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1.
Nat Med ; 26(1): 98-109, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31932796

RESUMO

Discovery of genotype-phenotype relationships remains a major challenge in clinical medicine. Here, we combined three sources of phenotypic data to uncover a new mechanism for rare and common diseases resulting from collagen secretion deficits. Using a zebrafish genetic screen, we identified the ric1 gene as being essential for skeletal biology. Using a gene-based phenome-wide association study (PheWAS) in the EHR-linked BioVU biobank, we show that reduced genetically determined expression of RIC1 is associated with musculoskeletal and dental conditions. Whole-exome sequencing identified individuals homozygous-by-descent for a rare variant in RIC1 and, through a guided clinical re-evaluation, it was discovered that they share signs with the BioVU-associated phenome. We named this new Mendelian syndrome CATIFA (cleft lip, cataract, tooth abnormality, intellectual disability, facial dysmorphism, attention-deficit hyperactivity disorder) and revealed further disease mechanisms. This gene-based, PheWAS-guided approach can accelerate the discovery of clinically relevant disease phenome and associated biological mechanisms.


Assuntos
Anormalidades Múltiplas/patologia , Bancos de Espécimes Biológicos , Fatores de Troca do Nucleotídeo Guanina/genética , Fenômica , Proteínas de Peixe-Zebra/genética , Animais , Comportamento Animal , Condrócitos/patologia , Condrócitos/ultraestrutura , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/ultraestrutura , Humanos , Modelos Biológicos , Sistema Musculoesquelético/patologia , Osteogênese , Fenótipo , Pró-Colágeno/metabolismo , Transporte Proteico , Via Secretória , Síndrome , Peixe-Zebra
2.
Nat Biomed Eng ; 4(3): 343-354, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31768001

RESUMO

Imaging techniques for quantifying changes in the hierarchical structure of deforming joints are constrained by destructive sample treatments, sample-size restrictions and lengthy scan times. Here, we report the use of fast low-dose pink-beam synchrotron X-ray tomography in combination with mechanical loading at nanometric precision for in situ imaging, at resolutions below 100 nm, of the mechanical strain in intact untreated joints under physiologically realistic conditions. We show that in young, older and osteoarthritic mice, hierarchical changes in tissue structure and mechanical behaviour can be simultaneously visualized, and that the tissue structure at the cellular level correlates with the mechanical performance of the whole joint. We also use the tomographic approach to study the colocalization of tissue strains to specific chondrocyte lacunar organizations within intact loaded joints and to explore the role of calcified-cartilage stiffness on the biomechanics of healthy and pathological joints.


Assuntos
Articulações/diagnóstico por imagem , Síncrotrons , Tomografia por Raios X/métodos , Animais , Condrócitos/ultraestrutura , Imageamento Tridimensional , Articulações/ultraestrutura , Masculino , Camundongos , Nanoestruturas , Osteoartrite/diagnóstico por imagem , Osteoartrite/patologia , Estresse Mecânico
3.
Int. j. morphol ; 37(4): 1450-1455, Dec. 2019. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1040152

RESUMO

SUMMARY: The normal sequential development of the hip joint (HJ) was considered for the evaluation of the morphological and ultrastructural aspects of the joint cartilage of the proximal femoral head epiphysis in human fetuses between 16 to 31 weeks of intra uterine life (IUL). Twenty human fetuses were fixed in 10 % formalin solution. Fetuses were divided into 4 groups (n=5): Group 1 (G1): 16-19 weeks IUL; Group 2 (G2): 20-23 weeks IUL; Group 3 (G3): 24-27 weeks IUL and Group 4 (G4): 28-31 weeks IUL. The right moieties of the HJ were subjected to light microscopy to determine the chondrocyte area, volume, and density and the extracellular matrix (ECM) density. The collagen component in ECM was qualitatively evaluated using Safranin-O and picrosirius techniques under polarized light. The left portions were analyzed using scanning electron microscopy (SEM). The advance of age revealed a gradual increase in chondrocyte area and volume and in ECM density, and a decrease in chondrocyte density. The apparent prevalence of type II collagen fibers in G1 and type III collagen fibers in G4, as well as a balance between type I and III collagen fibers in G2 and G3 suggest a process of cartilaginous evolution and repair. The pantographic organization of the collagen fiber meshes from the depth to the cartilage surface of the femoral head suggests that the arcade collagen network architecture starts at the fetal stage, regardless of the compressive forces applied. The morphological data may contribute not only to a better understanding of the maturation and cartilage organization in this area but also to serve as a theoretical basis for aspects related to diseases and joint malformations.


RESUMEN: El desarrollo secuencial normal de la articulación de la cadera (AC) se consideró para la evaluación de los aspectos morfológicos y ultraestructurales del cartílago articular de la epífisis proximal y de la cabeza femoral en fetos humanos entre 16 y 31 semanas de vida intrauterina (SVIU). Veinte fetos humanos fueron fijados en solución de formalina al 10 %. Los fetos se dividieron en 4 grupos (n = 5): Grupo 1 (G1), 1619 semanas de IUL; Grupo 2 (G2), 20-23 semanas SVIU; Grupo 3 (G3), 24-27 semanas SVIU y Grupo 4 (G4), 28-31 semanas SVIU. Las muestras derechas de la AC se sometieron a microscopía óptica para determinar el área, el volumen y la densidad de los condrocitos y la densidad de la matriz extracelular (MEC). El componente de colágeno en la MEC se evaluó cualitativamente utilizando técnicas de safranina-O y picrosirius bajo luz polarizada. Las muestras de la AC izquierda se analizaron utilizando microscopía electrónica de barrido (MEB). El avance de la edad reveló un aumento gradual en el área y el volumen de los condrocitos y en la densidad de la MEB, y una disminución en la densidad de los condrocitos. La aparente prevalencia de las fibras de colágeno tipo II en G1 y tipo III en G4, así como el equilibrio entre las fibras de colágeno tipo I y III en G2 y G3 sugieren un proceso de evolución y reparación cartilaginosa. La organización pantográfica de las mallas de fibra de colágeno desde la profundidad a la superficie del cartílago de la cabeza femoral sugiere que la arquitectura de la red de colágeno comienza en la etapa fetal, independientemente de las fuerzas compresivas aplicadas. Los datos morfológicos pueden contribuir no solo a una mejor comprensión de la organización de la maduración y el cartílago en esta área, sino también servir de base teórica para los aspectos relacionados con enfermedades y malformaciones articulares.


Assuntos
Humanos , Feto , Articulação do Quadril/ultraestrutura , Microscopia Eletrônica de Varredura , Colágeno/ultraestrutura , Condrócitos/ultraestrutura , Matriz Extracelular , Articulação do Quadril/embriologia
4.
Mater Sci Eng C Mater Biol Appl ; 105: 110018, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31546346

RESUMO

Osteoarthritis is caused by injuries and cartilage degeneration. Cartilage tissue engineering provides new ideas for the treatment of osteoarthritis. Herein, the different ratios composite membranes of silk fibroin/collagen type II were constructed (SF50-50:50, SF70-70:30, SF90-90:10). The surface properties of the composite membranes and chondrocyte morphology were observed by SEM (scanning electron microscopy). Physical functionality as well as stability of composite membranes was evaluated from tensile mechanical properties, the percentage of swelling and degradation. The tensile mechanical behavior of SF70 composite membranes was also predicted based on the constitutive model established in this study, and it is found that the experimental results and predictions were in good agreement. Biocompatibility was evaluated using chondrocytes (ADTC-5) culture. Cell proliferation was analyzed and the treatment of live/dead double staining was performed to assess the viability on chondrocytes. To sum up, SF70 showed the suitable morphology, physical stability, and biological functionality to promote proliferation of chondrocytes. This indicates that the mixing ratio of SF70 shows promise in the future as a scaffold material for cartilage repair.


Assuntos
Materiais Biocompatíveis/química , Colágeno Tipo II/química , Fibroínas/química , Teste de Materiais , Fenômenos Mecânicos , Membranas Artificiais , Animais , Bovinos , Adesão Celular , Linhagem Celular , Proliferação de Células , Forma Celular , Condrócitos/citologia , Condrócitos/ultraestrutura , Humanos , Resistência à Tração
5.
Micron ; 121: 1-7, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30861471

RESUMO

One of the most-scanned joints in preclinical animal models dealing with musculoskeletal pathologies is the mouse knee. While three-dimensional (3D) characterization of bone tissue porosity have previously been performed on cortical bone, it has not yet been comprehensively performed for the subchondral bone (SB) and the calcified cartilage (CC), which compose the subchondral mineralized zone (SMZ). Thus, it remains challenging to assess changes that occur in the SMZ of the mouse knee during pathologies such as osteoarthritis. One of the keys to addressing this challenge is to segment each layer to measure their morphologies, material properties, and porosity. Our study presents a novel approach for computing Tissue Mineral Density, 3D porosity, and the thickness of SB and CC in a mouse distal femur using High-Resolution Micro-Computed Tomography (HR-µCT). We have segmented the Vascular Porosity network, the osteocytes' lacunae of the SB, and the chondrocytes of the CC by using multi-thresholding and the percentage of chondrocytes porosity. Our results show a low intra- and inter-observer coefficient of variability. Regarding porosity and geometrical properties of both CC and SB, our results are within the range of the literature. Our approach opens new avenues for assessing porosity and vascular changes in the distal femur of preclinical animal models dealing with musculoskeletal pathologies such as osteoarthritis.


Assuntos
Densidade Óssea , Fêmur/diagnóstico por imagem , Imageamento Tridimensional/métodos , Microtomografia por Raio-X/métodos , Animais , Calcificação Fisiológica , Cartilagem Articular/citologia , Cartilagem Articular/diagnóstico por imagem , Condrócitos/citologia , Condrócitos/ultraestrutura , Fêmur/citologia , Camundongos , Osteócitos/citologia , Porosidade , Tíbia/citologia , Tíbia/diagnóstico por imagem
6.
Acta Otolaryngol ; 139(5): 439-443, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30806116

RESUMO

BACKGROUND: Scanning electron microscopy (SEM) study of the human incus bone is scanty whilst, to our knowledge, no information regarding human incudo-malleolar joint articular-cartilage morphology has previously been provided. AIMS/OBJECTIVES: Our aim was to shed some light on this morphological issue and to propose some theoretical perspectives on its functional role. MATERIAL AND METHODS: The human incudo-malleolar joint was documented with field emission SEM on samples recovered during ear surgery procedures after patients' informed consent. RESULTS: Normal articular cartilage chondrocytes, flattened cells with prominent nucleus and short microvilli were observed. Interestingly, cells provided with long cilia were identified. Type A cilia are arranged in a pyramidal formation with extra-long cilia stemming from the cluster, projecting upwards in an antenna-like formation ending with a dilated structure that as a whole, resembles the stereocilia with kinocilium. Types B, C and D cilia resemble those of the genital and respiratory tracts. CONCLUSIONS AND SIGNIFICANCE: It is therefore possible to hypothesize that the observed ciliated cells may be a new chondrocyte phenotype with sensory function. Motile cilia confer the ability to distinguish variations in synovial fluid chemical composition and, in addition, they perhaps may also play some role in the mechanism of sound transmission.


Assuntos
Cartilagem Articular/ultraestrutura , Condrócitos/ultraestrutura , Cílios/ultraestrutura , Bigorna , Martelo , Humanos , Fenótipo
7.
Int J Med Sci ; 16(2): 221-230, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30745802

RESUMO

Background: In a previous report, we demonstrated the presence of cells with a neural/glial phenotype on the concave side of the vertebral body growth plate in Idiopathic Scoliosis (IS) and proposed this phenotype alteration as the main etiological factor of IS. In the present study, we utilized the same specimens of vertebral body growth plates removed during surgery for Grade III-IV IS to analyse gene expression. We suggested that phenotype changes observed on the concave side of the vertebral body growth plate can be associated with altered expression of particular genes, which in turn compromise mechanical properties of the concave side. Methods: We used a Real-Time SYBR Green PCR assay to investigate gene expression in vertebral body growth plates removed during surgery for Grade III-IV IS; cartilage tissues from human fetal spine were used as a surrogate control. Special attention was given to genes responsible for growth regulation, chondrocyte differentiation, matrix synthesis, sulfation and transmembrane transport of sulfates. We performed morphological, histochemical, biochemical, and ultrastructural analysis of vertebral body growth plates. Results: Expression of genes that control chondroitin sulfate sulfation and corresponding protein synthesis was significantly lower in scoliotic specimens compared to controls. Biochemical analysis showed 1) a decrease in diffused proteoglycans in the total pool of proteoglycans; 2) a reduced level of their sulfation; 3) a reduction in the amount of chondroitin sulfate coinciding with raising the amount of keratan sulfate; and 4) reduced levels of sulfation on the concave side of the scoliotic deformity. Conclusion: The results suggested that altered expression of genes that control chondroitin sulfate sulfation and corresponding changes in protein synthesis on the concave side of vertebral body growth plates could be causal agents of the scoliotic deformity.


Assuntos
Condrócitos/fisiologia , Lâmina de Crescimento/metabolismo , Escoliose/metabolismo , Coluna Vertebral/metabolismo , Adolescente , Diferenciação Celular , Criança , Condrócitos/ultraestrutura , Sulfatos de Condroitina/metabolismo , Lâmina de Crescimento/patologia , Humanos , Biossíntese de Proteínas , Escoliose/genética , Escoliose/patologia
8.
Chin J Integr Med ; 25(9): 677-683, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30656598

RESUMO

OBJECTIVE: To illustrate the molecular mechanisms underlying the therapeutic effects of electroacupuncture (EA) on knee osteoarthritis (OA). METHODS: Twenty-seven six-month-old New Zealand white rabbits were allocated into three groups in accordance with a random number table: normal group (no surgery-induced OA; without treatment), model group (surgery-induced OA; without treatment) and EA group [surgery-induced OA; received treatment with EA at acupoints Dubi (ST 35) and Neixiyan (EX-LE 5), 30 min twice a day]. After eight consecutive weeks of treatment, the histopathological alterations in cartilage were observed using optical microscopy and transmission electron microscopy, cartilage degeneration was evaluated by modified Mankin's score principles, the synovial fluid concentration of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and matrix metalloproteinase-3 (MMP-3) were evaluated by enzyme-linked immunosorbent assay, and the protein expression levels of IL-1ß, IL-6, TNF-α, MMP-3, IκB kinase-ß (IKK-ß), nuclear factor of α light polypeptide gene enhancer in B-cells inhibitor α (IκB-α) and nuclear factor-κB (NF-κB) p65 were quantified by Western blot analysis. RESULTS: EA treatment significantly improved cartilage structure arrangement and reduced cellular degeneration. The IL-1ß, IL-6, TNF-α and MMP-3 of synovial fluid in the EA-treated group were significantly decreased compared with the model group (all P<0.01). Compared with the model group, the IL-1ß, IL-6, TNF-α, MMP-3, IKK-ß and NF-κB p65 protein expressions in cartilage of EA-treated group were significantly decreased (all P<0.01), whereas IκB-α expression was significantly up-regulated (P<0.01). CONCLUSION: EA treatment may delay cartilage degeneration by down-regulating inflammatory factors through NF-κB signaling pathway, which may, in part, explain its clinical efficacy in the treatment of knee OA.


Assuntos
Cartilagem Articular/patologia , Eletroacupuntura , NF-kappa B/metabolismo , Transdução de Sinais , Animais , Condrócitos/patologia , Condrócitos/ultraestrutura , Quinase I-kappa B/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Masculino , Metaloproteinase 3 da Matriz/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , Coelhos , Líquido Sinovial/metabolismo , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
9.
Cell Prolif ; 52(2): e12544, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30444057

RESUMO

OBJECTIVES: Connexin-mediated functional gap junction intercellular communication (GJIC) has a vital role in development, homeostasis and pathology. Transforming growth factor-ß1 (TGF-ß1), as one of the most vital factors in chondrocytes, promotes cartilage precursor cell differentiation and chondrocyte proliferation, migration and metabolism. However, how TGF-ß1 mediates GJIC in chondrocytes remains unclear. This study aims to determine the influence of TGF-ß1 on GJIC in mouse chondrocytes and its underlying mechanism. METHODS: qPCR and mRNA microarray were used to verify the expression of genes in the TGF-ß and connexin families in cartilage and chondrocytes. A scrape loading/dye transfer assay was performed to explore GJIC. Western blot analysis was used to detect connexin43 (Cx43) and Smad signalling components. Immunofluorescence staining was performed to characterize protein distribution. RESULTS: The TGF-ß1 mRNA was the highest expressed member of the TGFß super family in cartilage. TGF-ß1 promoted functional GJIC through increased expression of Cx43. TGF-ß1-mediated GJIC required the participation of TGF-ß type I receptor. TGF-ß1 activated Smad3 and Smad4 signalling to facilitate their nuclear translocation. The Smad3 and Smad4 signalling proteins bound to the promoter of Gja1 and thus initiated Cx43 gene expression. CONCLUSIONS: For the first time, these results revealed a vital role of TGF-ß1 in cell-cell communication in chondrocytes via gap junction formation. We describe the regulatory mechanism, the involvement of TGF-ß type I receptor and the nuclear translocation of Smad3/4.


Assuntos
Condrócitos/metabolismo , Junções Comunicantes/metabolismo , Transdução de Sinais , Proteína Smad3/metabolismo , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Células Cultivadas , Condrócitos/ultraestrutura , Junções Comunicantes/ultraestrutura , Camundongos Endogâmicos C57BL
10.
Bioelectrochemistry ; 126: 1-11, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30471483

RESUMO

The growth plate is a cartilaginous layer present from the gestation period until the end of puberty where it ossifies joining diaphysis and epiphysis. During this period several endocrine, autocrine, and paracrine processes within the growth plate are carried out by chondrocytes; therefore, a disruption in cellular functions may lead to pathologies affecting bone development. It is known that electric fields impact the growth plate; however, parameters such as stimulation time and electric field intensity are not well documented. Accordingly, this study presents a histomorphometrical framework to assess the effect of electric fields on chondroepiphysis explants. Bones were stimulated with 3.5 and 7 mV/cm, and for each electric field two exposure times were tested for 30 days (30 min and 1 h). Results evidenced that electric fields increased the hypertrophic zones compared with controls. In addition, a stimulation of 3.5 mV/cm applied for 1 h preserved the columnar cell density and its orientation. Moreover, a pre-hypertrophy differentiation in the center of the chondroepiphysis was observed when explants were stimulated during 1 h with both electric fields. These findings allow the understanding of the effect of electrical stimulation over growth plate organization and how the stimulation modifies chondrocytes morphophysiology.


Assuntos
Condrócitos/citologia , Estimulação Elétrica , Lâmina de Crescimento/crescimento & desenvolvimento , Animais , Proliferação de Células , Células Cultivadas , Condrócitos/patologia , Condrócitos/ultraestrutura , Estimulação Elétrica/instrumentação , Desenho de Equipamento , Fêmur/citologia , Fêmur/crescimento & desenvolvimento , Fêmur/patologia , Fêmur/ultraestrutura , Lâmina de Crescimento/citologia , Lâmina de Crescimento/patologia , Lâmina de Crescimento/ultraestrutura , Úmero/citologia , Úmero/crescimento & desenvolvimento , Úmero/patologia , Úmero/ultraestrutura , Hipertrofia , Osteogênese , Ratos , Ratos Wistar
11.
Cartilage ; 10(3): 364-369, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-29468902

RESUMO

OBJECTIVE: In orthopedic joint injection, the most frequently used local anesthetics are ropivacaine, bupivacaine, and 1% or 2% lidocaine. The aim of this study was to examine effects of these various anesthetics on the viability of human chondrocytes. Our hypothesis was that all local anesthetics tested damage human chondrocytes in vitro. METHODS: Primary human chondrocytes were isolated and cultured from 6 donated human knee joints (mean age of donors 61.2 years). Local anesthetics were added to these cultures. Toxicity analysis was performed by visualization of cell structure using light microscopy. Determination of vital chondrocytes was performed by use of a Casy cell counter. Chondrocytes' cell death was examined by fluorescence microscopy and an XTT ELISA assay. RESULTS: Light microscope and fluorescence microscope data revealed a defect cell structure and increased number of dead cells after addition of 1% or 2% lidocaine and bupivacaine but not ropivacaine. We were able to show an increased level of XTT activity after treatment with bupivacaine, 2% lidocaine or ropivacaine. The count of vital chondrocytes was significantly decreased after treatment with bupivacaine, 1% or 2% lidocaine, and ropivacaine. CONCLUSIONS: The data show that treatment with local anesthetics induces cell damage of human chondrocytes in vitro. Ropivacaine seems to be a local anesthetic with the lowest toxic potential on human chondrocytes, a feature that may favor its preference for use in joint injection.


Assuntos
Anestésicos Locais/toxicidade , Bupivacaína/toxicidade , Células Cultivadas/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Lidocaína/toxicidade , Ropivacaina/toxicidade , Morte Celular , Células Cultivadas/ultraestrutura , Condrócitos/ultraestrutura , Humanos , Injeções Intra-Articulares , Microscopia de Fluorescência/métodos , Pessoa de Meia-Idade , Doadores de Tecidos
12.
J Trace Elem Med Biol ; 50: 123-129, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30262269

RESUMO

OBJECTIVES: To investigate the effect of low selenium diet on rat´s knee cartilage and expression of chondroitin sulfate (CS) sulfated enzymes in articular and epiphyseal-plate cartilage of rats' femur and tibia. METHODS: Twenty-four SD rats were randomly divided into two groups with six female and six male in each group: control group (selenium 0.18 mg/kg), and low selenium group (selenium 0.02 mg/kg). After 109 days, the rats were sacrificed. The ultrastructural changes in chondrocytes of rat knee cartilage were observed by transmission electron microscopy (TEM). The morphology and pathology changes of knee cartilage were examined by hematoxylin-eosin (HE) and toluidine blue (TB) staining. The localization and expression of enzymes involved in CS sulfation, including chondroitin 6-O-sulfotransferase 1 (CHST-3), chondroitin 4-O-sulfotransferase 2 (CHST-12) and uronyl 2-O-sulfotransferase (UST) were examined by immunohistochemical staining and semi-quantitative analysis. RESULTS: In low selenium group, ultrastructural changes of chondrocytes were observed in articular cartilage of femur (AF), articular cartilage of tibia (AT), epiphyseal-plate cartilage of femur (EF) and epiphyseal-plate cartilage of tibia (ET); however, no significant changes in chondrocytes number were observed in the above AF, AT, EF or ET. Moreover, reduced thickness of cartilage layer in AF, EF and ET was detected along with reduced staining areas of sulfated glycosaminoglycan in EF and ET in low selenium group. In addition, positive staining rate of CHST-3 was lower in AF, AT and EF, while positive staining rates of CHST-12 and UST were lower in AF, AT, EF and ET in low selenium group when compared with control group. CONCLUSIONS: Low selenium undermines the ultrastructure of chondrocytes, inhibits the normal development of cartilage and the expression of CS sulfated enzymes.


Assuntos
Cartilagem Articular/metabolismo , Articulação do Joelho/metabolismo , Selênio/metabolismo , Sulfotransferases/metabolismo , Animais , Cartilagem Articular/ultraestrutura , Condrócitos/metabolismo , Condrócitos/ultraestrutura , Feminino , Masculino , Microscopia Eletrônica de Transmissão , Ratos , Ratos Sprague-Dawley , Selênio/deficiência
13.
Exp Mol Med ; 50(9): 123, 2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-30232322

RESUMO

Ageing is the primary risk factor for osteoarthritis (OA). A decline in the ageing-associated process of autophagy is suggested as a potential contributor to OA development. Polyamines such as spermidine decrease during ageing, contributing to impaired autophagy and reduced cellular function. However, the role of polyamines and their effect on the regulatory mechanism governing autophagy in aged and arthritic cartilage tissue has not been established. Elucidating if polyamine regulation of autophagy is impaired during ageing and OA in chondrocytes may lead to improved treatment approaches to protect against cartilage degradation. Our results indicate that polyamine synthesis was decreased in aged and OA cartilage, along with reduced autophagy activity, evidenced by decreased autophagy-related gene and protein expression and autophagosome formation. Importantly, spermidine treatment increased the expression of the acetyltransferase EP300, which binds to crucial autophagy proteins, Beclin1 and LC3, and elevates chondrocyte autophagy. Our data indicate spermidine prevents the ageing- and OA-related decrease in autophagy and may protect against OA development.


Assuntos
Autofagia/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Condrócitos/metabolismo , Condrócitos/patologia , Proteína p300 Associada a E1A/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Espermidina/farmacologia , Animais , Biomarcadores/metabolismo , Condrócitos/efeitos dos fármacos , Condrócitos/ultraestrutura , Condrogênese/efeitos dos fármacos , Camundongos Endogâmicos C57BL
14.
Biomed Pharmacother ; 106: 1696-1704, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30119244

RESUMO

Chondrocyte is targeted for disruption in Osteoarthritis (OA) and Kashin-Beck Disease (KBD), and chondrocyte death in cartilage may contribute to the progression of OA and KBD. Oxidative stress leads to increased risk for OA. Previous work in our laboratory implicates oxidative stress as a potential mediator in children with KBD. While these studies suggest a role for oxidative stress in the modulation of OA and KBD, the direct effects of reactive oxygen species/reactive nitrogen species (ROS/RNS) on the stability of this domain remain unclear. Here, we demonstrate that oxidative stress, as induced through treatment with 3-morpholinosydnonimine (SIN-1), a spontaneous ROS/RNS generator, decreased the cell viability in hypertrophic chondrocytes in a dose- and time- dependent manner. SIN-1 induced necrosis in hypertrophic chondrocytes, whereas triggered apoptosis in non-hypertrophic cells of non-differentiated ATDC5 cells and C28/I2 cells. Ultrastructural analysis of hypertrophic chondrocyte treated with SIN-1 revealed morphological changes, such as plasma membrane breakdown, generalized swelling of the cytoplasm and organelles, even to disappearance. Moreover, SIN-1 induced chondronecrosis in the deep zone of engineered cartilage tissue, such as cell-free vacancy and "red ghost" cells. Overall, we demonstrate for the first time that oxidative stress, as induced through exogenous ROS/RNS, leads to necrosis in hypertrophic chondrocytes. Oxidative stress-mediated necrotic cell death contributes to chondronecrosis in the deep zone of cartilage in both OA and KBD.


Assuntos
Cartilagem/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Doença de Kashin-Bek/patologia , Molsidomina/análogos & derivados , Osteoartrite/patologia , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Cartilagem/metabolismo , Cartilagem/patologia , Estudos de Casos e Controles , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/metabolismo , Condrócitos/ultraestrutura , Relação Dose-Resposta a Droga , Humanos , Hipertrofia , Doença de Kashin-Bek/metabolismo , Camundongos , Molsidomina/farmacologia , Necrose , Osteoartrite/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Engenharia Tecidual
15.
Int J Biol Macromol ; 118(Pt B): 2014-2020, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-30009919

RESUMO

Injectable hydrogels are attractive and alternative scaffolds for cell delivery because they could form in situ, simulate natural tissue and fill any shape of defect. This study aimed at fabricating injectable, self-crosslinkable and biomimetic hydrogels based on collagen type II (Col II) and activated chondroitin sulfate (CS-sNHS) under physiological conditions without the addition of any catalysts or crosslinking agents. The inner morphology of hydrogels was detected by scanning electron microscopy, and it showed that fibrous structure formed in the hydrogels. The gelation time, water absorption capacity and the mechanical property of hydrogels were closely related to the weight ratio of Col II and CS-sNHS in hydrogels. Chondrocytes were encapsulated into these hydrogels, and the effect of hydrogels on survival, proliferation, morphology of cells and remolding of extracellular matrix was investigated. The results demonstrated that chondrocytes survived well and showed round or oval morphology in these hydrogels, in addition, the matrix in hydrogels had been remolded and the collagen fibers displayed periodic alternation of light and shade. These results implied that the injectable and self-crosslinkable hydrogels were alternative carriers for chondrocyte delivery.


Assuntos
Sulfatos de Condroitina/química , Colágeno Tipo II/química , Reagentes para Ligações Cruzadas/química , Hidrogéis/química , Injeções , Engenharia Tecidual/métodos , Animais , Proliferação de Células , Forma Celular , Sobrevivência Celular , Junções Célula-Matriz , Condrócitos/citologia , Condrócitos/ultraestrutura , Sulfatos de Condroitina/síntese química , Módulo de Elasticidade , Matriz Extracelular/metabolismo , Coelhos , Succinimidas/química , Sus scrofa , Água
16.
Eur Rev Med Pharmacol Sci ; 22(12): 3656-3662, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29949137

RESUMO

OBJECTIVE: The purpose of the present study was to explore the mechanism of action of the adipokine chemerin in osteoarthritis (OA) by means of an in vitro OA model. MATERIALS AND METHODS: Primary chondrocytes were isolated from normal rats. The chondrocytes were stimulated with interleukin 1 beta (IL-1ß, 10 µg/L) to establish a model of induced OA. Chemerin was administered to cells of this model. After culture of the chondrocytes in the presence of chemerin for 48 h, the expression of the genes related to OA occurrence and protection, matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-3 (MMP-3), and matrix metalloproteinase-13 (MMP-13) was examined. Western blot was then performed to analyze the phosphorylation of the AKT and extracellular signal-regulated kinase (ERK) proteins in chondrocytes. RESULTS: Stimulation of chondrocytes with IL-1ß markedly reduced the proliferative capability of chondrocytes. Chemerin (5 µM) also significantly decreased the proliferative capability of chondrocytes. The combined administration of IL-1ß and chemerin induced an even greater reduction in the proliferative capability of chondrocytes. Polymerase chain reaction (PCR) results showed that both IL-1ß and chemerin reduced the expression of the protective genes in OA (MMP-1, MMP-3, and MMP-13). Also, the stimulation with IL-1ß and chemerin significantly enhanced the phosphorylation of AKT/ERK in chondrocytes. CONCLUSIONS: This adipokine induces changes in the metabolic and proliferative capabilities of chondrocytes by increasing the phosphorylation of AKT/ERK, thereby inducing OA or aggravating the symptoms of OA.


Assuntos
Quimiocinas/genética , Condrócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Sistema de Sinalização das MAP Quinases/genética , Proteína Oncogênica v-akt/genética , Proteína Oncogênica v-akt/metabolismo , Osteoartrite/genética , Animais , Cartilagem Articular/citologia , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Proliferação de Células/genética , Células Cultivadas , Condrócitos/ultraestrutura , Interleucina-1beta/farmacologia , Masculino , Metaloproteinases da Matriz/metabolismo , Osteoartrite/metabolismo , Fosforilação , Ratos , Ratos Wistar
17.
Biomed Pharmacother ; 102: 1209-1220, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29710540

RESUMO

BACKGROUND: Rheumatoid arthritis (RA) is a long-term autoimmune disorder that primarily results in warm, swollen, and painful joints. In this study, we investigate the potentially therapeutic role of artesunate (Art) on chondrocyte proliferation, apoptosis and autophagy in rheumatoid arthritis (RA) via the PI3K/AKT/mTOR signaling pathway. METHODS: Rat model of RA was successfully established through subcutaneous injection of emulsion. Positive protein expression rates of PI3K, AKT and mTOR were determined by immunohistochemistry. RA chondrocytes were initially randomized into five different groups. The mRNA expressions of PI3K, AKT, mTOR, Bcl-2, Bcl-xl, LC3-I, LC3-II and Becline-1 were measured by RT-qPCR. Protein expressions of p-PI3K, p-AKT, p-mTOR, Bcl-2, Bcl-xl, Bax, LC3-I, LC3-II and Becline-1 were determined using western blotting. The chondrocytes of rats with RA and normal rats were isolated and cultured in vitro. Chondrocyte proliferation, apoptosis, cell cycle and autophagy were all determined by CCK-8 assay, flow cytometry and transmission electron microscope (TEM). RESULTS: Artesunate alleviated the inflammation and did not produce any form of hepatotoxicity in rats with RA. In addition, Artesunate decreased expressions of PI3K, AKT, mTOR, p-PI3K, p-AKT, p-mTOR Bcl-2 and Bcl-xl and increased Bax, LC3II/LC3I and Becline-1 protein expression. Artesunate also inhibited chondrocyte proliferation and accelerates cell apoptosis and autophagy via suppression of the PI3K/AKT/mTOR signaling pathway. CONCLUSION: Our study demonstrates that Art inhibits chondrocyte proliferation and accelerates apoptosis and autophagy in RA rats through the PI3K/AKT/mTOR signaling pathway.


Assuntos
Apoptose/efeitos dos fármacos , Artemisininas/farmacologia , Artrite Reumatoide/patologia , Autofagia/efeitos dos fármacos , Condrócitos/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose/genética , Artemisininas/uso terapêutico , Artesunato , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Autofagia/genética , Cartilagem/efeitos dos fármacos , Cartilagem/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Condrócitos/ultraestrutura , Colágeno Tipo II/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Inflamação/patologia , Fígado/patologia , Masculino , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
18.
Int J Med Sci ; 15(5): 436-446, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29559832

RESUMO

Idiopathic scoliosis is one of the most common disabling pathologies of children and adolescents. Etiology and pathogenesis of idiopathic scoliosis remain unknown. To study the etiology of this disease we identified the cells' phenotypes in the vertebral body growth plates in patients with idiopathic scoliosis. Materials and methods: The cells were isolated from vertebral body growth plates of the convex and concave sides of the deformity harvested intraoperatively in 50 patients with scoliosis. Cells were cultured and identified by methods of common morphology, neuromorphology, electron microscopy, immunohistochemistry and PCR analysis. Results: Cultured cells of convex side of deformation were identified as chondroblasts. Cells isolated from the growth plates of the concave side of the deformation showed numerous features of neuro- and glioblasts. These cells formed synapses, contain neurofilaments, and expressed neural and glial proteins. Conclusion: For the first time we demonstrated the presence of cells with neural/glial phenotype in the concave side of the vertebral body growth plate in scoliotic deformity. We hypothesized that neural and glial cells observed in the growth plates of the vertebral bodies represent derivatives of neural crest cells deposited in somites due to alterations in their migratory pathway during embryogenesis. We also propose that ectopic localization of cells derived from neural crest in the growth plate of the vertebral bodies is the main etiological factor of the scoliotic disease.


Assuntos
Lâmina de Crescimento/patologia , Crista Neural/patologia , Neuroglia/patologia , Escoliose/patologia , Adolescente , Criança , Condrócitos/metabolismo , Condrócitos/patologia , Condrócitos/ultraestrutura , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica/genética , Lâmina de Crescimento/metabolismo , Lâmina de Crescimento/ultraestrutura , Humanos , Masculino , Microscopia Eletrônica de Varredura , Crista Neural/metabolismo , Crista Neural/ultraestrutura , Neuroglia/metabolismo , Escoliose/etiologia , Escoliose/genética , Coluna Vertebral/metabolismo , Coluna Vertebral/patologia , Coluna Vertebral/ultraestrutura
19.
Int J Exp Pathol ; 99(6): 312-322, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30680829

RESUMO

The purpose of this paper was to investigate chondrocyte distribution and death in the cartilage in Kashin-Beck disease (KBD). Apoptotic chondrocytes were detected by TUNEL assay. Ultrastructural changes were examined by transmission electron microscope (TEM). Biochemical markers associated with apoptosis (eg, caspase-3) and necroptosis (eg, RIP3) were investigated by immunohistochemistry. In KBD cartilage chondrocyte death was characterized by paler staining of the cells. Multiple chondral cell clusters surrounded the areas lacking cells in the deep zone. The per cent of TUNEL-positive and RIP3-positive chondrocytes were higher in the middle zones of KBD samples; however, there was some positive staining for TUNEL but negative staining for caspase-3. Immunohistochemistry failed to detect significant differences in caspase-3 levels in KBD children compared to controls, suggesting that beside apoptosis necroptosis dominates as a cell death mechanism in the middle zone of cartilage from KBD children. To clarify further the presence of chondrocyte necroptosis in KBD, we performed TUNEL, caspase-3 and RIP3 staining in a rat KBD model which is based upon T-2 toxin treatment under selenium-deficient conditions. Apoptosis and necroptosis co-existed in the middle zone in this rat KBD model. Ultrastructural analysis of chondrocyte from deep cartilage revealed abnormal cells with numerous morphological changes, such as plasma membrane breakdown, generalized swelling of the cytoplasm and loss of identifiable organelles. Chondrocyte death by necrosis in the deep zone of cartilages in KBD may be a result of exposure to T-2 toxin from bone marrow or bloodstream under selenium-deficient nutrition status in KBD endemic areas. Chondrocyte death plays a key role in either the initiation or the progression of KBD pathogenesis.


Assuntos
Apoptose/fisiologia , Condrócitos/patologia , Doença de Kashin-Bek/patologia , Animais , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Cartilagem Articular/ultraestrutura , Caspase 3/metabolismo , Morte Celular/fisiologia , Criança , Pré-Escolar , Condrócitos/metabolismo , Condrócitos/ultraestrutura , Feminino , Humanos , Doença de Kashin-Bek/metabolismo , Masculino , Microscopia Eletrônica de Varredura , Necrose , Ratos Sprague-Dawley , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo
20.
Cartilage ; 9(3): 313-320, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29156946

RESUMO

Objectives During arthroscopic or open joint surgery, articular cartilage may be subjected to mechanical insults by accident or design. These may lead to chondrocyte death, cartilage breakdown and posttraumatic osteoarthritis. We have shown that increasing osmolarity of routinely used normal saline protected chondrocytes against injuries that may occur during orthopedic surgery. Often several liters of irrigation fluid are used during an orthopedic procedure, which is usually kept at room temperature, but is sometimes chilled. Here, we compared the effect of normal and hyperosmolar saline solution at different temperatures on chondrocyte viability following cartilage injury using in vitro and in vivo models of scalpel-induced injury. Design Cartilage injury was induced in bovine osteochondral explants and the patellar groove of rats in vivo by a single pass of a scalpel blade in the presence of normal saline (300 mOsm) or hyperosmolar saline solution (600 mOsm, sucrose addition) at 4°C, 21°C, or 37°C. Chondrocytes were fluorescently labeled and visualized by confocal microscopy to assess cell death. Results Hyperosmolar saline reduced scalpel-induced chondrocyte death in both bovine and rat cartilage by ~50% at all temperatures studied (4°C, 21°C, 37°C; P < 0.05). Raising temperature of both irrigation solutions to 37°C reduced scalpel-induced cell death ( P < 0.05). Conclusions Increasing the osmolarity of normal saline and raising the temperature of the irrigation solutions to 37°C reduced chondrocyte death associated with scalpel-induced injury in both in vitro and in vivo cartilage injury models. A hyperosmolar saline irrigation solution at 37°C may protect cartilage by decreasing the risk of chondrocyte death during mechanical injury.


Assuntos
Doenças das Cartilagens/tratamento farmacológico , Cartilagem Articular/lesões , Cartilagem/lesões , Sobrevivência Celular/fisiologia , Condrócitos/efeitos dos fármacos , Animais , Cartilagem/efeitos dos fármacos , Cartilagem/patologia , Cartilagem Articular/efeitos dos fármacos , Bovinos , Morte Celular/efeitos dos fármacos , Condrócitos/ultraestrutura , Microscopia Confocal/instrumentação , Modelos Animais , Procedimentos Ortopédicos/efeitos adversos , Procedimentos Ortopédicos/normas , Concentração Osmolar , Substâncias Protetoras/farmacologia , Ratos , Solução Salina/farmacologia , Temperatura , Irrigação Terapêutica/efeitos adversos , Irrigação Terapêutica/métodos
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