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1.
Nat Biomed Eng ; 5(8): 926-940, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34373601

RESUMO

Current protocols for the differentiation of human pluripotent stem cells (hPSCs) into chondrocytes do not allow for the expansion of intermediate progenitors so as to prospectively assess their chondrogenic potential. Here we report a protocol that leverages PRRX1-tdTomato reporter hPSCs for the selective induction of expandable and ontogenetically defined PRRX1+ limb-bud-like mesenchymal cells under defined xeno-free conditions, and the prospective assessment of the cells' chondrogenic potential via the cell-surface markers CD90, CD140B and CD82. The cells, which proliferated stably and exhibited the potential to undergo chondrogenic differentiation, formed hyaline cartilaginous-like tissue commensurate to their PRRX1-expression levels. Moreover, we show that limb-bud-like mesenchymal cells derived from patient-derived induced hPSCs can be used to identify therapeutic candidates for type II collagenopathy and we developed a method to generate uniformly sized hyaline cartilaginous-like particles by plating the cells on culture dishes coated with spots of a zwitterionic polymer. PRRX1+ limb-bud-like mesenchymal cells could facilitate the mass production of chondrocytes and cartilaginous tissues for applications in drug screening and tissue engineering.


Assuntos
Proteínas de Homeodomínio/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Pluripotentes/citologia , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Condrócitos/citologia , Condrócitos/metabolismo , Condrócitos/transplante , Condrogênese , Doenças do Colágeno/terapia , Meios de Cultura/química , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Pluripotentes/metabolismo , Polímeros/química , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Antígenos Thy-1/metabolismo , Engenharia Tecidual
2.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445320

RESUMO

Meniscus injuries can be highly debilitating and lead to knee osteoarthritis. Progenitor cells from the meniscus could be a superior cell type for meniscus repair and tissue-engineering. The purpose of this study is to characterize meniscus progenitor cells isolated by differential adhesion to fibronectin (FN-prog). Human osteoarthritic menisci were digested, and FN-prog were selected by differential adhesion to fibronectin. Multilineage differentiation, population doubling time, colony formation, and MSC surface markers were assessed in the FN-prog and the total meniscus population (Men). Colony formation was compared between outer and inner zone meniscus digest. Chondrogenic pellet cultures were performed for redifferentiation. FN-prog demonstrated multipotency. The outer zone FN-prog formed more colonies than the inner zone FN-prog. FN-prog displayed more colony formation and a higher proliferation rate than Men. FN-prog redifferentiated in pellet culture and mostly adhered to the MSC surface marker profile, except for HLA-DR receptor expression. This is the first study that demonstrates differential adhesion to fibronectin for the isolation of a progenitor-like population from the meniscus. The high proliferation rates and ability to form meniscus extracellular matrix upon redifferentiation, together with the broad availability of osteoarthritis meniscus tissue, make FN-prog a promising cell type for clinical translation in meniscus tissue-engineering.


Assuntos
Adesão Celular , Fibronectinas/metabolismo , Menisco/citologia , Células-Tronco Mesenquimais/citologia , Engenharia Tecidual/métodos , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Condrócitos/fisiologia , Condrogênese , Humanos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Pessoa de Meia-Idade , Tecidos Suporte/química
3.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360718

RESUMO

Besides the direct effects of radiations, indirect effects are observed within the surrounding non-irradiated area; irradiated cells relay stress signals in this close proximity, inducing the so-called radiation-induced bystander effect. These signals received by neighboring unirradiated cells induce specific responses similar with those of direct irradiated cells. To understand the cellular response of bystander cells, we performed a 2D gel-based proteomic study of the chondrocytes receiving the conditioned medium of low-dose irradiated chondrosarcoma cells. The conditioned medium was directly analyzed by mass spectrometry in order to identify candidate bystander factors involved in the signal transmission. The proteomic analysis of the bystander chondrocytes highlighted 20 proteins spots that were significantly modified at low dose, implicating several cellular mechanisms, such as oxidative stress responses, cellular motility, and exosomes pathways. In addition, the secretomic analysis revealed that the abundance of 40 proteins in the conditioned medium of 0.1 Gy irradiated chondrosarcoma cells was significantly modified, as compared with the conditioned medium of non-irradiated cells. A large cluster of proteins involved in stress granules and several proteins involved in the cellular response to DNA damage stimuli were increased in the 0.1 Gy condition. Several of these candidates and cellular mechanisms were confirmed by functional analysis, such as 8-oxodG quantification, western blot, and wound-healing migration tests. Taken together, these results shed new lights on the complexity of the radiation-induced bystander effects and the large variety of the cellular and molecular mechanisms involved, including the identification of a new potential actor, namely the stress granules.


Assuntos
Neoplasias Ósseas/metabolismo , Efeito Espectador/efeitos da radiação , Condrócitos/metabolismo , Condrossarcoma/metabolismo , Grânulos Citoplasmáticos/metabolismo , Proteômica , Raios X , Neoplasias Ósseas/radioterapia , Linhagem Celular Tumoral , Condrossarcoma/radioterapia , Humanos
4.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208464

RESUMO

Piezo channels are mechanosensitive ion channels located in the cell membrane and function as key cellular mechanotransducers for converting mechanical stimuli into electrochemical signals. Emerged as key molecular detectors of mechanical forces, Piezo channels' functions in bone have attracted more and more attention. Here, we summarize the current knowledge of Piezo channels and review the research advances of Piezo channels' function in bone by highlighting Piezo1's role in bone cells, including osteocyte, bone marrow mesenchymal stem cell (BM-MSC), osteoblast, osteoclast, and chondrocyte. Moreover, the role of Piezo channels in bone diseases is summarized.


Assuntos
Osso e Ossos/metabolismo , Canais Iônicos/fisiologia , Animais , Doenças Ósseas , Condrócitos/metabolismo , Suscetibilidade a Doenças , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos/agonistas , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/química , Mecanotransdução Celular , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteócitos/metabolismo , Relação Estrutura-Atividade
5.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208590

RESUMO

Osteoarthritis (OA) is the most common musculoskeletal disorder causing a great disability and a reduction in the quality of life. In OA, articular chondrocytes (AC) and synovial fibroblasts (SF) release innate-derived immune mediators that initiate and perpetuate inflammation, inducing cartilage extracellular matrix (ECM) degradation. Given the lack of therapies for the treatment of OA, in this study, we explore biomarkers that enable the development of new therapeutical approaches. We analyze the set of secreted proteins in AC and SF co-cultures by stable isotope labeling with amino acids (SILAC). We describe, for the first time, 115 proteins detected in SF-AC co-cultures stimulated by fibronectin fragments (Fn-fs). We also study the role of the vasoactive intestinal peptide (VIP) in this secretome, providing new proteins involved in the main events of OA, confirmed by ELISA and multiplex analyses. VIP decreases proteins involved in the inflammatory process (CHI3L1, PTX3), complement activation (C1r, C3), and cartilage ECM degradation (DCN, CTSB and MMP2), key events in the initiation and progression of OA. Our results support the anti-inflammatory and anti-catabolic properties of VIP in rheumatic diseases and provide potential new targets for OA treatment.


Assuntos
Condrócitos/metabolismo , Fibroblastos/metabolismo , Osteoartrite/metabolismo , Proteoma , Proteômica , Membrana Sinovial/citologia , Peptídeo Intestinal Vasoativo/metabolismo , Biomarcadores , Condrócitos/efeitos dos fármacos , Técnicas de Cocultura , Citocinas/metabolismo , Suscetibilidade a Doenças , Matriz Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Osteoartrite/etiologia , Osteoartrite/patologia , Proteômica/métodos , Peptídeo Intestinal Vasoativo/farmacologia
6.
Int J Mol Sci ; 22(14)2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34299264

RESUMO

Inflammation has a fundamental impact on the pathophysiology of osteoarthritis (OA), a common form of degenerative arthritis. It has previously been established that curcumin, a component of turmeric (Curcuma longa), has anti-inflammatory properties. This research evaluates the potentials of curcumin on the pathophysiology of OA in vitro. To explore the anti-inflammatory efficacy of curcumin in an inflamed joint, an osteoarthritic environment (OA-EN) model consisting of fibroblasts, T-lymphocytes, 3D-chondrocytes is constructed and co-incubated with TNF-α, antisense oligonucleotides targeting NF-kB (ASO-NF-kB), or an IkB-kinase (IKK) inhibitor (BMS-345541). Our results show that OA-EN, similar to TNF-α, suppresses chondrocyte viability, which is accompanied by a significant decrease in cartilage-specific proteins (collagen II, CSPG, Sox9) and an increase in NF-kB-driven gene proteins participating in inflammation, apoptosis, and breakdown (NF-kB, MMP-9, Cox-2, Caspase-3). Conversely, similar to knockdown of NF-kB at the mRNA level or at the IKK level, curcumin suppresses NF-kB activation, NF-kB-promotes gene proteins derived from the OA-EN, and stimulates collagen II, CSPG, and Sox9 expression. Furthermore, co-immunoprecipitation assay shows that curcumin reduces OA-EN-mediated inflammation and chondrocyte apoptosis, with concomitant chondroprotective effects, due to modulation of Sox-9/NF-kB signaling axis. Finally, curcumin selectively hinders the interaction of p-NF-kB-p65 directly with DNA-this association is disrupted through DTT. These results suggest that curcumin suppresses inflammation in OA-EN via modulating NF-kB-Sox9 coupling and is essential for maintaining homeostasis in OA by balancing chondrocyte survival and inflammatory responses. This may contribute to the alternative treatment of OA with respect to the efficacy of curcumin.


Assuntos
Curcumina/farmacologia , Osteoartrite/tratamento farmacológico , Osteoartrite/metabolismo , Apoptose/efeitos dos fármacos , Cartilagem/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Curcuma/metabolismo , Curcumina/metabolismo , Ciclo-Oxigenase 2/metabolismo , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/metabolismo , Imidazóis/farmacologia , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Interleucina-1beta/metabolismo , NF-kappa B/metabolismo , Osteoartrite/fisiopatologia , Cultura Primária de Células , Quinoxalinas/farmacologia , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo
7.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201564

RESUMO

Obesity increases the risk of hip osteoarthritis (OA). Recent studies have shown that adipokine extracellular nicotinamide phosphoribosyltransferase (eNAMPT or visfatin) induces the production of IL-6 and matrix metalloproteases (MMPs) in chondrocytes, suggesting it may promote articular cartilage degradation. However, neither the functional effects of extracellular visfatin on human articular cartilage tissue, nor its expression in the joint of hip OA patients of varying BMI, have been reported. Hip OA joint tissues were collected from patients undergoing joint replacement surgery. Cartilage explants were stimulated with recombinant human visfatin. Pro-inflammatory cytokines and MMPs were measured by ELISA and Luminex. Localisation of visfatin expression in cartilage tissue was determined by immunohistochemistry. Cartilage matrix degradation was determined by quantifying proteoglycan release. Expression of visfatin was elevated in the synovial tissue of hip OA patients who were obese, and was co-localised with MMP-13 in areas of cartilage damage. Visfatin promoted the degradation of hip OA cartilage proteoglycan and induced the production of pro-inflammatory cytokines (IL-6, MCP-1, CCL20, and CCL4) and MMPs. The elevated expression of visfatin in the obese hip OA joint, and its functional effects on hip cartilage tissue, suggests it plays a central role in the loss of cartilage integrity in obese patients with hip OA.


Assuntos
Cartilagem Articular/patologia , Citocinas/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Osteoartrite do Quadril/metabolismo , Idoso , Idoso de 80 Anos ou mais , Cartilagem Articular/metabolismo , Quimiocinas/metabolismo , Condrócitos/metabolismo , Citocinas/sangue , Articulação do Quadril/metabolismo , Articulação do Quadril/fisiopatologia , Humanos , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinases da Matriz/metabolismo , Pessoa de Meia-Idade , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/sangue , Obesidade/metabolismo , Técnicas de Cultura de Órgãos , Osteoartrite do Quadril/patologia , Proteoglicanas/metabolismo
8.
Int J Mol Sci ; 22(13)2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34281278

RESUMO

Osteoarthritis (OA) is hallmarked by a progressive degradation of articular cartilage. One major driver of OA is inflammation, in which cytokines such as IL-6, TNF-α and IL-1ß are secreted by activated chondrocytes, as well as synovial cells-including macrophages. Intra-articular injection of blood products-such as citrate-anticoagulated plasma (CPRP), hyperacute serum (hypACT), and extracellular vesicles (EVs) isolated from blood products-is gaining increasing importance in regenerative medicine for the treatment of OA. A co-culture system of primary OA chondrocytes and activated M1 macrophages was developed to model an OA joint in order to observe the effects of EVs in modulating the inflammatory environment. Primary OA chondrocytes were obtained from patients undergoing total knee replacement. Primary monocytes obtained from voluntary healthy donors and the monocytic cell line THP-1 were differentiated and activated into proinflammatory M1 macrophages. EVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis and Western blot. Gene expression analysis of chondrocytes by RT-qPCR revealed increased type II collagen expression, while cytokine profiling via ELISA showed lower TNF-α and IL-1ß levels associated with EV treatment. In conclusion, the inflammation model provides an accessible tool to investigate the effects of blood products and EVs in the inflammatory context of OA.


Assuntos
Condrócitos/imunologia , Vesículas Extracelulares/imunologia , Osteoartrite/terapia , Condrócitos/metabolismo , Técnicas de Cocultura , Feminino , Perfilação da Expressão Gênica , Humanos , Inflamação/imunologia , Inflamação/terapia , Injeções Intra-Articulares , Interleucina-1beta/metabolismo , Masculino , Modelos Biológicos , Monócitos/imunologia , Osteoartrite/genética , Osteoartrite/imunologia , Medicina Regenerativa/métodos , Fator de Necrose Tumoral alfa/metabolismo
9.
Int J Mol Sci ; 22(13)2021 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-34199089

RESUMO

The meniscus possesses low self-healing properties. A perfect regenerative technique for this tissue has not yet been developed. This work aims to evaluate the role of hypoxia in meniscal development in vitro. Menisci from neonatal pigs (day 0) were harvested and cultured under two different atmospheric conditions: hypoxia (1% O2) and normoxia (21% O2) for up to 14 days. Samples were analysed at 0, 7 and 14 days by histochemical (Safranin-O staining), immunofluorescence and RT-PCR (in both methods for SOX-9, HIF-1α, collagen I and II), and biochemical (DNA, GAGs, DNA/GAGs ratio) techniques to record any possible differences in the maturation of meniscal cells. Safranin-O staining showed increments in matrix deposition and round-shape "fibro-chondrocytic" cells in hypoxia-cultured menisci compared with controls under normal atmospheric conditions. The same maturation shifting was observed by immunofluorescence and RT-PCR analysis: SOX-9 and collagen II increased from day zero up to 14 days under a hypoxic environment. An increment of DNA/GAGs ratio typical of mature meniscal tissue (characterized by fewer cells and more GAGs) was observed by biochemical analysis. This study shows that hypoxia can be considered as a booster to achieve meniscal cell maturation, and opens new opportunities in the field of meniscus tissue engineering.


Assuntos
Diferenciação Celular , Hipóxia/metabolismo , Menisco/citologia , Menisco/metabolismo , Animais , Biomarcadores , Células Cultivadas , Condrócitos/metabolismo , Expressão Gênica , Glicosaminoglicanos/metabolismo , Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Suínos , Engenharia Tecidual/métodos
10.
Nat Commun ; 12(1): 4148, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230481

RESUMO

Osteoarthritis (OA), the most common aging-related joint disease, is caused by an imbalance between extracellular matrix synthesis and degradation. Here, we discover that both strands of microRNA-455 (miR-455), -5p and -3p, are up-regulated by Sox9, an essential transcription factor for cartilage differentiation and function. Both miR-455-5p and -3p are highly expressed in human chondrocytes from normal articular cartilage and in mouse primary chondrocytes. We generate miR-455 knockout mice, and find that cartilage degeneration mimicking OA and elevated expression of cartilage degeneration-related genes are observed at 6-months-old. Using a cell-based miRNA target screening system, we identify hypoxia-inducible factor-2α (HIF-2α), a catabolic factor for cartilage homeostasis, as a direct target of both miR-455-5p and -3p. In addition, overexpression of both miR-455-5p and -3p protect cartilage degeneration in a mouse OA model, demonstrating their potential therapeutic value. Furthermore, knockdown of HIF-2α in 6-month-old miR-455 knockout cartilage rescues the elevated expression of cartilage degeneration-related genes. These data demonstrate that both strands of a miRNA target the same gene to regulate articular cartilage homeostasis.


Assuntos
Cartilagem/metabolismo , Homeostase , Hipóxia/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Fatores de Transcrição/metabolismo , Animais , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Osteoartrite/genética , Fatores de Transcrição SOX9
11.
Int J Mol Sci ; 22(14)2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34298911

RESUMO

Osteoarthritis (OA) is the most common articular degenerative disease characterized by chronic pain, joint inflammation, and movement limitations, which are significantly influenced by aberrant epigenetic modifications of numerous OA-susceptible genes. Recent studies revealed that both the abnormal activation and differential expression of histone deacetylases (HDACs) might contribute to OA pathogenesis. In this study, we investigated the chondroprotective effects of a marine-derived HDAC inhibitor, panobinostat, on anterior cruciate ligament transection (ACLT)-induced experimental OA rats. The intra-articular administration of 2 or 10 µg of panobinostat (each group, n = 7) per week from the 6th to 17th week attenuates ACLT-induced nociceptive behaviors, including secondary mechanical allodynia and weight-bearing distribution. Histopathological and microcomputed tomography analysis showed that panobinostat significantly prevents cartilage degeneration after ACLT. Moreover, intra-articular panobinostat exerts hypertrophic effects in the chondrocytes of articular cartilage by regulating the protein expressions of HDAC4, HDAC6, HDAC7, runt-domain transcription factor-2, and matrix metalloproteinase-13. The study indicated that HDACs might have different modulations on the chondrocyte phenotype in the early stages of OA development. These results provide new evidence that panobinostat may be a potential therapeutic drug for OA.


Assuntos
Ligamento Cruzado Anterior/efeitos dos fármacos , Cartilagem Articular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Osteoartrite do Joelho/tratamento farmacológico , Dor/tratamento farmacológico , Panobinostat/farmacologia , Animais , Ligamento Cruzado Anterior/metabolismo , Lesões do Ligamento Cruzado Anterior/tratamento farmacológico , Lesões do Ligamento Cruzado Anterior/metabolismo , Doenças das Cartilagens/tratamento farmacológico , Doenças das Cartilagens/metabolismo , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Modelos Animais de Doenças , Masculino , Osteoartrite do Joelho/metabolismo , Dor/metabolismo , Ratos , Ratos Wistar , Suporte de Carga
12.
Int J Mol Sci ; 22(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34298867

RESUMO

The hexosamine biosynthetic pathway (HBP) is essential for the production of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), the building block of glycosaminoglycans, thus playing a crucial role in cartilage anabolism. Although O-GlcNAcylation represents a protective regulatory mechanism in cellular processes, it has been associated with degenerative diseases, including osteoarthritis (OA). The present study focuses on HBP-related processes as potential therapeutic targets after cartilage trauma. Human cartilage explants were traumatized and treated with GlcNAc or glucosamine sulfate (GS); PUGNAc, an inhibitor of O-GlcNAcase; or azaserine (AZA), an inhibitor of GFAT-1. After 7 days, cell viability and gene expression analysis of anabolic and catabolic markers, as well as HBP-related enzymes, were performed. Moreover, expression of catabolic enzymes and type II collagen (COL2) biosynthesis were determined. Proteoglycan content was assessed after 14 days. Cartilage trauma led to a dysbalanced expression of different HBP-related enzymes, comparable to the situation in highly degenerated tissue. While GlcNAc and PUGNAc resulted in significant cell protection after trauma, only PUGNAc increased COL2 biosynthesis. Moreover, PUGNAc and both glucosamine derivatives had anti-catabolic effects. In contrast, AZA increased catabolic processes. Overall, "fueling" the HBP by means of glucosamine derivatives or inhibition of deglycosylation turned out as cells and chondroprotectives after cartilage trauma.


Assuntos
Vias Biossintéticas/efeitos dos fármacos , Doenças das Cartilagens/tratamento farmacológico , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Glucosamina/farmacologia , Hexosaminas/metabolismo , Uridina Difosfato N-Acetilglicosamina/farmacologia , Biomarcadores/metabolismo , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Doenças das Cartilagens/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo II/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Glicosilação/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite/tratamento farmacológico , Osteoartrite/metabolismo , Fosforilação/efeitos dos fármacos
13.
Int J Mol Sci ; 22(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34281202

RESUMO

Autologous chondrocyte implantation (ACI) is a cell therapy for the treatment of focal cartilage defects. The ACI product that is currently approved for use in the European Union (EU) consists of spheroids of autologous matrix-associated chondrocytes. These spheroids are spherical aggregates of ex vivo expanded human autologous chondrocytes and their self-synthesized extracellular matrix. The aim is to provide an overview of the preclinical and nonclinical studies that have been performed to ensure reproducible quality, safety, and efficacy of the cell therapy, and to evaluate the clinical data on ACI with spheroids. A systematic review was performed to include all English publications on self-aggregated spheroids of chondrocytes cultured in autologous serum without other supplements. A total of 20 publications were included, 7 pre- and nonclinical and 13 clinical research publications. The pre- and nonclinical research publications describe the development from concept to in vivo efficacy and quality- and safety-related aspects such as biodistribution, tumorigenicity, genetic stability, and potency. The evaluation of clinical research shows short- to mid-term safety and efficacy for the ACI with spheroid-based treatment of cartilage defects in both randomized clinical trials with selected patients, as well as in routine treatment providing real-world data in more complex patients.


Assuntos
Condrócitos/transplante , Animais , Doenças das Cartilagens/cirurgia , Doenças das Cartilagens/terapia , Cartilagem Articular/cirurgia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Condrócitos/citologia , Condrócitos/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Humanos , Traumatismos do Joelho/cirurgia , Traumatismos do Joelho/terapia , Articulação do Joelho/metabolismo , Articulação do Joelho/patologia , Procedimentos Ortopédicos/métodos , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/patologia , Osteoartrite do Joelho/terapia , Esferoides Celulares , Transplante Autólogo
14.
Arch Gerontol Geriatr ; 96: 104477, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34280597

RESUMO

BACKGROUND: CircRNA circFADS2 has been reported to protect cells from LPS (Lipopolysaccharide)-induced cell injury, which contributes to osteoarthritis (OA). This study was carried out to study the role of circFADS2 in OA. METHODS: The expression of circFADS2 and miR-195-5p in synovial fluid samples from 63 OA patients and 63 healthy controls were detected by RT-qPCRs. The effects of overexpression of circFADS2 on the expression of miR-195-5p and the methylation of miR-195-5p gene in chondrocytes were evaluated by RT-qPCRs and MSPs, respectively. The roles of circFADS2 and miR-195-5p in regulating the LPS-induced apoptosis of chondrocytes were explored by cell apoptosis assay. RESULTS: CircFADS2 was downregulated in OA and inversely correlated with miR-195-5p. In chondrocytes, overexpression of circFADS2 and downregulation of miR-195-5p were observed after LPS treatment. In chondrocytes, overexpression of circFADS2 decreased the expression levels of miR-195-5p, and increased the methylation of miR-195-5p gene. Cell apoptosis assay showed that overexpression of circFADS2 reduced the apoptosis of chondrocytes under LPS treatment. Overexpression of miR-195-5p increased cell apoptosis and attenuated the effects of overexpression of circFADS2 on cell apoptosis. CONCLUSION: CircFADS2 may downregulate miR-19-5p through methylation to suppress LPS-induced apoptosis of chondrocytes, thereby suppressing OA.


Assuntos
MicroRNAs , Osteoartrite , Apoptose/genética , Condrócitos/metabolismo , Humanos , Lipopolissacarídeos , Metilação , MicroRNAs/genética , Osteoartrite/genética
15.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070455

RESUMO

Chondrosarcomas are malignant bone tumors. Their abundant cartilage-like extracellular matrix and their hypoxic microenvironment contribute to their resistance to chemotherapy and radiotherapy, and no effective therapy is currently available. MicroRNAs (miRNAs) may be an interesting alternative in the development of therapeutic options. Here, for the first time in chondrosarcoma cells, we carried out high-throughput functional screening using impedancemetry, and identified five miRNAs with potential antiproliferative or chemosensitive effects on SW1353 chondrosarcoma cells. The cytotoxic effects of miR-342-5p and miR-491-5p were confirmed on three chondrosarcoma cell lines, using functional validation under normoxia and hypoxia. Both miRNAs induced apoptosis and miR-342-5p also induced autophagy. Western blots and luciferase reporter assays identified for the first time Bcl-2 as a direct target of miR-342-5p, and also Bcl-xL as a direct target of both miR-342-5p and miR-491-5p in chondrosarcoma cells. MiR-491-5p also inhibited EGFR expression. Finally, only miR-342-5p induced cell death on a relevant 3D chondrosarcoma organoid model under hypoxia that mimics the in vivo microenvironment. Altogether, our results revealed the tumor suppressive activity of miR-342-5p, and to a lesser extent of miR-491-5p, on chondrosarcoma lines. Through this study, we also confirmed the potential of Bcl-2 family members as therapeutic targets in chondrosarcomas.


Assuntos
Antineoplásicos/farmacologia , Apoptose/genética , Neoplasias Ósseas/metabolismo , Condrossarcoma/metabolismo , MicroRNAs/farmacologia , Organoides/metabolismo , Microambiente Tumoral/genética , Autofagia/genética , Neoplasias Ósseas/genética , Ciclo Celular/genética , Hipóxia Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Condrócitos/metabolismo , Condrossarcoma/genética , Cisplatino/farmacologia , Receptores ErbB/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Organoides/citologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína bcl-X/metabolismo
16.
Yonsei Med J ; 62(7): 650-659, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34164963

RESUMO

PURPOSE: Our previous work demonstrated that miRNA-495 targets SOX9 to inhibit chondrogenesis of mesenchymal stem cells. In this study, we aimed to investigate whether miRNA-495-mediated SOX9 regulation could be a novel therapeutic target for osteoarthritis (OA) using an in vitro cell culture model. MATERIALS AND METHODS: An in vitro model mimicking the OA environment was established using TC28a2 normal human chondrocyte cells. Interleukin-1ß (IL-1ß, 10 ng/mL) was utilized to induce inflammation-related changes in TC28a2 cells. Safranin O staining and glycosaminoglycan assay were used to detect changes in proteoglycans among TC28a2 cells. Expression levels of COX-2, ADAMTS5, MMP13, SOX9, CCL4, and COL2A1 were examined by qRT-PCR and/or Western blotting. Immunohistochemistry was performed to detect SOX9 and CCL4 proteins in human cartilage tissues obtained from patients with OA. RESULTS: miRNA-495 was upregulated in IL-1ß-treated TC28a2 cells and chondrocytes from damaged cartilage tissues of patients with OA. Anti-miR-495 abolished the effect of IL-1ß in TC28a2 cells and rescued the protein levels of SOX9 and COL2A1, which were reduced by IL-1ß. SOX9 was downregulated in the damaged cartilage tissues of patients with OA, and knockdown of SOX9 abolished the effect of anti-miR-495 on IL-1ß-treated TC28a2 cells. CONCLUSION: We demonstrated that inhibition of miRNA-495 alleviates IL-1ß-induced inflammatory responses in chondrocytes by rescuing SOX9 expression. Accordingly, miRNA-495 could be a potential novel target for OA therapy, and the application of anti-miR-495 to chondrocytes could be a therapeutic strategy for treating OA.


Assuntos
Condrócitos , Interleucina-1beta , MicroRNAs , Fatores de Transcrição SOX9 , Células Cultivadas , Condrócitos/metabolismo , Regulação para Baixo , Humanos , Interleucina-1beta/metabolismo , MicroRNAs/genética , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo
17.
FASEB J ; 35(7): e21721, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34118090

RESUMO

Mineral homeostasis is regulated by a complex network involving endocrine actions by calcitriol, parathyroid hormone (PTH), and FGF23 on several organs including kidney, intestine, and bone. Alterations of mineral homeostasis are found in chronic kidney disease and other systemic disorders. The interplay between the immune system and the skeletal system is not fully understood, but cytokines play a major role in modulating calcitriol production and function. One of the main cellular signaling pathways mediating cytokine function is the Janus kinase (JAK)--signal transducer and activator of transcription (STAT) pathway. Here, we used a mouse model (Jak1S645P+/- ) that resembles a constitutive activating mutation of the Jak1/Stat3 signaling pathway in humans, and shows altered mineral metabolism, with higher fibroblast growth factor 23 (FGF23) levels, lower PTH levels, and higher calcitriol levels. The higher calcitriol levels are probably due to extrarenal calcitriol production. Furthermore, systemic Jak1/Stat3 activation led to growth impairment and skeletal alterations. The growth plate in long bones showed decreased chondrocyte proliferation rates and reduced height of terminal chondrocytes. Furthermore, we demonstrate that Jak1 is also involved in bone remodeling early in life. Jak1S645P+/- animals have decreased bone and cortical volume, imbalanced bone remodeling, reduced MAP kinase signaling, and local inflammation. In conclusion, Jak1 plays a major role in bone health probably both, directly and systemically by regulating mineral homeostasis. Understanding the role of this signaling pathway will contribute to a better knowledge in bone growth and in mineral physiology, and to the development of selective Jak inhibitors as osteoprotective agents.


Assuntos
Osso e Ossos/metabolismo , Osso e Ossos/fisiologia , Calcitriol/metabolismo , Transtornos do Crescimento/metabolismo , Janus Quinase 1/metabolismo , Transdução de Sinais/fisiologia , Animais , Remodelação Óssea/fisiologia , Proliferação de Células/fisiologia , Condrócitos/metabolismo , Condrócitos/fisiologia , Citocinas/metabolismo , Feminino , Fatores de Crescimento de Fibroblastos , Lâmina de Crescimento/metabolismo , Lâmina de Crescimento/fisiologia , Homeostase/fisiologia , Humanos , Inflamação/metabolismo , Rim/metabolismo , Rim/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Mutação/genética , Hormônio Paratireóideo/metabolismo , Fator de Transcrição STAT3/metabolismo
18.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34073090

RESUMO

Nuclear magnetic resonance therapy (NMRT) is discussed as a participant in repair processes regarding cartilage and as an influence in pain signaling. To substantiate the application of NMRT, the underlying mechanisms at the cellular level were studied. In this study microRNA (miR) was extracted from human primary healthy and osteoarthritis (OA) chondrocytes after NMR treatment and was sequenced by the Ion PI Hi-Q™ Sequencing 200 system. In addition, T/C-28a2 chondrocytes grown under hypoxic conditions were studied for IL-1ß induced changes in expression on RNA and protein level. HDAC activity an NAD(+)/NADH was measured by luminescence detection. In OA chondrocytes miR-106a, miR-27a, miR-34b, miR-365a and miR-424 were downregulated. This downregulation was reversed by NMRT. miR-365a-5p is known to directly target HDAC and NF-ĸB, and a decrease in HDAC activity by NMRT was detected. NAD+/NADH was reduced by NMR treatment in OA chondrocytes. Under hypoxic conditions NMRT changed the expression profile of HIF1, HIF2, IGF2, MMP3, MMP13, and RUNX1. We conclude that NMRT changes the miR profile and modulates the HDAC and the NAD(+)/NADH signaling in human chondrocytes. These findings underline once more that NMRT counteracts IL-1ß induced changes by reducing catabolic effects, thereby decreasing inflammatory mechanisms under OA by changing NF-ĸB signaling.


Assuntos
Condrócitos , Espectroscopia de Ressonância Magnética/métodos , MicroRNAs/metabolismo , Osteoartrite , Linhagem Celular , Condrócitos/citologia , Condrócitos/metabolismo , Condrócitos/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Osteoartrite/metabolismo , Osteoartrite/terapia , Cultura Primária de Células
19.
Int J Mol Sci ; 22(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34072015

RESUMO

Osteoarthritis (OA) is a painful and disabling disease that affects millions of patients. Its etiology is largely unknown, but it is most likely multifactorial. OA pathogenesis involves the catabolism of the cartilage extracellular matrix and is supported by inflammatory and oxidative signaling pathways and marked epigenetic changes. To delay OA progression, a wide range of exercise programs and naturally derived compounds have been suggested. This literature review aims to analyze the main signaling pathways and the evidence about the synergistic effects of these two interventions to counter OA. The converging nutrigenomic and physiogenomic intervention could slow down and reduce the complex pathological features of OA. This review provides a comprehensive picture of a possible signaling approach for targeting OA molecular pathways, initiation, and progression.


Assuntos
Suplementos Nutricionais , Suscetibilidade a Doenças , Osteoartrite/etiologia , Osteoartrite/metabolismo , Transdução de Sinais , Animais , Antioxidantes , Biomarcadores , Condrócitos/metabolismo , Gerenciamento Clínico , Exercício Físico , Humanos , Nutrigenômica/métodos , Osteoartrite/diagnóstico , Osteoartrite/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
20.
Int J Biol Macromol ; 183: 2030-2043, 2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34097959

RESUMO

In this study, azide and alkyne moieties were introduced to the structure of citric acid-modified hydroxyethyl cellulose (HEC) and then through a bioorthogonal click chemistry method: Strain-promoted azide-alkyne cycloaddition, a novel crosslinked HEC scaffold (click sample) was obtained. Chemical modifications and successful crosslinking of the samples were assessed with FTIR and 1H NMR spectroscopy. Lyophilized samples exhibited a porous interconnected microarchitecture with desirable features for commensurate cartilage tissue engineering applications. As the stability of scaffolds improved upon crosslinking, considerable water uptake and swelling degree of ~650% could still be measured for the click sample. Offering Young's modulus of ~10 MPa and tensile strength of ~0.43 MPa, the mechanical characteristics of click sample were comparable with those of normal cartilage tissue. Various in vitro biological assays, including MTT analysis, cellular attachment, histological staining with safranin O, and real-time PCR decisively approved significant biocompatibility, chondrogenic ability, and bioorthogonal features of click sample.


Assuntos
Materiais Biocompatíveis/química , Cartilagem/fisiologia , Celulose/análogos & derivados , Condrócitos/fisiologia , Química Click , Reagentes para Ligações Cruzadas/química , Engenharia Tecidual , Tecidos Suporte , Cartilagem/metabolismo , Cartilagem/ultraestrutura , Adesão Celular , Linhagem Celular , Sobrevivência Celular , Celulose/química , Condrócitos/metabolismo , Condrócitos/ultraestrutura , Condrogênese , Ácido Cítrico/química , Módulo de Elasticidade , Humanos , Porosidade , Resistência à Tração
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