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1.
Yakugaku Zasshi ; 140(9): 1141-1150, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32879246

RESUMO

Osteoarthritis is the most common joint disorder worldwide and one of the leading causes of disability in the elderly. We have reported that the novel sodium hyaluronate derivative chemically linked with diclofenac (DF), diclofenac etalhyaluronate (SI-613), exerted a potent and long-lasting analgesic effect in experimental arthritis models. In this study, we evaluated the properties of residual SI-613 in the knee joint after an intra-articular (IA) administration. After IA administration of fluorescent labeled SI-613 (FA-SI-613) or fluorescent labeled hyaluronic acid (FA-HA) to rabbits, fluorescence intensities in the synovial membrane and cartilage were higher in the FA-SI-613 group until 7 d after administration than in the FA-HA group. After IA administration of radiolabeled SI-613 (14C-SI-613) to rabbits, the radioactivity remained in the joint cavity and the joint tissues such as synovial membrane and cartilage until 84 d after administration. This residual radioactivity was identified mainly as HA linked with DF, since 14C-SI-613 was labeled at the benzene ring of DF and since more DF-linked HA oligomer was detected on metabolite analysis than free DF in the synovial membrane and synovial lavage fluid up to 28 d after administration. These results suggested that intra-articularly administered SI-613 remained for a longer time in the joint as HA linked with DF than when HA was administered. Therefore, SI-613 was considered to prolong the pharmacological effects of both HA and DF by remaining in the joint as HA linked with DF.


Assuntos
Diclofenaco/administração & dosagem , Diclofenaco/metabolismo , Ácido Hialurônico/análogos & derivados , Articulação do Joelho , Osteoartrite/tratamento farmacológico , Animais , Cartilagem/metabolismo , Modelos Animais de Doenças , Ácido Hialurônico/administração & dosagem , Ácido Hialurônico/metabolismo , Injeções Intra-Articulares , Articulação do Joelho/metabolismo , Masculino , Coelhos , Membrana Sinovial/metabolismo , Fatores de Tempo
2.
PLoS One ; 15(9): e0238449, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32886713

RESUMO

Treatment of osteoarthritis (OA) is still a major clinical challenge due to the limited inherent healing capacity of cartilage. Recent studies utilising stem cells suggest that the therapeutic benefits of these cells are mediated through the paracrine mechanism of bioactive molecules. The present study evaluates the regenerative effect of stem cells from human exfoliated deciduous teeth (SHED) conditioned medium (CM) on OA chondrocytes. The CM was collected after the SHED were cultured in serum-free medium (SFM) for 48 or 72 h and the cells were characterised by the expression of MSC and pluripotency markers. Chondrocytes were stimulated with interleukin-1ß and treated with the CM. Subsequently, the expression of aggrecan, collagen type 2 (COL 2), matrix metalloproteinase-13 (MMP-13), nuclear factor-kB (NF-kB) and the level of inflammatory and anti-inflammatory markers were evaluated. SHED expressed mesenchymal stromal cell surface proteins but were negative for haematopoietic markers. SHED also showed protein expression of NANOG, OCT4 and SOX2 with differential subcellular localisation. Treatment of OA chondrocytes with CM enhanced anti-inflammation compared to control cells treated with SFM. Furthermore, the expression of MMP-13 and NF-kB was significantly downregulated in stimulated chondrocytes incubated in CM. The study also revealed that CM increased the expression of aggrecan and COL 2 in OA chondrocytes compared to SFM control. Both CM regenerate extracellular matrix proteins and mitigate increased MMP-13 expression through inhibition of NF-kB in OA chondrocytes due to the presence of bioactive molecules. The study underscores the potential of CM for OA treatment.


Assuntos
Condrócitos/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Osteoartrite/metabolismo , Agrecanas/metabolismo , Cartilagem/metabolismo , Técnicas de Cultura de Células/métodos , Células Cultivadas , Colágeno Tipo II/metabolismo , Humanos , Metaloproteinase 13 da Matriz/metabolismo , Células-Tronco Mesenquimais/metabolismo , NF-kappa B/metabolismo , Osteoartrite/terapia , Regeneração , Células-Tronco/metabolismo , Dente Decíduo/metabolismo
3.
PLoS One ; 15(8): e0237479, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790806

RESUMO

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


Assuntos
Proteína Morfogenética Óssea 2/genética , Hidrogéis/química , Fatores de Transcrição SOX9/genética , Fator de Crescimento Transformador beta1/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Cartilagem/citologia , Cartilagem/metabolismo , Cartilagem/patologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Condrogênese/genética , Colágeno Tipo I/química , Colágeno Tipo X/genética , Meios de Cultura Livres de Soro/química , Glicosaminoglicanos/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , RNA Mensageiro/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
4.
J Vis Exp ; (162)2020 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-32831304

RESUMO

Several negatively charged tissues in the body, like cartilage, present a barrier to the targeted drug delivery due to their high density of negatively charged aggrecans and, therefore, require improved targeting methods to increase their therapeutic response. Because cartilage has a high negative fixed charge density, drugs can be modified with positively charged drug carriers to take advantage of electrostatic interactions, allowing for enhanced intra-cartilage drug transport. Studying the transport of drug carriers is, therefore, crucial towards predicting the efficacy of drugs in inducing a biological response. We show the design of three experiments which can quantify the equilibrium uptake, depth of penetration and non-equilibrium diffusion rate of cationic peptide carriers in cartilage explants. Equilibrium uptake experiments provide a measure of the solute concentration within the cartilage compared to its surrounding bath, which is useful for predicting the potential of a drug carrier in enhancing therapeutic concentration of drugs in cartilage. Depth of penetration studies using confocal microscopy allow for the visual representation of 1D solute diffusion from the superficial to deep zone of cartilage, which is important for assessing whether solutes reach their matrix and cellular target sites. Non-equilibrium diffusion rate studies using a custom-designed transport chamber enables the measurement of the strength of binding interactions with the tissue matrix by characterizing the diffusion rates of fluorescently labeled solutes across the tissue; this is beneficial for designing carriers of optimal binding strength with cartilage. Together, the results obtained from the three transport experiments provide a guideline for designing optimally charged drug carriers which take advantage of weak and reversible charge interactions for drug delivery applications. These experimental methods can also be applied to evaluate the transport of drugs and drug-drug carrier conjugates. Further, these methods can be adapted for the use in targeting other negatively charged tissues such as meniscus, cornea and the vitreous humor.


Assuntos
Cartilagem/metabolismo , Portadores de Fármacos/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Peptídeos/farmacocinética , Animais , Cartilagem/efeitos dos fármacos , Cátions/química , Difusão , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Peptídeos/administração & dosagem , Peptídeos/química , Eletricidade Estática
5.
Medicine (Baltimore) ; 99(22): e20406, 2020 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-32481432

RESUMO

BACKGROUND: Structural and functional changes in subchondral bone have been recognized as a key factor in the development of related disease, and subchondral bone may be a new target for the treatment of osteoarthritis. The purpose of our present study is to investigate the global status and trends of subchondral bone research. METHOD: Publications related to the studies of subchondral bone from 1993 to 2018 were retrieved from the Science Citation Index-Expanded Web of Science database. The data source was studied and indexed by using bibliometric methodology. For visualized study, bibliographic coupling analysis, co-authorship analysis, co-citation analysis, co-occurrence analysis and the analysis of publication trends in subchondral bone research were conducted by VOS viewer and GraphPadPrism 5 software. RESULTS: A total of 4780 publications were included. There is an increasing trend of the relative research interests and number of publications per year globally. The cumulative number of publications about subchondral bone research followed the logistic growth model (Equation is included in full-text article.). The USA made the highest contributions to the global research with the most citations, the highest H-index, and the most total link strength, while Denmark had the highest average citation per item. The journal Osteoarthritis and Cartilage had the largest publication number. Boston University is the most contributive institution. Studies could be divided into 4 clusters: "Mechanism research", "Animal study", "Clinical study" and "Pathological features". Less efforts were put into clinical study. CONCLUSION: The number of publications about subchondral bone research would be increasing in the next years based on the current global trends. Attention should be drawn to the latest popular research, including "Mesenchymal stem-cells", "Autologous chondrocyte implantation", "Microfracture" and "Pain". Therefore, more and more efforts will be put into mechanism research on subchondral bone, which may inspire new clinical treatments for osteoarthritis and other related diseases based on subchondral bone.


Assuntos
Bibliometria , Pesquisa Biomédica/estatística & dados numéricos , Osso e Ossos/metabolismo , Cartilagem/metabolismo , Animais , Doenças Ósseas/metabolismo , Doenças das Cartilagens/metabolismo , Humanos
6.
PLoS Comput Biol ; 16(6): e1007998, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32584809

RESUMO

Post-traumatic osteoarthritis (PTOA) is associated with cartilage degradation, ultimately leading to disability and decrease of quality of life. Two key mechanisms have been suggested to occur in PTOA: tissue inflammation and abnormal biomechanical loading. Both mechanisms have been suggested to result in loss of cartilage proteoglycans, the source of tissue fixed charge density (FCD). In order to predict the simultaneous effect of these degrading mechanisms on FCD content, a computational model has been developed. We simulated spatial and temporal changes of FCD content in injured cartilage using a novel finite element model that incorporates (1) diffusion of the pro-inflammatory cytokine interleukin-1 into tissue, and (2) the effect of excessive levels of shear strain near chondral defects during physiologically relevant loading. Cytokine-induced biochemical cartilage explant degradation occurs near the sides, top, and lesion, consistent with the literature. In turn, biomechanically-driven FCD loss is predicted near the lesion, in accordance with experimental findings: regions near lesions showed significantly more FCD depletion compared to regions away from lesions (p<0.01). Combined biochemical and biomechanical degradation is found near the free surfaces and especially near the lesion, and the corresponding bulk FCD loss agrees with experiments. We suggest that the presence of lesions plays a role in cytokine diffusion-driven degradation, and also predisposes cartilage for further biomechanical degradation. Models considering both these cartilage degradation pathways concomitantly are promising in silico tools for predicting disease progression, recognizing lesions at high risk, simulating treatments, and ultimately optimizing treatments to postpone the development of PTOA.


Assuntos
Biofísica , Cartilagem/lesões , Cartilagem/metabolismo , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Estresse Mecânico , Animais , Humanos
7.
Life Sci ; 256: 117924, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32522568

RESUMO

AIMS: Osteoarthritis (OA) is a common degenerative joint disease characterized by cartilage degeneration and joint inflammation. As its pathogenesis remains unclear, there are no effective treatments established. Circular RNA (circRNA), microRNA (miRNA), and other noncoding RNAs participate in OA development; however, the effects and mechanisms of circRNA and miRNA in OA remain unknown. MAIN METHODS: Cartilage miRNA was examined in patients with and without OA. KEY FINDINGS: CircRNA-9119 and phosphatase and tensin homolog (PTEN) expression decreased in OA-affected cartilage and interleukin (IL)-1ß-induced chondrocytes, and miR-26a expression significantly decreased in normal cells and tissues. CircRNA-9119 overexpression restored chondrocyte growth, whereas IL-1ß treatment impaired chondrocyte growth. Annexin V-FITC & PI flow cytometry and Bcl-2/Bax ratio measurement indicated that the apoptosis of IL-1ß-treated articular chondrocytes was decreased by circRNA-9119 upregulation. Bioinformatic prediction and the dual-luciferase reporter assay indicated that circRNA-9119 served as a miR-26a sponge and that miR-26a targeted the 3'-UTR of PTEN. Transfection of chondrocytes with a circRNA-9119-overexpressing vector revealed downregulation of miR-26a expression. Furthermore, circRNA-9119 overexpression induced PTEN expression. In addition, a miR-26a mimic induced IL-1ß-induced chondrocyte apoptosis, and circRNA-9119 overexpression inhibited IL-1ß-induced chondrocyte apoptosis. SIGNIFICANCE: CircRNA-9119 is an important regulator of IL-1ß-treated chondrocytes through the miR-26a/PTEN axis, possibly contributing to OA development.


Assuntos
Condrócitos/metabolismo , Interleucina-1beta/metabolismo , Osteoartrite/tratamento farmacológico , Substâncias Protetoras/metabolismo , RNA Circular/metabolismo , Regiões 3' não Traduzidas , Apoptose/efeitos dos fármacos , Cartilagem/metabolismo , Cartilagem Articular/metabolismo , Linhagem Celular , Condrócitos/citologia , Regulação para Baixo , Matriz Extracelular/metabolismo , Humanos , MicroRNAs/genética , PTEN Fosfo-Hidrolase/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Regulação para Cima , Proteína X Associada a bcl-2/metabolismo
8.
PLoS One ; 15(5): e0232989, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32407402

RESUMO

Multi drug treatments are increasingly used in the clinic to combat complex and co-occurring diseases. However, most drug combination discovery efforts today are mainly focused on anticancer therapy and rarely examine the potential of using more than two drugs simultaneously. Moreover, there is currently no reported methodology for performing second- and higher-order drug combination analysis of secretomic patterns, meaning protein concentration profiles released by the cells. Here, we introduce COMBSecretomics (https://github.com/EffieChantzi/COMBSecretomics.git), the first pragmatic methodological framework designed to search exhaustively for second- and higher-order mixtures of candidate treatments that can modify, or even reverse malfunctioning secretomic patterns of human cells. This framework comes with two novel model-free combination analysis methods; a tailor-made generalization of the highest single agent principle and a data mining approach based on top-down hierarchical clustering. Quality control procedures to eliminate outliers and non-parametric statistics to quantify uncertainty in the results obtained are also included. COMBSecretomics is based on a standardized reproducible format and could be employed with any experimental platform that provides the required protein release data. Its practical use and functionality are demonstrated by means of a proof-of-principle pharmacological study related to cartilage degradation. COMBSecretomics is the first methodological framework reported to enable secretome-related second- and higher-order drug combination analysis. It could be used in drug discovery and development projects, clinical practice, as well as basic biological understanding of the largely unexplored changes in cell-cell communication that occurs due to disease and/or associated pharmacological treatment conditions.


Assuntos
Combinação de Medicamentos , Descoberta de Drogas/métodos , Metabolômica/métodos , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Simulação por Computador , Descoberta de Drogas/estatística & dados numéricos , Avaliação Pré-Clínica de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/estatística & dados numéricos , Humanos , Técnicas In Vitro , Metabolômica/estatística & dados numéricos , Modelos Biológicos , Osteoartrite/tratamento farmacológico , Osteoartrite/metabolismo , Proteômica/métodos , Proteômica/estatística & dados numéricos , Software
9.
Nat Methods ; 17(5): 531-540, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32371980

RESUMO

Single-molecule localization microscopy is a powerful tool for visualizing subcellular structures, interactions and protein functions in biological research. However, inhomogeneous refractive indices inside cells and tissues distort the fluorescent signal emitted from single-molecule probes, which rapidly degrades resolution with increasing depth. We propose a method that enables the construction of an in situ 3D response of single emitters directly from single-molecule blinking datasets, and therefore allows their locations to be pinpointed with precision that achieves the Cramér-Rao lower bound and uncompromised fidelity. We demonstrate this method, named in situ PSF retrieval (INSPR), across a range of cellular and tissue architectures, from mitochondrial networks and nuclear pores in mammalian cells to amyloid-ß plaques and dendrites in brain tissues and elastic fibers in developing cartilage of mice. This advancement expands the routine applicability of super-resolution microscopy from selected cellular targets near coverslips to intra- and extracellular targets deep inside tissues.


Assuntos
Encéfalo/metabolismo , Cartilagem/metabolismo , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Placa Amiloide/metabolismo , Imagem Individual de Molécula/métodos , Animais , Encéfalo/patologia , Cartilagem/patologia , Núcleo Celular/metabolismo , Células Cultivadas , Interpretação de Imagem Assistida por Computador/métodos , Masculino , Camundongos , Mitocôndrias/metabolismo , Imagem Molecular/métodos , Poro Nuclear/metabolismo , Placa Amiloide/patologia
10.
Gene ; 750: 144634, 2020 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-32240779

RESUMO

Lumbar degenerative disc disease (DDD) is a multifaceted progressive condition and often accompanied by disc herniation (DH) and/or degenerative spondylolisthesis (DS). Given the high prevalence of the disease (up to 20% according to some estimates) and the high costs associated with its care, there is a need to explore novel therapies such as regenerative medicine. Exploring these novel therapies first warrants investigation of molecular pathways underlying these disorders. Here, we show results from next generation RNA sequencing (RNA-seq) on mRNA isolated from 10 human nucleus pulposus (NP) samples of lumbar degenerated discs (DH and DS; n = 5 for each tissue) and other musculoskeletal tissues (Bone, cartilage, growth plate, and muscle; n = 7 for each tissue). Pathway and network analyses based on gene ontology (GO) terms were used to identify the biological functions of differentially expressed mRNAs. A total of 701 genes were found to be significantly upregulated in lumbar NP tissue compared to other musculoskeletal tissues. These differentially expressed mRNAs were primarily involved in DNA damage, immunity and G1/S transition of mitotic cell cycle. Interestingly, DH-specific signaling genes showed major network in chemotactic (e.g., CXCL10, CXCL11, IL1RL2 and IL6) and matrix-degrading pathway (e.g., MMP16, ADAMTSL1, 5, 8, 12, and 15), while DS-specific signaling genes were found to be those involved in cell adhesion (e.g., CDH1, EPHA1 and EFNA2) and inflammatory cytokines (e.g., CD19, CXCL5, CCL24, 25 and XCL2). Our findings provide new leads for therapeutic drug discovery that would permit optimization of medical or pharmacological intervention for cases of lumbar DDD.


Assuntos
Degeneração do Disco Intervertebral/genética , Deslocamento do Disco Intervertebral/genética , Espondilolistese/genética , Adulto , Cartilagem/metabolismo , Citocinas/metabolismo , Feminino , Ontologia Genética , Redes Reguladoras de Genes/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Disco Intervertebral/metabolismo , Vértebras Lombares/metabolismo , Masculino , Núcleo Pulposo/metabolismo , RNA Mensageiro/metabolismo
11.
Life Sci ; 253: 117636, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32251631

RESUMO

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


Assuntos
Fatores de Transcrição ARNTL/metabolismo , Desenvolvimento Ósseo/efeitos dos fármacos , Fatores de Transcrição ARNTL/genética , Animais , Densidade Óssea/efeitos dos fármacos , Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Cartilagem/metabolismo , Diferenciação Celular , Condrócitos/metabolismo , Condrogênese/efeitos dos fármacos , Relógios Circadianos/genética , Regulação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese , Dente/metabolismo
12.
Ann Agric Environ Med ; 27(1): 66-75, 2020 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-32208582

RESUMO

OBJECTIVE: The aim of the study was to determine the effect of nesfatin-1 on bone properties in female rats in the conditions of developing osteopenia induced by ovariectomy (OVX). MATERIAL AND METHODS: The experiment was performed on 21 female Wistar rats assigned to 3 groups receiving intraperitoneally physiological saline (SHO, OVX-PhS) and nesfatin-1 in dose 2 µg/kg BW of (OVX-NES) once a day for 8 wks. At the end of the experiment, the rats were scanned using the DXA method to determine the body composition, tBMC, and tBMD. The isolated femora and tibia were tested with the DXA method for BMD and BMC, and with the pQCT method for separate analysis of the cortical and trabecular bone tissue. The bone strength parameters were also determined. The immunohistochemical method was used for determination of nesfatin-1 localization in growth cartilage. Bone metabolism markers (osteocalcin, bALP, and NTx) were identified using an ELISA kit. RESULTS: OVX exerts a negative effect on bone tissue. The nesfatin-1 administration influenced positively the DXA parameters of tibia. TvBMD and TbvBMD measured by pQCT in metaphysis of bones were significantly higher in the OVX-NES group than in OVX-PhS. No differences were found in the values of bone strength parameters between SHO and OVX-NES females. Extra- and intracellular immunohistochemical reaction for nesfatin-1 was observed in all zones of growth cartilage, with the strongest reaction detected in the calcifying zone. Nesfatin-1 administration caused a significant increase in the osteocalcin and bALP concentration in relation to the OVX-PhS animals. CONCLUSIONS: The results of the experiment indicate that nesfatin-1 exerts a protective effect on bone tissue properties and can be used in the prevention of osteoporosis.


Assuntos
Densidade Óssea/efeitos dos fármacos , Doenças Ósseas Metabólicas/tratamento farmacológico , Nucleobindinas/farmacologia , Absorciometria de Fóton , Fosfatase Alcalina/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Doenças Ósseas Metabólicas/diagnóstico por imagem , Doenças Ósseas Metabólicas/metabolismo , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Feminino , Fêmur/diagnóstico por imagem , Fêmur/efeitos dos fármacos , Osteocalcina/metabolismo , Ovariectomia , Ratos Wistar , Tíbia/diagnóstico por imagem , Tíbia/efeitos dos fármacos
13.
Cell Prolif ; 53(3): e12765, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32034931

RESUMO

Cartilage is a connective tissue in the skeletal system and has limited regeneration ability and unique biomechanical reactivity. The growth and development of cartilage can be affected by different physical, chemical and biological factors, such as mechanical stress, inflammation, osmotic pressure, hypoxia and signalling transduction. Primary cilia are multifunctional sensory organelles that regulate diverse signalling transduction and cell activities. They are crucial for the regulation of cartilage development and act in a variety of ways, such as react to mechanical stress, mediate signalling transduction, regulate cartilage-related diseases progression and affect cartilage tumorigenesis. Therefore, research on primary cilia-mediated cartilage growth and development is currently extremely popular. This review outlines the role of primary cilia in cartilage development in recent years and elaborates on the potential regulatory mechanisms from different aspects.


Assuntos
Cartilagem/crescimento & desenvolvimento , Condrogênese , Cílios/metabolismo , Transdução de Sinais , Animais , Fenômenos Biomecânicos , Cartilagem/metabolismo , Cartilagem/ultraestrutura , Cílios/ultraestrutura , Humanos , Mecanotransdução Celular , Osteogênese
14.
Biomater Sci ; 8(6): 1711-1725, 2020 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-31994552

RESUMO

The encapsulation of cells into microgels is attractive for applications in tissue regeneration. While cells are protected against shear stress during injection, the assembly of microgels after injection into a tissue defect also forms a macroporous scaffold that allows effective nutrient transport throughout the construct. However, in most of current strategies that form microgel-based macroporous scaffold or higher-order structures, cells are seeded during or post the assembly process and not microencapsulated in situ. The objective of this study is to investigate the chondrogenic phenotype of microencapsulated fetal chondrocytes in a biocompatible, assembled microgel system vs. bulk gels and to test the stability of the constructs in vivo. Here, we demonstrate that cell microencapsulation leads to increased expression of cartilage-specific genes in a TGF-ß1-dependent manner. This correlates, as shown by histological staining, with the ability of microencapsulated cells to deposit cartilaginous matrix after migrating to the surface of the microgels, while keeping a macroscopic granular morphology. Implantation of precultured scaffolds in a subcutaneous mouse model results in vessel infiltration in bulk gels but not in assembled microgels, suggesting a higher stability of the matrix produced by the cells in the assembled microgel constructs. The cells are able to remodel the microgels as demonstrated by the gradual disappearance of the granular structure in vivo. The biocompatible microencapsulation and microgel assembly system presented in this article therefore hold great promise as an injectable system for cartilage repair.


Assuntos
Cartilagem/metabolismo , Encapsulamento de Células/métodos , Condrócitos/citologia , Condrogênese , Animais , Linhagem Celular , Condrócitos/metabolismo , Humanos , Camundongos , Modelos Animais , Transdução de Sinais , Tecidos Suporte , Fator de Crescimento Transformador beta1/metabolismo
15.
Int J Mol Sci ; 21(1)2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31935848

RESUMO

Aging and injury are two major risk factors for osteoarthritis (OA). Yet, very little is known about how aging and injury interact and contribute to OA pathogenesis. In the present study, we examined age- and injury-related molecular changes in mouse knee joints that could contribute to OA. Using RNA-seq, first we profiled the knee joint transcriptome of 10-week-old, 62-week-old, and 95-week-old mice and found that the expression of several inflammatory-response related genes increased as a result of aging, whereas the expression of several genes involved in cartilage metabolism decreased with age. To determine how aging impacts post-traumatic arthritis (PTOA) development, the right knee joints of 10-week-old and 62-week-old mice were injured using a non-invasive tibial compression injury model and injury-induced structural and molecular changes were assessed. At six-week post-injury, 62-week-old mice displayed significantly more cartilage degeneration and osteophyte formation compared with young mice. Although both age groups elicited similar transcriptional responses to injury, 62-week-old mice had higher activation of inflammatory cytokines than 10-week-old mice, whereas cartilage/bone metabolism genes had higher expression in 10-week-old mice, suggesting that the differential expression of these genes might contribute to the differences in PTOA severity observed between these age groups.


Assuntos
Envelhecimento/genética , Traumatismos do Joelho/complicações , Osteoartrite do Joelho/genética , Transcriptoma , Envelhecimento/metabolismo , Animais , Cartilagem/crescimento & desenvolvimento , Cartilagem/metabolismo , Células Cultivadas , Articulação do Joelho/crescimento & desenvolvimento , Articulação do Joelho/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoartrite do Joelho/etiologia , Osteoartrite do Joelho/metabolismo , RNA-Seq , Análise de Célula Única
16.
Magn Reson Chem ; 58(1): 65-76, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31323132

RESUMO

Cartilage-forming lesions include tumours that can vary in severity from benign enchondromas to high-grade malignant chondrosarcomas. Chondrosarcoma is the second most frequent malignant bone tumour, accounting for 20-30% of all malignant bone neoplasms. Surgery is the standard treatment for cartilage tumours (CTs); however, their incidental diagnosis and the difficult differentiation of low-grade lesions like chondrosarcoma grade I from benign entities like enchondroma are challenges for clinical management. In this sense, the search for circulating biomarkers for early detection and prognosis is an ongoing interest. Targeted metabolomics is a powerful tool that can propose potential biomarkers in biological fluids as well as help to discover disturbed metabolic pathways to reveal tumour pathogenesis. In this context, the aim of this study was to investigate the 1 H nuclear magnetic resonance metabolomic serum profile of patients with CTs contrasted with healthy controls. Forty-one metabolites were identified and quantified; the multivariate statistical methods principal component analysis and partial least squares discriminant analysis reveal a clear separation of the CT group, that is, the differential metabolites that were involved in two main metabolic pathways: the taurine and hypotaurine metabolism and synthesis and degradation of ketone bodies. Our results represent preliminary work for emergent serum-based diagnostics or prognostic methods for patients with chondrogenic tumours.


Assuntos
Biomarcadores Tumorais/sangue , Cartilagem/metabolismo , Condrossarcoma/diagnóstico , Espectroscopia de Ressonância Magnética/métodos , Soro/química , Adulto , Idoso , Condroma/metabolismo , Análise Discriminante , Feminino , Humanos , Masculino , Redes e Vias Metabólicas , Metabolômica/métodos , Pessoa de Meia-Idade , Simulação de Dinâmica Molecular , Análise Multivariada , Estadiamento de Neoplasias/métodos , Projetos Piloto , Soro/metabolismo
17.
Mater Sci Eng C Mater Biol Appl ; 107: 110291, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31761240

RESUMO

Electrospinning is a valuable technology for cartilage tissue engineering (CTE) due to its ability to produce fibrous scaffolds mimicking the nanoscale and alignment of collagen fibers present within the superficial zone of articular cartilage. Coaxial electrospinning allows the fabrication of core-shell fibers able to incorporate and release bioactive molecules (e.g., drugs or growth factors) in a controlled manner. Herein, we used coaxial electrospinning to produce coaxial poly(glycerol sebacate) (PGS)/poly(caprolactone) (PCL) aligned nanofibers (core:PGS/shell:PCL). The obtained scaffolds were characterized in terms of their structure, chemical composition, thermal properties, mechanical performance and in vitro degradation kinetics, in comparison to monoaxial PCL aligned fibers and respective non-aligned controls. All the electrospun scaffolds produced presented average fiber diameters within the nanometer-scale and the core-shell structure of the composite fibers was clearly confirmed by TEM. Additionally, fiber alignment significantly increased (>2-fold) the elastic modulus of both coaxial and monoxial scaffolds. Kartogenin (KGN), a small molecule known to promote mesenchymal stem/stromal cells (MSC) chondrogenesis, was loaded into the core PGS solution to generate coaxial PGS-KGN/PCL nanofibers. The KGN release kinetics and scaffold biological performance were evaluated in comparison to KGN-loaded monoaxial fibers and respective non-loaded controls. Coaxial PGS-KGN/PCL nanofibers showed a more controlled and sustained KGN release over 21 days than monoaxial PCL-KGN nanofibers. When cultured with human bone marrow MSC in incomplete chondrogenic medium (without TGF-ß3), KGN-loaded scaffolds enhanced significantly cell proliferation and chondrogenic differentiation, as suggested by the increased sGAG amounts and chondrogenic markers gene expression levels. Overall, these findings highlight the potential of using coaxial PGS-KGN/PCL aligned nanofibers as a bioactive scaffold for CTE applications.


Assuntos
Anilidas , Cartilagem , Nanofibras/química , Ácidos Ftálicos , Engenharia Tecidual , Tecidos Suporte , Anilidas/química , Anilidas/metabolismo , Anilidas/farmacocinética , Anilidas/farmacologia , Cartilagem/citologia , Cartilagem/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Decanoatos/química , Técnicas Eletroquímicas , Desenho de Equipamento , Glicerol/análogos & derivados , Glicerol/química , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Ácidos Ftálicos/química , Ácidos Ftálicos/metabolismo , Ácidos Ftálicos/farmacocinética , Ácidos Ftálicos/farmacologia , Poliésteres/química , Polímeros/química , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos
18.
Methods Mol Biol ; 2043: 75-91, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31463904

RESUMO

A disintegrin-like and metalloproteinase with thrombospondin type-1 motifs-4 (ADAMTS-4) and ADAMTS-5 are zinc-dependent metalloproteinases that are involved in the maintenance of cartilage extracellular matrix (ECM) and are currently considered the major aggrecanases in the development of osteoarthritis. In this chapter we describe the establishment and cultivation of cell lines expressing ADAMTS-4,-5 and their domain deletion mutants; the collection of medium containing expressed ADAMTS-4,-5; the subsequent purification of this medium through anti-FLAG affinity chromatography; and the characterization of ADAMTS-4,-5 activity using synthetic Förster resonance energy transfer (FRET) peptide substrates.


Assuntos
Proteína ADAMTS4/química , Proteína ADAMTS4/metabolismo , Proteína ADAMTS5/química , Proteína ADAMTS5/metabolismo , Mutação , Proteína ADAMTS4/genética , Proteína ADAMTS5/genética , Cartilagem/metabolismo , Domínio Catalítico , Técnicas de Cultura de Células/métodos , Cromatografia de Afinidade , Meios de Cultura/química , Matriz Extracelular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Domínios Proteicos
19.
J Cell Physiol ; 235(2): 979-992, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31267533

RESUMO

This study is carried out to investigate the role of microRNA-26a (miR-26a) in cartilage injury and chondrocyte proliferation and apoptosis in rats with rheumatoid arthritis (RA) by regulating expression of CTGF. A rat model of RA induced by type II collagen was established. The rats were assigned into normal, RA, RA + mimics negative control (NC), and RA + miR-26a mimics groups, and the cells were classified into blank, mimics NC, and miR-26a mimics groups. The degree of secondary joint swelling and arthritis index, expression of miR-26a, pathological changes, proliferation and apoptosis of chondrocytes, and expression of CTGF, interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor-α, Bax, and Bcl-2 were also determined through a series of experiments. The targeting relationship between miR-26a and CTGF was verified. Initially, downregulated miR-26a was found in cartilage tissues and inflammatory articular chondrocytes of RA rats. In addition, CTGF was determined as a direct target gene of miR-26a, and upregulation of miR-26a inhibited CTGF expression in cartilage tissues of RA rats. Furthermore, upregulation of miR-26a reduced swelling and inflammation of joints, inhibited cartilage damage, apoptosis of chondrocytes, inflammatory injury, promotes proliferation, and inhibited apoptosis of inflammatory articular chondrocytes, which may be correlated with the targeting inhibition of CTGF expression. Collectively, the results demonstrate that upregulating the expression of miR-26a could attenuate cartilage injury, stimulate the proliferation, and inhibit apoptosis of chondrocytes in RA rats.


Assuntos
Artrite Reumatoide/induzido quimicamente , Cartilagem/lesões , Proliferação de Células/fisiologia , Condrócitos/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , MicroRNAs/metabolismo , Animais , Apoptose/fisiologia , Cartilagem/metabolismo , Colágeno Tipo II/toxicidade , Fator de Crescimento do Tecido Conjuntivo/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Ratos , Ratos Sprague-Dawley
20.
Dis Model Mech ; 12(12)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848143

RESUMO

Idiopathic scoliosis (IS) is the most common type of musculoskeletal defect affecting children worldwide, and is classified by age of onset, location and degree of spine curvature. Although rare, IS with onset during infancy is the more severe and rapidly progressive form of the disease, associated with increased mortality due to significant respiratory compromise. The pathophysiology of IS, in particular for infantile IS, remains elusive. Here, we demonstrate the role of PRMT5 in the infantile IS phenotype in mouse. Conditional genetic ablation of PRMT5 in osteochondral progenitors results in impaired terminal hypertrophic chondrocyte differentiation and asymmetric defects of endochondral bone formation in the perinatal spine. Analysis of these several markers of endochondral ossification revealed increased type X collagen (COLX) and Ihh expression, coupled with a dramatic reduction in Mmp13 and RUNX2 expression, in the vertebral growth plate and in regions of the intervertebral disc in the Prmt5 conditional mutant mice. We also demonstrate that PRMT5 has a continuous role in the intervertebral disc and vertebral growth plate in adult mice. Altogether, our results establish PRMT5 as a critical promoter of terminal hypertrophic chondrocyte differentiation and endochondral bone formation during spine development and homeostasis.This article has an associated First Person interview with the first author of the paper.


Assuntos
Proteína Morfogenética Óssea 4/genética , Condrócitos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Escoliose/genética , Alelos , Animais , Proteína Morfogenética Óssea 4/metabolismo , Cartilagem/metabolismo , Diferenciação Celular , Proliferação de Células , Condrogênese , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Cruzamentos Genéticos , Deleção de Genes , Proteínas Hedgehog/metabolismo , Homeostase , Metaloproteinase 13 da Matriz/metabolismo , Camundongos , Mutação , Osteogênese/genética , Fenótipo , Regiões Promotoras Genéticas , Células-Tronco/citologia
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