracellular ERK1/2-RUNX2 signaling pathway, suggesting that PALI might have therapeutic action and aid the treatment and prevention of bone diseases, such as osteoporosis and periodontitis.


Assuntos
Acetofenonas/farmacologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteoblastos/metabolismo , Osteogênese , Animais , Proteína Morfogenética Óssea 2/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Proteína Wnt3/metabolismo
6.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34065020

RESUMO

Although optical hyperthermia could be a promising anticancer therapy, the need for high concentrations of light-absorbing metal nanoparticles and high-intensity lasers, or large exposure times, could discourage its use due to the toxicity that they could imply. In this article, we explore a possible role of silica microparticles that have high biocompatibility and that scatter light, when used in combination with conventional nanoparticles, to reduce those high concentrations of particles and/or those intense laser beams, in order to improve the biocompatibility of the overall procedure. Our underlying hypothesis is that the scattering of light caused by the microparticles would increase the optical density of the irradiated volume due to the production of multiple reflections of the incident light: the nanoparticles present in the same volume would absorb more energy from the laser than without the presence of silica particles, resulting either in higher heat production or in the need for less laser power or absorbing particles for the same required temperature rise. Testing this new optical hyperthermia procedure, based on the use of a mixture of silica and metallic particles, we have measured cell mortality in vitro experiments with murine glioma (CT-2A) and mouse osteoblastic (MC3T3-E1) cell lines. We have used gold nanorods (GNRs) that absorb light with a wavelength of 808 nm, which are conventional in optical hyperthermia, and silica microparticles spheres (hereinafter referred to as SMSs) with a diameter size to scatter the light of this wavelength. The obtained results confirm our initial hypothesis, because a high mortality rate is achieved with reduced concentrations of GNR. We found a difference in mortality between CT2A cancer cells and cells considered non-cancer MC3T3, maintaining the same conditions, which gives indications that this technique possibly improves the efficiency in the cell survival. This might be related with differences in the proliferation rate. Since the experiments were carried out in the 2D dimensions of the Petri dishes, due to sedimentation of the silica particles at the bottom, whilst light scattering is a 3D phenomenon, a large amount of the energy provided by the laser escapes outside the medium. Therefore, better results might be expected when applying this methodology in tissues, which are 3D structures, where the multiple reflections of light we believe will produce higher optical density in comparison to the conventional case of no using scattering particles. Accordingly, further studies deserve to be carried out in this line of work in order to improve the optical hyperthermia technique.


Assuntos
Glioblastoma/terapia , Hipertermia Induzida , Nanopartículas Metálicas/administração & dosagem , Osteoblastos/citologia , Dióxido de Silício/química , Animais , Sobrevivência Celular , Células Cultivadas , Glioblastoma/patologia , Lasers , Nanopartículas Metálicas/química , Camundongos
7.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34073015

RESUMO

Pentraxin 3 (PTX3) is a glycoprotein belonging to the humoral arm of innate immunity that participates in the body's defence mechanisms against infectious diseases. It has recently been defined as a multifunctional protein, given its involvement in numerous physiological and pathological processes, as well as in the pathogenesis of age-related diseases such as osteoporosis. Based on this evidence, the aim of our study was to investigate the possible role of PTX3 in both the osteoblastic differentiation and calcification process: to this end, primary osteoblast cultures from control and osteoporotic patients were incubated with human recombinant PTX3 (hrPTX3) for 72 h. Standard osteinduction treatment, consisting of ß-glycerophosphate, dexamethasone and ascorbic acid, was used as control. Our results showed that treatment with hrPTX3, as well as with the osteogenic cocktail, induced cell differentiation towards the osteoblastic lineage. We also observed that the treatment not only promoted an increase in cell proliferation, but also the formation of calcification-like structures, especially in primary cultures from osteoporotic patients. In conclusion, the results reported here suggest the involvement of PTX3 in osteogenic differentiation, highlighting its osteoinductive capacity, like the standard osteoinduction treatment. Therefore, this study opens new and exciting perspectives about the possible role of PTX3 as biomarker and therapeutic agent for osteoporosis.


Assuntos
Proteína C-Reativa/fisiologia , Osteoblastos , Osteogênese , Osteoporose/metabolismo , Componente Amiloide P Sérico/fisiologia , Calcificação Fisiológica , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos , Pessoa de Meia-Idade , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoblastos/patologia , Cultura Primária de Células
8.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069142

RESUMO

Bone healing is a complex, well-organized process. Multiple factors regulate this process, including growth factors, hormones, cytokines, mechanical stimulation, and aging. One of the most important signaling pathways that affect bone healing is the Notch signaling pathway. It has a significant role in controlling the differentiation of bone mesenchymal stem cells and forming new bone. Interventions to enhance the healing of critical-sized bone defects are of great importance, and stem cell transplantations are eminent candidates for treating such defects. Understanding how Notch signaling impacts pluripotent stem cell differentiation can significantly enhance osteogenesis and improve the overall healing process upon transplantation. In Rancourt's lab, mouse embryonic stem cells (ESC) have been successfully differentiated to the osteogenic cell lineage. This study investigates the role of Notch signaling inhibition in the osteogenic differentiation of mouse embryonic and induced pluripotent stem cells (iPS). Our data showed that Notch inhibition greatly enhanced the differentiation of both mouse embryonic and induced pluripotent stem cells.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Diferenciação Celular/efeitos dos fármacos , Osteogênese/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Animais , Osso e Ossos/metabolismo , Proteínas de Ciclo Celular/genética , Diferenciação Celular/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Dexametasona/farmacologia , Diaminas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/fisiologia , Mesoderma/citologia , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Osteogênese/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Receptores Notch/metabolismo , Tiazóis/farmacologia , Fatores de Transcrição HES-1/genética , Vitamina D/farmacologia
9.
Cell Prolif ; 54(7): e13058, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34053135

RESUMO

OBJECTIVES: To clarify the possible role and mechanism of Cathepsin K (CTSK) in alveolar bone regeneration mediated by jaw bone marrow mesenchymal stem cells (JBMMSC). MATERIALS AND METHODS: Tooth extraction models of Ctsk knockout mice (Ctsk-/- ) and their wildtype (WT) littermates were used to investigate the effect of CTSK on alveolar bone regeneration. The influences of deletion or inhibition of CTSK by odanacatib (ODN) on proliferation and osteogenic differentiation of JBMMSC were assessed by CCK-8, Western blot and alizarin red staining. To explore the differently expressed genes, RNA from WT and Ctsk-/- JBMMSC was sent to RNA-seq. ECAR, glucose consumption and lactate production were measured to identify the effect of Ctsk deficiency or inhibition on glycolysis. At last, we explored whether Ctsk deficiency or inhibition promoted JBMMSC proliferation and osteogenic differentiation through glycolysis. RESULTS: We found out that Ctsk knockout could promote alveolar bone regeneration in vivo. In vitro, we confirmed that both Ctsk knockout and inhibition by ODN could promote proliferation of JBMMSC, up-regulate expression of Runx2 and ALP, and enhance matrix mineralization. RNA-seq results showed that coding genes of key enzymes in glycolysis were significantly up-regulated in Ctsk-/- JBMMSC, and Ctsk deficiency or inhibition could promote glycolysis in JBMMSC. After blocking glycolysis by 3PO, the effect of Ctsk deficiency or inhibition on JBMMSC's regeneration was blocked subsequently. CONCLUSIONS: Our findings revealed that Ctsk knockout or inhibition could promote alveolar bone regeneration by enhancing JBMMSC regeneration via glycolysis. These results shed new lights on the regulatory mechanism of CTSK on bone regeneration.


Assuntos
Regeneração Óssea , Catepsina K/genética , Diferenciação Celular , Proliferação de Células , Células-Tronco Mesenquimais/metabolismo , Animais , Células da Medula Óssea/citologia , Catepsina K/deficiência , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Glucose/metabolismo , Glicólise , Arcada Osseodentária/citologia , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese
10.
Int J Nanomedicine ; 16: 3429-3456, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34040372

RESUMO

Purpose: Osteoporosis results in a severe decrease in the life quality of many people worldwide. The latest data shows that the number of osteoporotic fractures is becoming an increasing international health service problem. Therefore, a new kind of controllable treatment methods for osteoporotic fractures is extensively desired. For that reason, we have manufactured and evaluated nanohydroxyapatite (nHAp)-based composite co-doped with iron oxide (IO) nanoparticles. The biomaterial was used as a matrix for the controlled delivery of miR-21-5p and miR-124-3p, which have a proven impact on bone cell metabolism. Methods: The nanocomposite Ca5(PO4)3OH/Fe3O4 (later called nHAp/IO) was obtained by the wet chemistry method and functionalised with microRNAs (nHAp/IO@miR-21/124). Its physicochemical characterization was performed using XRPD, FT-IR, SEM-EDS and HRTEM and SAED methods. The modulatory effect of the composite was tested in vitro using murine pre-osteoblasts MC3T3-E1 and pre-osteoclasts 4B12. Moreover, the anti-inflammatory effects of biomaterial were analysed using a model of LPS-treated murine macrophages RAW 264.7. We have analysed the cells' viability, mitochondria membrane potential and oxidative stress under magnetic field (MF+) and without (MF-). Moreover, the results were supplemented with RT-qPCR and Western blot assays to evaluate the expression profile for master regulators of bone metabolism. Results: The results indicated pro-osteogenic effects of nHAp/IO@miR-21/124 composite enhanced by exposure to MF. The enhanced osteogenesis guided by nHAp/IO@miR-21/124 presence was associated with increased metabolism of progenitor cells and activation of osteogenic markers (Runx-2, Opn, Coll-1). Simultaneously, nanocomposite decreased metabolism and differentiation of pre-osteoclastic 4B12 cells accompanied by reduced expression of CaII and Ctsk. Obtained composite regulated viability of bone progenitor cells and showed immunomodulatory properties inhibiting the expression of inflammatory markers, ie, TNF-α, iNOs or IL-1ß, in LPS-stimulated RAW 264.7 cells. Conclusion: We have described for the first time a new concept of osteoporosis treatment based on nHAp/IO@miR-21/124 application. Obtained results indicated that fabricated nanocomposite might impact proper regeneration of osteoporotic bone, restoring the balance between osteoblasts and osteoclast.


Assuntos
Durapatita/química , Nanopartículas Magnéticas de Óxido de Ferro/química , MicroRNAs/química , Osteoblastos/citologia , Osteoclastos/citologia , Osteoporose/patologia , Células 3T3 , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Portadores de Fármacos/química , Inflamação/terapia , Campos Magnéticos , Camundongos , MicroRNAs/genética , Nanocompostos/química , Osteoblastos/patologia , Osteoclastos/patologia , Osteogênese/genética , Osteoporose/genética , Osteoporose/terapia
11.
Nature ; 593(7857): 61-66, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33953410

RESUMO

In only a few decades, lithium-ion batteries have revolutionized technologies, enabling the proliferation of portable devices and electric vehicles1, with substantial benefits for society. However, the rapid growth in technology has highlighted the ethical and environmental challenges of mining lithium, cobalt and other mineral ore resources, and the issues associated with the safe usage and non-hazardous disposal of batteries2. Only a small fraction of lithium-ion batteries are recycled, further exacerbating global material supply of strategic elements3-5. A potential alternative is to use organic-based redox-active materials6-8 to develop rechargeable batteries that originate from ethically sourced, sustainable materials and enable on-demand deconstruction and reconstruction. Making such batteries is challenging because the active materials must be stable during operation but degradable at end of life. Further, the degradation products should be either environmentally benign or recyclable for reconstruction into a new battery. Here we demonstrate a metal-free, polypeptide-based battery, in which viologens and nitroxide radicals are incorporated as redox-active groups along polypeptide backbones to function as anode and cathode materials, respectively. These redox-active polypeptides perform as active materials that are stable during battery operation and subsequently degrade on demand in acidic conditions to generate amino acids, other building blocks and degradation products. Such a polypeptide-based battery is a first step to addressing the need for alternative chemistries for green and sustainable batteries in a future circular economy.


Assuntos
Fontes de Energia Elétrica , Eletroquímica , Peptídeos/química , Animais , Bovinos , Linhagem Celular , Sobrevivência Celular , Óxidos N-Cíclicos/química , Camundongos , Osteoblastos/citologia , Oxirredução , Peptídeos/síntese química , Desenvolvimento Sustentável , Viologênios/química
12.
Int J Nanomedicine ; 16: 3329-3342, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34012262

RESUMO

Background and Purpose: Micro-/nano-tubes (TNTs) and micro-/nano-nets (TNNs) are the common and sensible choice in the first step of combined modifications of titanium surface for further functionalization in the purpose of extended indications and therapeutic effect. It is important to recognize the respective biologic reactions of these two substrates for guiding a biologically based first-step selection. Materials and Methods: TNTs were produced by anodic oxidation and TNNs were formed by alkali-heat treatment. The original selective laser melting (SLM) titanium surface was set as control. Surface characterization was evaluated by scanning electron microscopy, surface roughness, and water contact angle measurements. Osteoclastogenesis and osteogenesis were measured. MC3T3-E1 cells and RAW 264.7 cells were used for in vitro assay in terms of adhesion, proliferation, and differentiation. In vivo assessments were taken on Beagle dogs with micro-CT and histological analysis. Results: TNN and TNT groups performed decreased roughness and increased hydrophilicity compared with SLM group. For biological detections, the highest ALP activity and osteogenesis-related genes expression were observed in TNT group followed by TNN group (P <0.05). Interestingly, when it comes to the osteoclastogenesis, TNNs displayed lowest TRAP activity and osteoclastogenesis-related genes expression and TNTs were lower than SLM but higher than TNNs (P <0.05). BV/TV around implants was highest in TNT group after 4 weeks (P <0.05). HE, ALP and TRAP staining showed that osteogenic and osteoclastic activity around TNTs were both higher than TNNs (P <0.05). Conclusion: TNNs and TNTs have dual advantages in promotion of osteogenesis and inhibition of osteoclastogenesis. Furthermore, TNNs showed better capability in inhibiting osteoclast activity while TNTs facilitated stronger osteogenesis. Our results implied that TNT substrates would take advantage in early application after implantation, while diseases with inappropriate osteoclast activity would prefer TNN substrates, which will guide a biologically based first-step selection on combined modification for different clinical purposes.


Assuntos
Lasers , Nanotubos/química , Osseointegração/efeitos dos fármacos , Osseointegração/efeitos da radiação , Titânio/farmacologia , Células 3T3 , Animais , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cães , Camundongos , Microscopia Eletrônica de Varredura , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Próteses e Implantes , Células RAW 264.7 , Propriedades de Superfície , Titânio/química
13.
Biochem Biophys Res Commun ; 555: 175-181, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-33819748

RESUMO

Microgravity and radiation exposure-induced bone damage is one of the most significant alterations in astronauts after long-term spaceflight. However, the underlying mechanism is still largely unknown. Recent ground-based simulation studies have suggested that this impairment is likely mediated by increased production of reactive oxygen species (ROS) during spaceflight. The small Maf protein MafG is a basic-region leucine zipper-type transcription factor, and it globally contributes to regulation of antioxidant and metabolic networks. Our research investigated the role of MafG in the process of apoptosis induced by simulated microgravity and radiation in MC3T3-E1 cells. We found that simulated microgravity or radiation alone decreased MafG expression and elevated apoptosis in MC3T3-E1 cells, and combined simulated microgravity and radiation treatment aggravated apoptosis. Meanwhile, under normal conditions, increased ROS levels facilitated apoptosis and downregulated the expression of MafG in MC3T3-E1 cells. Overexpression of MafG decreased apoptosis induced by simulated microgravity and radiation. These findings provide new insight into the mechanism of bone damage induced by microgravity and radiation during space flight.


Assuntos
Apoptose/efeitos da radiação , Fator de Transcrição MafG/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos da radiação , Proteínas Repressoras/metabolismo , Apoptose/fisiologia , Linhagem Celular , Regulação para Baixo , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Fator de Transcrição MafG/genética , Osteoblastos/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/genética , Simulação de Ausência de Peso , Raios X
14.
Int J Mol Sci ; 22(8)2021 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-33924333

RESUMO

Bone formation starts near the end of the embryonic stage of development and continues throughout life during bone modeling and growth, remodeling, and when needed, regeneration. Bone-forming cells, traditionally termed osteoblasts, produce, assemble, and control the mineralization of the type I collagen-enriched bone matrix while participating in the regulation of other cell processes, such as osteoclastogenesis, and metabolic activities, such as phosphate homeostasis. Osteoblasts are generated by different cohorts of skeletal stem cells that arise from different embryonic specifications, which operate in the pre-natal and/or adult skeleton under the control of multiple regulators. In this review, we briefly define the cellular identity and function of osteoblasts and discuss the main populations of osteoprogenitor cells identified to date. We also provide examples of long-known and recently recognized regulatory pathways and mechanisms involved in the specification of the osteogenic lineage, as assessed by studies on mice models and human genetic skeletal diseases.


Assuntos
Osteoblastos/citologia , Osteogênese , Células-Tronco/citologia , Animais , Osso e Ossos/citologia , Osso e Ossos/embriologia , Epigênese Genética , Humanos , Osteogênese/genética , Transdução de Sinais
15.
Int J Mol Sci ; 22(9)2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33919218

RESUMO

Metallothioneins (MTs) are intracellular cysteine-rich proteins, and their expressions are enhanced under stress conditions. MTs are recognized as having the ability to regulate redox balance in living organisms; however, their role in regulating osteoblast differentiation is still unclear. In this research, we found that the expression of MT3, one member of the MT protein family, was specifically upregulated in the differentiation process of C2C12 myoblasts treated with bone morphogenetic protein 4 (BMP4). Transfection with MT3-overexpressing plasmids in C2C12 cells enhanced their differentiation to osteoblasts, together with upregulating the protein expression of bone specific transcription factors runt-related gene 2 (Runx2), Osterix, and distal-less homeobox 5 (Dlx5). Additionally, MT3 knockdown performed the opposite. Further studies revealed that overexpression of MT3 decreased reactive oxygen species (ROS) production in C2C12 cells treated with BMP4, and MT3 silencing enhanced ROS production. Treating C2C12 cells with antioxidant N-acetylcysteine also promoted osteoblast differentiation, and upregulated Runx2/Osterix/Dlx5, while ROS generator antimycin A treatment performed the opposite. Finally, antimycin A treatment inhibited osteoblast differentiation and Runx2/Osterix/Dlx5 expression in MT3-overexpressing C2C12 cells. These findings identify the role of MT3 in osteoblast differentiation and indicate that MT3 may have interesting potential in the field of osteogenesis research.


Assuntos
Diferenciação Celular , Regulação da Expressão Gênica , Mioblastos/citologia , Proteínas do Tecido Nervoso/metabolismo , Osteoblastos/citologia , Osteogênese , Estresse Oxidativo , Animais , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Células Cultivadas , Camundongos , Mioblastos/metabolismo , Proteínas do Tecido Nervoso/genética , Osteoblastos/metabolismo
16.
Int J Mol Sci ; 22(9)2021 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-33923149

RESUMO

In addition to their chemical composition various physical properties of synthetic bone substitute materials have been shown to influence their regenerative potential and to influence the expression of cytokines produced by monocytes, the key cell-type responsible for tissue reaction to biomaterials in vivo. In the present study both the regenerative potential and the inflammatory response to five bone substitute materials all based on ß-tricalcium phosphate (ß-TCP), but which differed in their physical characteristics (i.e., granule size, granule shape and porosity) were analyzed for their effects on monocyte cytokine expression. To determine the effects of the physical characteristics of the different materials, the proliferation of primary human osteoblasts growing on the materials was analyzed. To determine the immunogenic effects of the different materials on human peripheral blood monocytes, cells cultured on the materials were evaluated for the expression of 14 pro- and anti-inflammatory cytokines, i.e., IL-6, IL-10, IL-1ß, VEGF, RANTES, IL-12p40, I-CAM, IL-4, V-CAM, TNF-α, GM-CSF, MIP-1α, Il-8 and MCP-1 using a Bio-Plex® Multiplex System. The granular shape of bone substitutes showed a significant influence on the osteoblast proliferation. Moreover, smaller pore sizes, round granular shape and larger granule size increased the expression of GM-CSF, RANTES, IL-10 and IL-12 by monocytes, while polygonal shape and the larger pore sizes increased the expression of V-CAM. The physical characteristics of a bone biomaterial can influence the proliferation rate of osteoblasts and has an influence on the cytokine gene expression of monocytes in vitro. These results indicate that the physical structure of a biomaterial has a significant effect of how cells interact with the material. Thus, specific characteristics of a material may strongly affect the regenerative potential in vivo.


Assuntos
Materiais Biocompatíveis/farmacologia , Substitutos Ósseos/farmacologia , Citocinas/metabolismo , Macrófagos/metabolismo , Osteoblastos/metabolismo , Proliferação de Células , Células Cultivadas , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos
17.
Biomed Res Int ; 2021: 6697749, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33816629

RESUMO

Long noncoding RNAs (lncRNAs) play very important roles in cell differentiation. Our recent study has demonstrated that a novel lncRNA named lnc-OAD modulated 3T3-L1 adipocyte differentiation. In the present study, we examined the roles of lnc-OAD in bone morphogenetic protein 2- (BMP-2-) induced osteoblast differentiation. Lnc-OAD expression was increased during BMP-2-induced osteoblast differentiation in C3H10T1/2 mesenchymal stem cells and MC3T3-E1 preosteoblast cells. Knockdown of lnc-OAD expression by specific siRNA remarkably decreased early osteoblast differentiation. In addition, stable knockdown of lnc-OAD by lentivirus vector also significantly inhibited late osteoblast differentiation and matrix mineralization in vitro. Conversely, stably overexpressed lnc-OAD with lentiviral vector accelerated osteoblast differentiation. Mechanistically, knockdown of lnc-OAD reduced significantly the phosphorylation of AKT and the expression of Osterix induced by BMP-2, while overexpression of lnc-OAD enhanced the phosphorylation of AKT and the expression of Osterix. Taken together, our study suggests that lnc-OAD plays an important role in modulating BMP-2-induced osteoblast differentiation via, at least in part, regulating the AKT-Osterix signaling axis.


Assuntos
Proteína Morfogenética Óssea 2/biossíntese , Diferenciação Celular , Osteoblastos/metabolismo , RNA Longo não Codificante/biossíntese , Transdução de Sinais , Células 3T3-L1 , Animais , Proteína Morfogenética Óssea 2/genética , Camundongos , Osteoblastos/citologia , RNA Longo não Codificante/genética
18.
Int J Mol Sci ; 22(8)2021 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-33919677

RESUMO

Platelet-rich fibrin (PRF) is a natural fibrin meshwork material with multiple functions that are suitable for tissue engineering applications. PRF provides a suitable scaffold for critical-size bone defect treatment due to its platelet cytokines and rich growth factors. However, the structure of PRF not only promotes cell attachment but also, due to its density, provides a pool for cell migration into the PRF to facilitate regeneration. In our study, we used repeated freeze drying to enlarge the pores of PRF to engineer large-pore PRF (LPPRF), a type of PRF that has expanded pores for cell migration. Moreover, a biodegradable Mg ring was used to provide stability to bone defects and the release of Mg ions during degradation may enhance osteoconduction and osteoinduction. Our results revealed that cell migration was more extensive when LPPRF was used rather than when PRF was used and that LPPRF retained the growth factors present in PRF. Moreover, the Mg ions released from the Mg ring during degradation significantly enhanced the calcium deposition of MC3T3-E1 preosteoblasts. In the present study, a bone substitute comprising LPPRF combined with a Mg ring was demonstrated to have much potential for critical-size bone defect repair.


Assuntos
Osso e Ossos/patologia , Movimento Celular/efeitos dos fármacos , Magnésio/farmacologia , Osteoblastos/citologia , Osteogênese/efeitos dos fármacos , Fibrina Rica em Plaquetas/metabolismo , Cicatrização , Animais , Osso e Ossos/efeitos dos fármacos , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/ultraestrutura , Coelhos , Tecidos Suporte/química , Titânio/farmacologia , Cicatrização/efeitos dos fármacos
19.
Int J Mol Sci ; 22(5)2021 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-33801166

RESUMO

Root bark of Dictamnus dasycarpus Turcz. has been widely used as a traditional medicine and is a well-known anti-inflammatory agent. We isolated limonoid triterpene, obacunone (Obac) from the dried root bark of D. dasycarpus. Obac has been reported to exhibit varieties of biological activities including anti-inflammatory, anti-cancer, and anti-oxidant effects. This study aimed to investigate the beneficial effects and biological mechanisms of Obac in osteoblast differentiation and bone matrix mineralization. In the present study, Obac at concentrations ranging from 1 to 100 µM showed no proliferation effects in MC3T3-E1. The treatment of Obac (1 and 10 µM) increased wound healing and migration rates in a dose-dependent manner. Alkaline phosphatase (ALP) staining and activity showed that Obac (1 and 10 µM) enhanced early osteoblast differentiation in a dose-dependent manner. Obac also increased late osteoblast differentiation in a dose-dependent manner, as indicated by the mineralized nodule formation of ARS staining. The effects of Obac on osteoblast differentiation was validated by the levels of mRNAs encoding the bone differentiation markers, including Alp, bone sialoprotein (Bsp), osteopontin (Opn), and osteocalcin (Ocn). Obac increased the expression of bone morphogenetic protein (BMP), and the phosphorylation of smad1/5/8, and the expression of runt-related transcription factor 2 (RUNX2); Obac also inhibited GSK3ß and upregulated the protein level of ß-catenin in a dose-dependent manner during osteoblast differentiation. Obac-mediated osteoblast differentiation was attenuated by a BMP2 inhibitor, Noggin and a Wnt/ß-catenin inhibitor, Dickkopf-1 (Dkk1) with the abolishment of RUNX2 expression and nuclear accumulation by Obac. Taken together, the findings of this study demonstrate that Obac has pharmacological and biological activates to promote osteoblast differentiation and bone mineralization through BMP2, ß-catenin, and RUNX2 pathways, and suggest that Obac might be a therapeutic effect for the treatment and prevention of bone diseases such as osteoporosis and periodontitis.


Assuntos
Benzoxepinas/farmacologia , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Limoninas/farmacologia , Osteoblastos/citologia , Osteoblastos/fisiologia , Osteogênese , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Camundongos , Osteoblastos/efeitos dos fármacos , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismo
20.
Int J Mol Sci ; 22(6)2021 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-33805785

RESUMO

Osteoporosis is a worldwide chronic disease characterized by increasing bone fragility and fracture likelihood. In the treatment of bone defects, materials based on calcium phosphates (CaPs) are used due to their high resemblance to bone mineral, their non-toxicity, and their affinity to ionic modifications and increasing osteogenic properties. Moreover, CaPs, especially hydroxyapatite (HA), can be successfully used as a vehicle for local drug delivery. Therefore, the aim of this work was to fabricate hydroxyapatite-based composite beads for potential use as local carriers for raloxifene. HA powder, modified with magnesium and silicon ions (Mg,Si-HA) (both of which play beneficial roles in bone formation), was used to prepare composite beads. As an organic matrix, sodium alginate with chondroitin sulphate and/or keratin was applied. Cross-linking of beads containing raloxifene hydrochloride (RAL) was carried out with Mg ions in order to additionally increase the concentration of this element on the material surface. The morphology and porosity of three different types of beads obtained in this work were characterized by scanning electron microscopy (SEM) and mercury intrusion porosimetry, respectively. The Mg and Si released from the Mg,Si-HA powder and from the beads were measured by inductively coupled plasma optical emission spectrometry (ICP-OES). In vitro RAL release profiles were investigated for 12 weeks and studied using UV/Vis spectroscopy. The beads were also subjected to in vitro biological tests on osteoblast and osteosarcoma cell lines. All the obtained beads revealed a spherical shape with a rough, porous surface. The beads based on chondroitin sulphate and keratin (CS/KER-RAL) with the lowest porosity resulted in the highest resistance to crushing. Results revealed that these beads possessed the most sustained drug release and no burst release effect. Based on the results, it was possible to select the optimal bead composition, consisting of a mixture of chondroitin sulphate and keratin.


Assuntos
Alginatos/química , Conservadores da Densidade Óssea/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Durapatita/química , Silicatos de Magnésio/química , Cloridrato de Raloxifeno/farmacologia , Conservadores da Densidade Óssea/metabolismo , Regeneração Óssea/fisiologia , Osso e Ossos/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sulfatos de Condroitina/química , Liberação Controlada de Fármacos , Humanos , Queratinas/química , Cinética , Nanopartículas/química , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Porosidade , Cloridrato de Raloxifeno/metabolismo
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