Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.627
Filtrar
1.
Arch Biochem Biophys ; 685: 108333, 2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32194044

RESUMO

This study summarizes the available evidence from systematic reviews on the in vitro effects of photobiomodulation on the proliferation and differentiation of human bone and stromal cells by appraising their methodological quality. Improvements for future studies are also highlighted, with particular emphasis on in vitro protocols and cell-related characteristics. Six reviews using explicit eligibility criteria and methods selected in order to minimize bias were included. There was no compelling evidence on the cellular mechanisms of action or treatment parameters of photobiomodulation; compliance with quality assessment was poor. A rigorous description of laser parameters (wavelength, power, beam spot size, power density, energy density, repetition rate, pulse duration or duty cycle, exposure duration, frequency of treatments, and total radiant energy), exposure conditions (methods to ensure a uniform irradiation and to avoid cross-irradiation, laser-cell culture surface distance, lid presence during irradiation) and cell-related characteristics (cell type or line, isolation and culture conditions, donor-related factors where applicable, tissue source, cell phenotype, cell density, number of cell passages in culture) should be included among eligibility criteria for study inclusion. These methodological improvements will maximize the contribution of in vitro studies on the effects of photobiomodulation on human bone and stromal cells to evidence-based translational research.


Assuntos
Terapia com Luz de Baixa Intensidade , Osteócitos/metabolismo , Células Estromais/metabolismo , Animais , Diferenciação Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Humanos , Osteócitos/efeitos da radiação , Células Estromais/efeitos da radiação , Revisões Sistemáticas como Assunto
2.
Am J Pathol ; 190(6): 1151-1163, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32194053

RESUMO

Osteomyelitis is an inflammation of the bone and bone marrow that is most commonly caused by a Staphylococcus aureus infection. Much of our understanding of the underlying pathophysiology of osteomyelitis, from the perspective of both host and pathogen, has been revised in recent years, with notable discoveries including the role played by osteocytes in the recruitment of immune cells, the invasion and persistence of S. aureus in submicron channels of cortical bone, and the diagnostic role of polymorphonuclear cells in implant-associated osteomyelitis. Advanced in vitro cell culture models, such as ex vivo culture models or organoids, have also been developed over the past decade, and have become widespread in many fields, including infectious diseases. These models better mimic the in vivo environment, allow the use of human cells, and can reduce our reliance on animals in osteomyelitis research. In this review, we provide an overview of the main pathologic concepts in osteomyelitis, with a focus on the new discoveries in recent years. Furthermore, we outline the value of modern in vitro cell culture techniques, with a focus on their current application to infectious diseases and osteomyelitis in particular.


Assuntos
Osteomielite/imunologia , Osteomielite/patologia , Infecções Estafilocócicas/patologia , Animais , Modelos Animais de Doenças , Humanos , Osteócitos/patologia , Projetos de Pesquisa , Infecções Estafilocócicas/imunologia , Staphylococcus aureus
3.
Oral Dis ; 26(3): 621-629, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31943597

RESUMO

OBJECTIVE: Regulation of bone metabolism by the sympathetic nervous system has recently been clarified. Tooth movement is increased by increased bone metabolic turnover due to sympathetic activation. This study aimed to compare the effects of the ß-adrenergic receptor (ß-AR) blockers atenolol (ß1-AR blocker), butoxamine (ß2-AR blocker) and propranolol (non-selective ß-AR blocker) on tooth movement in spontaneously hypertensive rats (SHR) with sympathicotonia. MATERIALS AND METHODS: Spontaneously hypertensive rats were divided into the following four groups: an SHR control group and groups treated with 0.1 mg/kg atenolol, 1 mg/kg butoxamine or 1 mg/kg propranolol (n = 6 rats/group). Atenolol, butoxamine or propranolol was administered daily to each treatment group, and orthodontic force was applied using a closed-coil spring. Finally, immunohistochemical analysis was performed for receptor activator of nuclear factor kappa-B ligand (RANKL) and sclerostin (SOST). RESULTS: Atenolol, butoxamine and propranolol inhibited tooth movement and increased maxillary alveolar bone volume. Histological analysis revealed that these ß-AR blockers decreased osteoclast activity on the compression side. Furthermore, immunohistochemical analysis revealed that atenolol, butoxamine and propranolol decreased the number of RANKL- and SOST-positive osteocytes on the compression side. CONCLUSIONS: ß-AR blockers decreased tooth movement and downregulated SOST in osteocytes, accompanied by increasing alveolar bone resorption.


Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Proteínas Morfogenéticas Ósseas/metabolismo , Técnicas de Movimentação Dentária , Animais , Atenolol , Remodelação Óssea , Reabsorção Óssea , Butoxamina , Marcadores Genéticos , Osteoclastos , Osteócitos/efeitos dos fármacos , Osteócitos/fisiologia , Propranolol , Ligante RANK , Ratos , Ratos Endogâmicos SHR
4.
Commun Biol ; 3(1): 45, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31988398

RESUMO

Intraflagellar transport (IFT) proteins are essential for cilia assembly and function. IFT protein mutations lead to ciliopathies, which manifest as variable skeletal abnormalities. However, how IFT proteins regulate cell alignment during bone development is unknown. Here, we show that the deletion of IFT20 in osteoblast lineage using Osterix-Cre and inducible type I Collagen-CreERT cause a compromised cell alignment and a reduced bone mass. This finding was validated by the disorganized collagen fibrils and decreased bone strength and stiffness in IFT20-deficient femurs. IFT20 maintains cilia and cell alignment in osteoblasts, as the concentric organization of three-dimensional spheroids was disrupted by IFT20 deletion. Mechanistically, IFT20 interacts with the ceramide-PKCζ complex to promote PKCζ phosphorylation in cilia and induce the apical localization of ß-catenin in osteoblasts, both of which were disrupted in the absence of IFT20. These results reveal that IFT20 regulates polarity and cell alignment via ceramide-pPKCζ-ß-catenin signaling during bone development.


Assuntos
Desenvolvimento Ósseo/genética , Proteínas de Transporte/metabolismo , Ceramidas/metabolismo , Cílios/metabolismo , Osteócitos/metabolismo , Proteína Quinase C/metabolismo , beta Catenina/metabolismo , Animais , Proteínas de Transporte/genética , Diferenciação Celular/genética , Linhagem da Célula , Polaridade Celular/genética , Fêmur/metabolismo , Camundongos Transgênicos , Fosforilação/genética , Proteína cdc42 de Ligação ao GTP/metabolismo
5.
Biochimie ; 168: 92-99, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31676316

RESUMO

As osteogenesis is a multifactorial mechanism, we wonder whether osteoblast-induced extracellular matrix (ECM) remodeling might be modulated by trophic factors released by fibroblasts in a paracrine signaling manner. To address this issue, fibroblasts were cultured for 72 h under conventional conditions when their conditioned medium was harvested and used to challenge pre-osteoblasts (MC3T3-E1 cells) for 14 days. Preliminarily, we validated the potential effect of fibroblasts in contributing to osteocyte phenotype, which specifically requires significant expression of Dentin Matrix Protein 1 (DMP1; about 10-fold changes) and Sclerostin (SOST; about 7-fold changes), both biomarkers of osteocyte. Fibroblasts also seem contributing to ECM remodeling in osteoblasts, because we detected a high level of both mRNA and enzyme activities of matrix metalloproteinase -9 (MMP-9) as well as a high level of reversion inducing cysteine rich protein with kazal motifs (RECK) transcripts (about 13-fold changes), a membrane-anchored MMP inhibitor, which seems to be a constitutive pathway in osteoblasts. Considering inflammatory panorama and using RTqPCR technology, both IL-13 (about 13-fold changes) and IL-33 (about 5-fold changes) genes were up-expressed in response to the fibroblast-secreted trophic factors, as were the receptor activator of NF-κB ligand (RANKL; about 8-fold changes) and osteoprotegerin (OPG; about 3-fold changes). Although preliminary, these data suggest a stimulus to finely control osteoclastogenesis, and this mechanism reinforces the role of fibroblasts in bone remodeling and homeostasis. Moreover, these results suggest an important crosstalk between fibroblast and osteoblast, when fibroblast-secreted trophic factors upmodulate osteocyte gene markers and contribute to ECM remodeling stimulus in osteoblast.


Assuntos
Matriz Extracelular/metabolismo , Osteócitos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Remodelação Óssea , Diferenciação Celular/efeitos dos fármacos , Meios de Cultivo Condicionados , Fibroblastos/metabolismo , Camundongos , Células NIH 3T3 , Osteócitos/citologia
6.
Ann N Y Acad Sci ; 1460(1): 68-76, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31646646

RESUMO

The biological effect of ultrasound on bone regeneration has been well documented, yet the underlying mechanotransduction mechanism is largely unknown. In relation to the mechanobiological modulation of the cytoskeleton and Ca2+ influx by short-term focused acoustic radiation force (FARF), the current study aimed to visualize and quantify Ca2+ oscillations in real-time of in situ and in vivo osteocytes in response to focused low-intensity pulsed ultrasound (FLIPUS). For in situ studies, fresh mice calvaria were subjected to FLIPUS stimulation at 0.05, 0.2, 0.3, and 0.7 W. For the in vivo study, 3-month-old C57BL/6J Ai38/Dmp1-Cre mice were subjected to FLIPUS at 0.15, 1, and 1.5 W. As observed via real-time confocal imaging, in situ FLIPUS led to more than 80% of cells exhibiting Ca2+ oscillations at 0.3-0.7 W and led to a higher number of Ca2+ spikes with larger values at >0.3 W. In vivo FLIPUS at 1-1.5 W led to more than 90% of cells exhibiting Ca2+ oscillations. Higher FLIPUS energies led to larger Ca2+ spike magnitudes. In conclusion, this study provided a pilot study of both in situ and in vivo osteocytic Ca2+ oscillations under noninvasive FARF, which aids further exploration of the mechanosensing mechanism of the controlled bone cell motility response to the stimulus.


Assuntos
Acústica , Sinalização do Cálcio , Mecanotransdução Celular , Osteócitos/metabolismo , Radiação , Ultrassom , Estimulação Acústica , Animais , Feminino , Camundongos Endogâmicos C57BL , Crânio/diagnóstico por imagem
7.
Biomed Tech (Berl) ; 65(1): 107-111, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31348752

RESUMO

Osteocytes are of high importance in bone metabolism as they orchestrate bone remodeling, react to mechanosensory stimuli and have endocrine functions. In vitro investigations with osteocytes are therefore of high relevance for biomaterial and drug testing. The application of primary human cells instead of rodent osteocyte cell lines like MLOY4 and IDG SW3 is desirable but provides the challenge of isolating these cells, which are deeply embedded into the mineralized bone matrix. The present study describes an improved protocol for the isolation of human primary osteocytes. In contrast to an already established protocol, resting steps between the demineralization /digestion steps of the bone particles considerably improved the yield of osteocytes. Real-time polymerase chain reaction (PCR) analysis revealed the expression of typical osteocyte markers like osteocalcin, E11/podoplanin and dentin matrix protein 1 (DMP-1).


Assuntos
Materiais Biocompatíveis/metabolismo , Biomarcadores/química , Osso e Ossos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Osteócitos/citologia , Fosfoproteínas/metabolismo , Animais , Biomarcadores/metabolismo , Linhagem Celular , Proteínas da Matriz Extracelular/química , Humanos , Osteócitos/metabolismo , Osteócitos/fisiologia , Fosfoproteínas/química
8.
Acta Orthop ; 91(1): 115-120, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31762353

RESUMO

Background and purpose - Insufficient initial fixation or early micromotion of an implant is associated with a thin layer of fibrous tissue at the peri-implant interface. It is unknown if bone loss is induced by the fibrous tissue interface acting as an active biological membrane, or as a membrane that will produce supraphysiologic fluid flow conditions during gait, which activates the mechanosensitive osteocytes to mediate osteoclast differentiation. We investigated whether mechanically induced osteolysis is dependent on the fibrous tissue interface as a biologically active scaffold, or if it merely acts as a conduit for fluid flow, affecting the mechanosensitive osteocytes in the peri-prosthetic bone.Methods - Using a rat model of mechanically instability-induced aseptic loosening, we assessed whether the induction of osteoclast differentiation was dependent on the presence of a peri-implant fibrous interface. We analyzed the amount of osteoclast differentiation, osteocyte apoptosis, pro-resorptive cytokine expression and bone loss using immunohistochemistry, mRNA expression and micro-CT.Results - Osteoclast differentiation and bone loss were induced by mechanical instability but were not affected by the presence of the fibrous tissue membrane or associated with osteocyte apoptosis. There was no increased mRNA expression of any of the cytokines in the fibrous tissue membrane compared with the peri-implant bone.Interpretation - Our data show that the fibrous tissue membrane in the interface plays a minor role in inducing bone loss. This indicates that the peri-implant bone adjacent to loose bone implants might play an important role for osteoclast differentiation.


Assuntos
Apoptose , Diferenciação Celular , Citocinas/metabolismo , Instabilidade Articular/metabolismo , Osteoclastos/metabolismo , Osteócitos/metabolismo , Falha de Prótese , Tíbia/metabolismo , Animais , Reabsorção Óssea/diagnóstico por imagem , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Interface Osso-Implante/diagnóstico por imagem , Citocinas/genética , Modelos Animais de Doenças , Imuno-Histoquímica , Instabilidade Articular/diagnóstico por imagem , Instabilidade Articular/genética , Osteoclastos/citologia , Osteócitos/citologia , RNA Mensageiro/metabolismo , Ratos , Tíbia/diagnóstico por imagem , Microtomografia por Raio-X
9.
Int Endod J ; 53(1): 84-96, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31429089

RESUMO

AIM: To evaluate the effect of alendronate (ALN) on the development of periapical lesions induced in ovariectomized rats. METHODOLOGY: Twenty-five rats were divided into three groups: sham (control), ovariectomy (OVX) and OVX + ALN. One day after OVX, animals from the OVX + ALN group received the medication via gavage. After 9 weeks, the first molars of all animals were submitted to periapical lesion induction. After 21 days, the animals were euthanized. Femurs were analysed for bone mineral density. The blocks of bone tissue containing the mandibular first molars were submitted to histotechnical processing and staining with haematoxylin and eosin (HE) for periapical lesion analysis under conventional microscopy. At the same time, the morphometric analysis of the periapical lesion area was performed in the fluorescence mode, as well as the histoenzimology for the quantification of osteoclasts and 4'-6-diamidino-2-phenylindole staining for the quantification of apoptotic osteocytes. In addition, the first maxillary molars were used for analysis of the gene expression of proinflammatory cytokines (IL-1ß, IL-6 and TNF-α) and osteoclastogenesis markers (RANKL/OPG). The results were submitted to ANOVA and Kruskal-Wallis tests and Tukey and Dunn post-tests (significance level of 5%). RESULTS: Ovariectomy reduced bone mineral density of the femur, and treatment with ALN was able to prevent bone loss (P < 0.001). Regarding the microscopic analysis of the periapical region, the sham and OVX + ALN groups had moderately increased periodontal ligament and inflammatory infiltrate, while the OVX group had these parameters increased intensely. The periapical lesions of the OVX group were significantly larger in area in comparison to the other groups (P < 0.001). The OVX group had the largest amount of apoptotic osteocytes, and ALN was able to prevent the apoptosis of these cells, in addition to significantly reducing IL-6 expression (P < 0.05). OVX and ALN had no effect on RANKL/OPG expression and did not influence the number of osteoclasts around the periapical lesion (P > 0.05). CONCLUSION: The hypoestrogenic condition induced by OVX aggravated bone resorption, inducing the death of osteocytes and provoking larger periapical lesions. ALN treatment inhibited osteocyte apoptosis and inflammation via IL-6, inhibiting bone resorption in periapical lesions of ovariectomized rats.


Assuntos
Conservadores da Densidade Óssea , Reabsorção Óssea , Alendronato , Animais , Apoptose , Feminino , Humanos , Inflamação , Interleucina-6 , Osteócitos , Ovariectomia , Ratos
10.
Oral Dis ; 26(3): 590-596, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31863612

RESUMO

OBJECTIVE: Fibroblast growth factor 8 (FGF8) signaling is essential in regulating craniofacial osteogenesis. This study aims to explore the effect of altered FGF8 signaling in maxillomandibular development during embryogenesis. MATERIALS AND METHODS: Dmp1Cre ;R26RmTmG mice were generated to trace Dmp1+ cell lineage, and Dmp1Cre ;R26RFgf8 mice were generated to explore the effects of augmented FGF8 signaling in Dmp1+ cells on osteogenesis with a focus on maxillomandibular development during embryogenesis, as assessed by whole mount skeletal staining, histology, and immunostaining. Additionally, cell proliferation rate and the expression of osteogenic genes were examined. RESULTS: Osteocytes of maxillomandibular bones were found Dmp1-positive prenatally, and Fgf8 over-expression in Dmp1+ cells led to mandibular hypoplasia. While Dmp1Cre allele functions in the osteocytes of the developing mandibular bone at as early as E13.5, and enhanced cell proliferation rate is observed in the bone forming region of the mandible in Dmp1Cre ;R26RFgf8 mice at E14.5, histological examination showed that osteogenesis was initially impacted at E15.5, along with an inhibition of osteogenic differentiation markers. CONCLUSIONS: Augmented FGF8 signaling in Dmp1+ cells lead to osteogenic deficiency in the mandibular bones, resulting in mandibular hypoplasia.


Assuntos
Desenvolvimento Embrionário , Fator 8 de Crescimento de Fibroblasto/fisiologia , Mandíbula/patologia , Osteócitos/patologia , Osteogênese , Transdução de Sinais , Animais , Embrião de Mamíferos , Proteínas da Matriz Extracelular/genética , Mandíbula/embriologia , Camundongos , Camundongos Transgênicos
11.
Ann Anat ; 227: 151422, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31563568

RESUMO

The knowledge of bone biology has largely changed in the last few decades. Osteocytes are multifunctional bone cells that are surrounded by mineralized bone matrix and for decades it was considered that they might be relatively inactive cells. However, nowadays it is known that osteocytes are highly active cells which are indispensable for the normal function of the skeleton, playing main roles in several physiological processes, both within and beyond the bone microenvironment. This review highlights and updates the current state of knowledge of the osteocyte and focuses on its roles in bone remodeling and mineral homeostasis, and also reviews its recently discovered endocrine function. Osteocytes secrete sclerostin (a protein that works as a negative regulator of bone mass), and FGF-23, the most important osteocyte-secreted endocrine factor, since it is able to regulate the phosphate metabolism. Moreover, osteocytes can act as mechanosensory cells, transforming the mechanical strain into chemical signaling towards the effector cells (osteoblasts and osteoclasts). Therefore, the osteocyte plays an important role in bone biology, specifically in the remodeling process, since it regulates both the osteoblast and osteoclast activity. Finally, the paper discusses the clinical application of the bone biology, updating the new therapies against bone-loss disorders.


Assuntos
Osso e Ossos/citologia , Osteócitos/fisiologia , Apoptose/fisiologia , Remodelação Óssea/fisiologia , Reabsorção Óssea/fisiopatologia , Cálcio/sangue , Células Endócrinas/fisiologia , Humanos , Mecanorreceptores/fisiologia , Mecanotransdução Celular/fisiologia , Osteócitos/citologia , Osteogênese/fisiologia
12.
Ann Anat ; 227: 151427, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31614180

RESUMO

Mandibular/alveolar (m/a) bone, as a component of the periodontal apparatus, allows for the proper tooth anchorage and function of dentition. Bone formation around the tooth germs starts prenatally and, in the mouse model, the mesenchymal condensation turns into a complex vascularized bone (containing osteo-blasts, -cytes, -clasts) within only two days. This very short but critical period is characterized by synchronized cellular and molecular events. The m/a bone, as others, is subjected to endocrine regulations. This not only requires vasculature to allow the circulation of active molecules (ligands), but also the expression of corresponding cell receptors to define target tissues. This contribution aimed at following the dynamics of calciotropic receptors´ expression during morphological transformation of a mesenchymal condensation into the initial m/a bone structure. Receptors for all three calciotropic systemic regulators: parathormone, calcitonin and activated vitamin D (calcitriol), were localized on serial histological sections using immunochemistry and their relative expression was quantified by q-PCR. The onset of calciotropic receptors was followed along with bone cell differentiation (as checked using osteocalcin, sclerostin, RANK and TRAP) and vascularization (CD31) during mouse prenatal/embryonic (E) days 13-15 and 18. Additionally, the timing of calciotropic receptor appearance was compared with that of estrogen receptors (ESR1, ESR2). PTH receptor (PTH1r) appeared in the bone already at E13, when the first osteocalcin-positive cells were detected within the mesenchymal condensation forming the bone anlage. At this stage, blood vessels were only lining the condensation. At E14, the osteoblasts started to express the receptor for activated vitamin D (VDR). At this stage, the vasculature just penetrated the forming bone. On the same day, the first TRAP-positive (but not yet multinucleated) osteoclastic cells were identified. However, calcitonin receptor was detected only one day later. The first Sost-positive osteocytes, present at E15, were PTH1r and VDR positive. ESR1 almost copied the expression pattern of PTH1r, and ESR2 appearance was similar with VDR with a significant increase between E15 and E18. This report focuses on the in vivo situation and links morphological transformation of the mesenchymal cell condensation into a bone structure with dynamics of cell differentiation/maturation, vascularization and onset of receptors for calciotropic endocrine signalling in developing m/a bone.


Assuntos
Mandíbula/crescimento & desenvolvimento , Osteogênese/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Diferenciação Celular , Imuno-Histoquímica , Camundongos , Osteoblastos/fisiologia , Osteocalcina/análise , Osteocalcina/genética , Osteoclastos/fisiologia , Osteócitos/fisiologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Receptores da Calcitonina/metabolismo
13.
Biomed Res Int ; 2019: 7623562, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31828128

RESUMO

As the initial part in the development of osteoarthritis (OA), subchondral bone sclerosis has been considered to be initiated by excess mechanical loading and proven to be correlated to other pathological changes. Sclerostin, which is an essential mechanical stress response protein, is encoded by the SOST gene. It is expressed in osteocytes and mature chondrocytes and has been proven to be closely correlated to OA. However, the relationship and mechanism between the SOST gene and the development of OA remain unclear. The aim of the present study was to investigate the role of the SOST gene in OA pathogenesis in the subchondral bone. A knee anterior cruciate ligament transection (ACLT) mouse osteoarthritis (OA) model on SOST-knockout (SOST KO) and wild-type (WT) mice was established. The pathogenic and phenotypic changes in the subchondral bone were investigated by histology, micro-CT, immunohistochemistry, TRAP staining, Masson staining, and Toluidine blue staining. It was found that sclerostin expression decreased in both the calcified cartilage and mineralized subchondral structures during the development of OA. Joint instability induced a severe cartilage degradation phenotype, with higher OARSI scores in SOST KO mice, when compared to WT mice. SOST KO mice with OA exhibited a higher BMD and BV/TV ratio, as well as a higher rate of bone remodeling and TRAP-positive cell number, when compared to the WT counterparts, but the difference was not significant between the sham-operation groups. It was concluded that loss of sclerostin aggravates knee OA in mice by promoting subchondral bone sclerosis and increasing catabolic activity of cartilage.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Hiperostose/genética , Osteoartrite/genética , Esclerose/genética , Sindactilia/genética , Animais , Densidade Óssea/genética , Remodelação Óssea/genética , Osso e Ossos/metabolismo , Osso e Ossos/fisiopatologia , Condrócitos/metabolismo , Condrócitos/patologia , Modelos Animais de Doenças , Fêmur/diagnóstico por imagem , Fêmur/metabolismo , Fêmur/fisiopatologia , Expressão Gênica/genética , Humanos , Hiperostose/diagnóstico por imagem , Hiperostose/fisiopatologia , Instabilidade Articular/diagnóstico por imagem , Instabilidade Articular/fisiopatologia , Camundongos , Camundongos Knockout , Osteoartrite/diagnóstico por imagem , Osteoartrite/fisiopatologia , Osteócitos/metabolismo , Osteócitos/patologia , Esclerose/diagnóstico por imagem , Esclerose/fisiopatologia , Sindactilia/diagnóstico por imagem , Sindactilia/fisiopatologia
14.
Arch Pharm Res ; 42(12): 1052-1062, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31802425

RESUMO

Due to a rapidly expanding aging population, the incidence of age-related or degenerative diseases has increased, and efforts to handle the issue with regenerative medicine via adult stem cells have become more important. And it is now clear that the mitochondrial energy metabolism is important for stem cell differentiation. When stem cells commit to differentiate, glycolytic metabolism is being shifted to mitochondrial oxidative phosphorylation (OXPHOS) to meet an increased cellular energy demand required for differentiated cells. However, the nature of cellular metabolisms during the differentiation process of periosteum-derived mesenchymal stem cells (POMSC) is still unclear. In the present study, we investigated mitochondrial biogenesis during the adipogenic, chondrogenic, and osteogenic differentiation of POMSCs. Both mitochondrial DNA (mtDNA) contents and mitochondrial proteins (VDAC and mitochondrial OXPHOS complex subunits) were increased during all of these mesenchymal lineage differentiations of POMSCs. Interestingly, glycolytic metabolism is reduced as POMSCs undergo osteogenic differentiation. Furthermore, reducing mtDNA contents by ethidium bromide treatments prevents osteogenic differentiation of POMSCs. In conclusion, these results indicate that mitochondrial biogenesis and OXPHOS metabolism play important roles in the differentiation of POMCS and suggest that pharmaceutical modulation of mitochondrial biogenesis and/or function can be a novel regulation for POMSC differentiation and regenerative medicine.


Assuntos
Adipócitos/citologia , Condrócitos/citologia , Células-Tronco Mesenquimais/citologia , Mitocôndrias/metabolismo , Osteócitos/citologia , Adipócitos/metabolismo , Biomarcadores/análise , Diferenciação Celular , Células Cultivadas , Condrócitos/metabolismo , DNA Mitocondrial/genética , Citometria de Fluxo , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteócitos/metabolismo
15.
Sci Adv ; 5(11): eaaw7215, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31799389

RESUMO

Mitochondrial transfer plays a crucial role in the regulation of tissue homeostasis and resistance to cancer chemotherapy. Osteocytes have interconnecting dendritic networks and are a model to investigate its mechanism. We have demonstrated, in primary murine osteocytes with photoactivatable mitochondria (PhAM)floxed and in MLO-Y4 cells, mitochondrial transfer in the dendritic networks visualized by high-resolution confocal imaging. Normal osteocytes transferred mitochondria to adjacent metabolically stressed osteocytes and restored their metabolic function. The coordinated movement and transfer of mitochondria within the dendritic network rely on contact between the endoplasmic reticulum (ER) and mitochondria. Mitofusin 2 (Mfn2), a GTPase that tethers ER to mitochondria, predominantly mediates the transfer. A decline in Mfn2 expression with age occurs concomitantly with both impaired mitochondrial distribution and transfer in the osteocyte dendritic network. These data show a previously unknown function of ER-mitochondrial contact in mediating mitochondrial transfer and provide a mechanism to explain the homeostasis of osteocytes.


Assuntos
Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/metabolismo , Osteócitos/metabolismo , Animais , Linhagem Celular , Homeostase/fisiologia , Camundongos , Camundongos Knockout , Microscopia Confocal
16.
J Diabetes Res ; 2019: 6757428, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31886284

RESUMO

Osteocyte plays an essential role in bone metabolism by regulating osteoblast and osteoclast activities. Dysfunction or apoptosis of osteocyte will severely endanger the bone homeostasis and result in bone diseases such as osteoporosis. Osteoporosis has been considered as one of the diabetes complications; however, the mechanism is still to be discovered. Advanced glycation end products (AGEs), as the main pathogenic factor of diabetes mellitus, have the capacity to induce osteocyte apoptosis thus sabotaging bone homeostasis. Here, we examined the role of AGE during osteocyte apoptosis and how this effect would affect osteocyte's regulation of osteoblast and osteoclast. Mouse osteocyte-like MLO-Y4 cells were used to study the properties of osteocyte and to examine its biological and pathological function. MTT assay and Annexin V assay showed that AGE significantly induce MLO-Y4 cell apoptosis. qPCR and Western blot results have shown that AGE upregulates proapoptotic gene p53 and its downstream target gene Bax, which leads to enhanced activation of caspase-3, thus inducing apoptosis in MLO-Y4 cells. Increased expression of sclerostin and RANKL in osteocytes has shown that AGE induces osteocyte dysfunction thus severely damaging the bone homeostasis by decreasing osteoblast and increasing osteoclast activities. Furthermore, the role of the transcription factor FOXO1, which is intensely associated with apoptosis, has been determined. Western blot has shown that AGE significantly decreases Akt activities. Immunofluorescence has shown that AGE promotes FOXO1 nuclei localization and enhances FOXO1 expression. Silencing of FOXO1 suppressed AGE-enhanced apoptosis; mRNA and protein expressions of cleaved caspase-3, sclerostin, and RANKL were downregulated as well. Moreover, exogenous FOXO1 increased caspase-3 mRNA levels and caspase-3 transcriptional activity. Lastly, ChIP assay has established the capacity of FOXO1 binding directly on the caspase-3, sclerostin, and RANKL promoter region in AGE environment, providing the mechanism of the AGE-induced osteocyte apoptosis and dysfunction. Our results have shown that FOXO1 plays a crucial role in AGE-induced osteocyte dysfunction and apoptosis through its regulation of caspase-3, sclerostin, and RANKL. This study provides new insight into diabetes-enhanced risk of osteoporosis given the critical role of AGE in the pathogenesis of diabetes and the essential part of osteocyte in bone metabolism.


Assuntos
Apoptose/efeitos dos fármacos , Proteína Forkhead Box O1/metabolismo , Produtos Finais de Glicação Avançada/toxicidade , Osteócitos/efeitos dos fármacos , Soroalbumina Bovina/toxicidade , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sítios de Ligação , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular , Proteína Forkhead Box O1/genética , Regulação da Expressão Gênica , Camundongos , Osteócitos/metabolismo , Osteócitos/patologia , Regiões Promotoras Genéticas , Ligante RANK/genética , Ligante RANK/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
17.
J Orthop Surg Res ; 14(1): 442, 2019 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-31842947

RESUMO

BACKGROUND: Bone tissue is one of the tissues that are capable of self-regeneration. However, bone self-regeneration is defeated in the case of broad lesion of bone structure. Isolated stem cells from wisdom tooth follicles can potentially differentiate into ectodermal and mesodermal cells. Saghez is a natural substance that has been extracted from Pistacia terebinthus with unique features, such as high temperature and mechanical stability, adhesive structure, biocompatibility, and anti-neoplastic properties. METHODS: In this study, Saghez-encapsulated BMP2 was applied as a scaffold for wisdom tooth follicle stem cell differentiation into the osteocyte. A total of three wisdom tooth follicles were obtained for stem cell isolation. For verification of differentiation of the isolated stem cells into osteocyte and adipocyte, Oil Red and Alizarin staining were applied, respectively. Moreover, mesenchymal stem cells were distinguished by profiling their cell surface markers, includingCD73, CD90, CD44, and CD105, by flow cytometry. Saghez scaffold loaded with BMP2 factor was prepared using sol-gel method. Four experimental groups were considered in this study: cells seeded on BMP2 encapsulated in Saghez scaffold, Saghez scaffold, osteogenic medium, and DMEM medium. RESULTS: Mechanical properties of Saghez scaffold, including tensile Young's modulus, ultimate tensile stress, compression Young's modulus, and complex shear modulus, were 19 MPa, 32 MPa, 0.42 MPa, and 0.9 MPa, respectively. The porosity of the scaffold was 70-140 µm, and the percentage of porosity was 75-98%. The results of flow cytometry studies indicated that CD44, CD73, CD90, and CD105 were positively expressed on the membrane of the tooth follicles' stem cell. The results indicated that the rate of differentiation of the follicle stem cells into osteocyte was the highest in the Saghez-BMP2 scaffold containing differentiation medium groups. These findings were verified by morphological studies, osteoblast and osteocalcin gene and protein expression investigations, and alkaline phosphatase activity measurement. The highest osteopontin and osteocalcin genes expression levels (1.7 and 1.9) were seen in positive control, followed by DMEM + differentiation factor (1.5 and 1.6), scaffold + BMP2 (1.2 and 1.4), DMEM + stem cell (1 and 1) and scaffold (0.4 and 0.5), and negative control respectively. CONCLUSION: This study provides a novel system for differentiation of the stem cell into osteocytes. The results of this study suggest that loaded BMP2 in Saghez scaffold possibly acts as an osteocyte differentiator factor.


Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Saco Dentário/citologia , Osteócitos/citologia , Células-Tronco/citologia , Tecidos Suporte/química , Fenômenos Biomecânicos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura , Saco Dentário/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Dente Serotino/citologia , Osteócitos/efeitos dos fármacos , Osteogênese , Porosidade , Células-Tronco/efeitos dos fármacos
18.
Hua Xi Kou Qiang Yi Xue Za Zhi ; 37(5): 463-468, 2019 Oct 01.
Artigo em Chinês | MEDLINE | ID: mdl-31721490

RESUMO

OBJECTIVE: To investigate the mechanism of the participation of osteocytes in the formation of osteoclasts under hypoxia. METHODS: The hypoxia culture system of osteocyte-like cell line MLO-Y4 was established by deferoxamine mesylate (DFO) in vitro. The proliferation of MLO-Y4 cells was examined by CCK-8 cell proliferation/toxicity assay. RAW264.7 cells were induced to osteoclasts by the conditioned medium containing the cultured MLO-Y4. Tartrate-resistant acid phosphatase (TRAP) staining was performed on day 7. Quantitative real-time fluorescence polymerase chain reaction, immunofluorescence, and Western blot were used to detect the expression levels of hypoxia-inducible factor (HIF)-1α and receptor activator of nuclear factor-κB ligand (RANKL) in MLO-Y4 under hypoxia. The effects of siHIF-1α on the expression levels of HIF-1α and RANKL in MLO-Y4 under the same conditions were detected. RESULTS: DFO (100 µmol·L⁻¹) promoted the proliferation of MLO-Y4 at 24 h, which decreased with time (P<0.01). After the addition of soluble sRANKL, the formation of osteoclasts was significantly increased in the DFO group (P<0.001). The expression of RANKL mRNA in MLO-Y4 under 100 µmol·L⁻¹ DFO increased first and then decreased with the duration of hypoxia. This expression reached a peak at 24 h (P<0.01). Hypoxia up-regulated the expression of HIF-1α and RANKL protein (P<0.01). Under hypoxia, siHIF-1α downregulated the expression of HIF-1α and RANKL (P<0.01). siHIF-1α also decreased the number of osteoclasts (P<0.01). CONCLUSIONS: Under hypoxia, MLO-Y4 could facilitate the formation of RANKL through upre-gulating the expression of HIF-1α protein, thereby accelerate the differentiation of RAW264.7 cells into osteoclasts.


Assuntos
Osteoclastos , Osteócitos , Diferenciação Celular , Linhagem Celular , Humanos , Hipóxia
19.
J Craniofac Surg ; 30(8): e776-e780, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31689739

RESUMO

OBJECTIVE: Granulocyte colony-stimulating factor (G-CSF) is the critical regulator of the proliferation, differentiation, and survival of granulocytes. Recently, it has been shown that G-CSF can adversely affect bone health in both animal models and patients. Here, the authors aimed to investigate whether G-CSF could inhibit the growth of osteoblasts and osteocytes by regulating nitric oxide. METHODS: The C57BL/6 mice were divided into the control group, G-CSF treatment group and recovery group (G-CSF+L-NAME). The morphology of femurs was assessed by histology and immunohistochemistry. The expression of apoptosis-related molecules in femurs was detected by immunohistochemistry and quantitative RT-PCR, respectively. To examine if neutrophil-secreted factors can induce apoptosis in osteoblasts, Gr1-positive (Gr1+) neutrophils from the bone marrow of wild-type mice were sorted and co-cultured with MC3T3 pre-osteoblasts for 2 days. RESULTS: The number of osteoblasts and newly embedding osteocytes significantly decreased and markers related to osteoblasts and osteocytes were downregulated in the G-CSF treatment compared to the control group. Moreover, G-CSF treatment did not change proliferation markers but induced apoptosis in osteoblast-lineage cells. The combined treatment of mice with G-CSF and a nitric oxide inhibitor partially restored the number of osteoblasts and osteocyte parameters. CONCLUSIONS: The G-CSF can inhibit osteoblasts and osteocytes by upregulating nitric oxide.


Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Neutrófilos/efeitos dos fármacos , Óxido Nítrico/biossíntese , Osteoblastos/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/citologia , Neutrófilos/metabolismo , Osteoblastos/citologia , Osteócitos/citologia , Osteócitos/metabolismo , Regulação para Cima/efeitos dos fármacos
20.
Biomed Res Int ; 2019: 5025398, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31737666

RESUMO

Debridement of the bone surface during a surgical fusion procedure initiates an injury response promoting a healing cascade of molecular mediators released over time. Autologous grafts offer natural scaffolding to fill the bone void and to provide local bone cells. Commercial bone grafting products such as allografts, synthetic bone mineral products, etc., are used to supplement or to replace autologous grafts by supporting osteoinductivity, osteoconductivity, and osteogenesis at the surgical site. To assure osteogenic potential, preservation of allogeneic cells with cryoprotectants has been developed to allow for long-term storage and thus delivery of viable bone cells to the surgical site. Dimethyl sulfoxide (DMSO) is an intracellular cryoprotectant commonly used because it provides good viability of the cells post-thaw. However, there is known cytotoxicity reported for DMSO when cells are stored above cryogenic temperatures. For most cellular bone graft products, the cryoprotectant is incorporated with the cells into the other mineralized bone and demineralized bone components. During thawing, the DMSO may not be sufficiently removed from allograft products compared to its use in a cell suspension where removal by washing and centrifugation is available. Therefore, both the allogeneic cell types in the bone grafting product and the local cell types at the bone grafting site could be affected as cytotoxicity varies by cell type and by DMSO content according to reported studies. Overcoming cytotoxicity may be an additional challenge in the formation of bone at a wound or surgical site. Other extracellular cryoprotectants have been explored as alternatives to DMSO which preserve without entering the cell membrane, thereby providing good cellular viability post-thaw and might abrogate the cytotoxicity concerns.


Assuntos
Transplante Ósseo/métodos , Criopreservação/métodos , Crioprotetores/farmacologia , Osteogênese/efeitos dos fármacos , Aloenxertos/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Osteócitos/efeitos dos fármacos , Osteogênese/fisiologia , Transplante Homólogo/métodos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA