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1.
Nat Commun ; 12(1): 4939, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400627

RESUMO

Pain is a central feature of soft tissue trauma, which under certain contexts, results in aberrant osteochondral differentiation of tissue-specific stem cells. Here, the role of sensory nerve fibers in this abnormal cell fate decision is investigated using a severe extremity injury model in mice. Soft tissue trauma results in NGF (Nerve growth factor) expression, particularly within perivascular cell types. Consequently, NGF-responsive axonal invasion occurs which precedes osteocartilaginous differentiation. Surgical denervation impedes axonal ingrowth, with significant delays in cartilage and bone formation. Likewise, either deletion of Ngf or two complementary methods to inhibit its receptor TrkA (Tropomyosin receptor kinase A) lead to similar delays in axonal invasion and osteochondral differentiation. Mechanistically, single-cell sequencing suggests a shift from TGFß to FGF signaling activation among pre-chondrogenic cells after denervation. Finally, analysis of human pathologic specimens and databases confirms the relevance of NGF-TrkA signaling in human disease. In sum, NGF-mediated TrkA-expressing axonal ingrowth drives abnormal osteochondral differentiation after soft tissue trauma. NGF-TrkA signaling inhibition may have dual therapeutic use in soft tissue trauma, both as an analgesic and negative regulator of aberrant stem cell differentiation.


Assuntos
Diferenciação Celular , Fator de Crescimento Neural/metabolismo , Receptor trkA/metabolismo , Transdução de Sinais , Ferimentos e Lesões/metabolismo , Animais , Axônios/metabolismo , Cartilagem/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Neural/genética , Osteogênese , Células-Tronco/metabolismo , Ferimentos e Lesões/patologia
2.
Biomolecules ; 11(7)2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209859

RESUMO

Cartilage-derived stem/progenitor cells (CSPCs) are a potential choice for seed cells in osteal and chondral regeneration, and the outcomes of their survival and position distribution in vivo form the basis for the investigation of their mechanism. However, the current use of in vivo stem cell tracing techniques in laboratories is relatively limited, owing to their high operating costs and cytotoxicity. Herein, we performed tri-modal in vivo imaging of CSPCs during subcutaneous chondrogenesis using upconversion nanoparticles (UCNPs) for 28 days. Distinctive signals at accurate positions were acquired without signal noise from X-ray computed tomography, magnetic resonance imaging, and upconversion luminescence. The measured intensities were all significantly proportional to the cell numbers, thereby enabling real-time in vivo quantification of the implanted cells. However, limitations of the detectable range of cell numbers were also observed, owing to the imaging shortcomings of UCNPs, which requires further improvement of the nanoparticles. Our study explores the application value of upconversion nanomaterials in the tri-modal monitoring of implanted stem cells and provides new perspectives for future clinical translation.


Assuntos
Cartilagem/metabolismo , Rastreamento de Células/métodos , Células-Tronco/metabolismo , Animais , Sobrevivência Celular , Humanos , Luminescência , Imageamento por Ressonância Magnética/métodos , Nanopartículas/química , Nanoestruturas/química , Tomografia Computadorizada por Raios X/métodos
3.
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
4.
FASEB J ; 35(8): e21779, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34314047

RESUMO

The incredible mechanical strength and durability of mature fibrous tissues and their extremely limited turnover and regenerative capacity underscores the importance of proper matrix assembly during early postnatal growth. In tissues with composite extracellular matrix (ECM) structures, such as the adult knee meniscus, fibrous (Collagen-I rich), and cartilaginous (Collagen-II, proteoglycan-rich) matrix components are regionally segregated to the outer and inner portions of the tissue, respectively. While this spatial variation in composition is appreciated to be functionally important for resisting complex mechanical loads associated with gait, the establishment of these specialized zones is poorly understood. To address this issue, the following study tracked the growth of the murine meniscus from its embryonic formation through its first month of growth, encompassing the critical time-window during which animals begin to ambulate and weight bear. Using histological analysis, region specific high-throughput qPCR, and Col-1, and Col-2 fluorescent reporter mice, we found that matrix and cellular features defining specific tissue zones were already present at birth, before continuous weight-bearing had occurred. These differences in meniscus zones were further refined with postnatal growth and maturation, resulting in specialization of mature tissue regions. Taken together, this work establishes a detailed timeline of the concurrent spatiotemporal changes that occur at both the cellular and matrix level throughout meniscus maturation. The findings of this study provide a framework for investigating the reciprocal feedback between cells and their evolving microenvironments during assembly of a mechanically robust fibrocartilage tissue, thus providing insight into mechanisms of tissue degeneration and effective regenerative strategies.


Assuntos
Cartilagem , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Menisco , Animais , Cartilagem/embriologia , Cartilagem/crescimento & desenvolvimento , Cartilagem/metabolismo , Diferenciação Celular , Proliferação de Células , Menisco/embriologia , Menisco/crescimento & desenvolvimento , Menisco/metabolismo , Camundongos , Camundongos Transgênicos
5.
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
6.
Molecules ; 26(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200590

RESUMO

The aim of the presented research was to obtain reconstituted atelocollagen fibers after extraction from poultry cartilage using the pepsin-acidic method in order to remove telopeptides from the tropocollagen. Firstly, we examined the extraction of collagen from the cartilage extracellular matrix (ECM) after proteoglycans (PG) had been removed by the action of salts, i.e., NaCl or chaotropic MgCl2. Additionally, the effects of the salt type used for PG and hyaluronic acid removal on the properties of self-assembled fibers in solutions at pH 7.4 and freeze-dried matrices were investigated. The basic features of the obtained fibers were characterized, including thermal properties using scanning calorimetry, rheological properties using dynamic oscillatory rheometry, and the structure by scanning electron microscopy. The fibers obtained after PG removal with both analyzed types of salts had similar thermal denaturation characteristics. However, the fibers after PG removal with NaCl, in contrast to those obtained after MgCl2 treatment, showed different rheological properties during gelatinization and smaller diameter size. Moreover, the degree of fibrillogenesis of collagens after NaCl treatment was complete compared to that with MgCl2, which was only partial (70%). The structures of fibers after lyophilization were fundamentally different. The matrices obtained after NaCl pretreatment form regular scaffolds in contrast to the thin, surface structures of the cartilage matrix after proteoglycans removal using MgCl2.


Assuntos
Cartilagem/metabolismo , Galinhas/metabolismo , Colágeno/metabolismo , Proteoglicanas/metabolismo , Cloreto de Sódio/metabolismo , Animais , Matriz Extracelular/metabolismo , Ácido Hialurônico/metabolismo , Pepsina A/metabolismo
7.
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
8.
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
9.
Carbohydr Polym ; 263: 117927, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33858586

RESUMO

There is inconsistent information regarding the size effects of exogenously given hyaluronan on its in vivo fate. The data are often biased by the poor quality of hyaluronan and non-ideal labelling strategies used for resolving exogenous/endogenous hyaluronan, which only monitor the label and not hyaluronan itself. To overcome these drawbacks and establish the pharmacokinetics of intravenous hyaluronan in relation to its Mw, 13C-labelled HA of five Mws from 13.6-1562 kDa was prepared and administered to mice at doses 25-50 mg kg-1. The elimination efficiency increased with decreasing Mw. Low Mw hyaluronan was rapidly eliminated as small hyaluronan fragments in urine, while high Mw hyaluronan exhibited saturable kinetics and complete metabolization within 48 h. All tested Mws exhibited a similar uptake by liver cells and metabolization into activated sugars. 13C-labelling combined with LC-MS provides an excellent approach to elucidating in vivo fate and biological activities of hyaluronan.


Assuntos
Ácido Hialurônico/farmacocinética , Marcação por Isótopo/métodos , Administração Intravenosa , Animais , Osso e Ossos/metabolismo , Isótopos de Carbono/química , Isótopos de Carbono/metabolismo , Isótopos de Carbono/farmacocinética , Cartilagem/metabolismo , ADP-Ribose Cíclica/metabolismo , Vias de Eliminação de Fármacos , Feminino , Ácido Hialurônico/química , Ácido Hialurônico/metabolismo , Camundongos Endogâmicos BALB C , Peso Molecular , Distribuição Tecidual , Uridina Difosfato Glucose/metabolismo , Uridina Difosfato N-Acetilglicosamina/metabolismo
10.
PLoS Genet ; 17(4): e1009275, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33819267

RESUMO

Mammalian Hedgehog (HH) signalling pathway plays an essential role in tissue homeostasis and its deregulation is linked to rheumatological disorders. UBR5 is the mammalian homologue of the E3 ubiquitin-protein ligase Hyd, a negative regulator of the Hh-pathway in Drosophila. To investigate a possible role of UBR5 in regulation of the musculoskeletal system through modulation of mammalian HH signaling, we created a mouse model for specific loss of Ubr5 function in limb bud mesenchyme. Our findings revealed a role for UBR5 in maintaining cartilage homeostasis and suppressing metaplasia. Ubr5 loss of function resulted in progressive and dramatic articular cartilage degradation, enlarged, abnormally shaped sesamoid bones and extensive heterotopic tissue metaplasia linked to calcification of tendons and ossification of synovium. Genetic suppression of smoothened (Smo), a key mediator of HH signalling, dramatically enhanced the Ubr5 mutant phenotype. Analysis of HH signalling in both mouse and cell model systems revealed that loss of Ubr5 stimulated canonical HH-signalling while also increasing PKA activity. In addition, human osteoarthritic samples revealed similar correlations between UBR5 expression, canonical HH signalling and PKA activity markers. Our studies identified a crucial function for the Ubr5 gene in the maintenance of skeletal tissue homeostasis and an unexpected mode of regulation of the HH signalling pathway.


Assuntos
Artrite Reumatoide/genética , Proteínas de Drosophila/genética , Músculo Esquelético/metabolismo , Receptor Smoothened/genética , Ubiquitina-Proteína Ligases/genética , Animais , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Cartilagem/crescimento & desenvolvimento , Cartilagem/metabolismo , Cartilagem/patologia , Condrócitos/metabolismo , Modelos Animais de Doenças , Drosophila melanogaster/genética , Proteínas Hedgehog/genética , Homeostase/genética , Humanos , Articulação do Joelho/metabolismo , Articulação do Joelho/patologia , Camundongos , Músculo Esquelético/patologia , Osteogênese/genética , Transdução de Sinais/genética , Tendões/metabolismo , Tendões/patologia
11.
Molecules ; 26(7)2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33915775

RESUMO

Although the anti-tumor and anti-infective properties of ß-glucans have been well-discussed, their role in bone metabolism has not been reviewed so far. This review discusses the biological effects of ß-glucans on bone metabolisms, especially on bone-resorbing osteoclasts, which are differentiated from hematopoietic precursors. Multiple immunoreceptors that can recognize ß-glucans were reported to be expressed in osteoclast precursors. Coordinated co-stimulatory signals mediated by these immunoreceptors are important for the regulation of osteoclastogenesis and bone remodeling. Curdlan from the bacterium Alcaligenes faecalis negatively regulates osteoclast differentiation in vitro by affecting both the osteoclast precursors and osteoclast-supporting cells. We also showed that laminarin, lichenan, and glucan from baker's yeast, as well as ß-1,3-glucan from Euglema gracilisas, inhibit the osteoclast formation in bone marrow cells. Consistent with these findings, systemic and local administration of ß-glucan derived from Aureobasidium pullulans and Saccharomyces cerevisiae suppressed bone resorption in vivo. However, zymosan derived from S. cerevisiae stimulated the bone resorption activity and is widely used to induce arthritis in animal models. Additional research concerning the relationship between the molecular structure of ß-glucan and its effect on osteoclastic bone resorption will be beneficial for the development of novel treatment strategies for bone-related diseases.


Assuntos
Glucanos/metabolismo , Osteogênese/fisiologia , Animais , Regeneração Óssea , Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Diferenciação Celular/efeitos dos fármacos , Glucanos/farmacologia , Humanos , Imunomodulação , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Receptores Imunológicos/metabolismo
12.
Int J Mol Sci ; 22(8)2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33916928

RESUMO

Osteoarthritis (OA), a degenerative joint disorder, has been reported as the most common cause of disability worldwide. The production of inflammatory cytokines is the main factor in OA. Previous studies have been reported that obeticholic acid (OCA) and OCA derivatives inhibited the release of proinflammatory cytokines in acute liver failure, but they have not been studied in the progression of OA. In our study, we screened our small synthetic library of OCA derivatives and found T-2054 had anti-inflammatory properties. Meanwhile, the proliferation of RAW 264.7 cells and ATDC5 cells were not affected by T-2054. T-2054 treatment significantly relieved the release of NO, as well as mRNA and protein expression levels of inflammatory cytokines (IL-6, IL-8 and TNF-α) in LPS-induced RAW 264.7 cells. Moreover, T-2054 promoted extracellular matrix (ECM) synthesis in TNF-α-treated ATDC5 chondrocytes. Moreover, T-2054 could relieve the infiltration of inflammatory cells and degeneration of the cartilage matrix and decrease the levels of serum IL-6, IL-8 and TNF-α in DMM-induced C57BL/6 mice models. At the same time, T-2054 showed no obvious toxicity to mice. Mechanistically, T-2054 decreased the extent of p-p65 expression in LPS-induced RAW 264.7 cells and TNF-α-treated ATDC5 chondrocytes. In summary, we showed for the first time that T-2054 effectively reduced the release of inflammatory mediators, as well as promoted extracellular matrix (ECM) synthesis via the NF-κB-signaling pathway. Our findings support the potential use of T-2054 as an effective therapeutic agent for the treatment of OA.


Assuntos
Anti-Inflamatórios/farmacologia , Ácido Quenodesoxicólico/análogos & derivados , NF-kappa B/metabolismo , Osteoartrite/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Biomarcadores , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Cartilagem/patologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ácido Quenodesoxicólico/química , Ácido Quenodesoxicólico/farmacologia , Ácido Quenodesoxicólico/uso terapêutico , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/efeitos adversos , Masculino , Camundongos , Óxido Nítrico/biossíntese , Osteoartrite/tratamento farmacológico , Osteoartrite/etiologia , Osteoartrite/patologia , Células RAW 264.7
13.
Int J Mol Sci ; 22(7)2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33807323

RESUMO

A continuing challenge in cartilage tissue engineering for cartilage regeneration is the creation of a suitable synthetic microenvironment for chondrocytes and tissue regeneration. The aim of this study was to develop a highly tunable hybrid scaffold based on a silk fibroin matrix (SM) and a hyaluronic acid (HA) hydrogel. Human articular chondrocytes were embedded in a porous 3-dimensional SM, before infiltration with tyramine modified HA hydrogel. Scaffolds were cultured in chondropermissive medium with and without TGF-ß1. Cell viability and cell distribution were assessed using CellTiter-Blue assay and Live/Dead staining. Chondrogenic marker expression was detected using qPCR. Biosynthesis of matrix compounds was analyzed by dimethylmethylene blue assay and immuno-histology. Differences in biomaterial stiffness and stress relaxation were characterized using a one-step unconfined compression test. Cell morphology was investigated by scanning electron microscopy. Hybrid scaffold revealed superior chondro-inductive and biomechanical properties compared to sole SM. The presence of HA and TGF-ß1 increased chondrogenic marker gene expression and matrix deposition. Hybrid scaffolds offer cytocompatible and highly tunable properties as cell-carrier systems, as well as favorable biomechanical properties.


Assuntos
Cartilagem Articular/metabolismo , Fibroínas/farmacologia , Engenharia Tecidual/métodos , Idoso , Materiais Biocompatíveis/metabolismo , Cartilagem/citologia , Cartilagem/metabolismo , Cartilagem Articular/citologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Condrócitos/metabolismo , Condrogênese , Fibroínas/metabolismo , Humanos , Ácido Hialurônico/farmacologia , Hidrogéis/metabolismo , Hidrogéis/farmacologia , Pessoa de Meia-Idade , Porosidade , Seda/metabolismo , Tecidos Suporte/química
14.
Int J Mol Sci ; 22(8)2021 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-33919985

RESUMO

Tissue engineering strategies promote bone regeneration for large bone defects by stimulating the osteogenesis route via intramembranous ossification in engineered grafts, which upon implantation are frequently constrained by insufficient integration and functional anastomosis of vasculature from the host tissue. In this study, we developed a hybrid biomaterial incorporating decellularized cartilage extracellular matrix (CD-ECM) as a template and silk fibroin (SF) as a carrier to assess the bone regeneration capacity of bone marrow-derived mesenchymal stem cells (hBMSC's) via the endochondral ossification (ECO) route. hBMSC's were primed two weeks for chondrogenesis, followed by six weeks for hypertrophy onto hybrid CD-ECM/SF or SF alone scaffolds and evaluated for the mineralized matrix formation in vitro. Calcium deposition biochemically determined increased significantly from 4-8 weeks in both SF and CD-ECM/SF constructs, and retention of sGAG's were observed only in CD-ECM/SF constructs. SEM/EDX revealed calcium and phosphate crystal localization by hBMSC's under all conditions. Compressive modulus reached a maximum of 40 KPa after eight weeks of hypertrophic induction. µCT scanning at eight weeks indicated a cloud of denser minerals in groups after hypertrophic induction in CD-ECM/SF constructs than SF constructs. Gene expression by RT-qPCR revealed that hBMSC's expressed hypertrophic markers VEGF, COL10, RUNX2, but the absence of early hypertrophic marker ChM1 and later hypertrophic marker TSBS1 and the presence of osteogenic markers ALPL, IBSP, OSX under all conditions. Our data indicate a new method to prime hBMSC'S into the late hypertrophic stage in vitro in mechanically stable constructs for ECO-mediated bone tissue regeneration.


Assuntos
Regeneração Óssea/genética , Condrogênese/genética , Células-Tronco Mesenquimais/citologia , Engenharia Tecidual , Tecido Adiposo/crescimento & desenvolvimento , Tecido Adiposo/metabolismo , Animais , Osso e Ossos/metabolismo , Cartilagem/crescimento & desenvolvimento , Cartilagem/metabolismo , Diferenciação Celular/genética , Condrócitos/metabolismo , Matriz Extracelular/genética , Fibroínas/química , Fibroínas/genética , Humanos , Osteogênese/genética , Tecidos Suporte
15.
Int J Mol Sci ; 22(7)2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810460

RESUMO

Osteoarthritis (OA) is the most common type of arthritis and is associated with wear and tear, aging, and inflammation. Previous studies revealed that several antimicrobial peptides are up-regulated in the knee synovium of patients with OA or rheumatoid arthritis. Here, we investigated the functional effects of cathelicidin-related antimicrobial peptide (Cramp) on OA pathogenesis. We found that Cramp is highly induced by IL-1ß via the NF-κB signaling pathway in mouse primary chondrocytes. Elevated Cramp was also detected in the cartilage and synovium of mice suffering from OA cartilage destruction. The treatment of chondrocytes with Cramp stimulated the expression of catabolic factors, and the knockdown of Cramp by small interfering RNA reduced chondrocyte catabolism mediated by IL-1ß. Moreover, intra-articular injection of Cramp into mouse knee joints at a low dose accelerated traumatic OA progression. At high doses, Cramp affected meniscal ossification and tears, leading to cartilage degeneration. These findings demonstrate that Cramp is associated with OA pathophysiology.


Assuntos
Peptídeos Catiônicos Antimicrobianos/efeitos adversos , Osteoartrite do Joelho/fisiopatologia , Animais , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Cartilagem/metabolismo , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Injeções Intra-Articulares , Interleucina-1beta/metabolismo , Articulação do Joelho/efeitos dos fármacos , Articulação do Joelho/fisiopatologia , Masculino , Menisco/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Osteoartrite do Joelho/induzido quimicamente , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Membrana Sinovial/metabolismo
16.
Molecules ; 26(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799537

RESUMO

Kaempferia parviflora Wall. ex Baker (KP) has been reported to attenuate cartilage destruction in rat model of osteoarthritis. Previously, we demonstrated that KP rhizome extract and its active components effectively suppressed mechanisms associated with RA in SW982 cells. Here, we further evaluated the anti-arthritis potential of KP extract by using multi-level models, including a complete Freund's adjuvant-induced arthritis and a cartilage explant culture model, and to investigate the effects of KP extract and its major components on related gene expressions and underlying mechanisms within cells. In arthritis rats, the KP extract reduced arthritis indexes, with no significant changes in biological parameters. In the cartilage explant model, the KP extract exerted chondroprotective potential by suppressing sulfated glycosaminoglycans release while preserving high accumulation of proteoglycans. In human chondrocyte cell line, a mixture of the major components equal to their amounts in KP extract showed strong suppression the expression of genes-associated inflammatory joint disease similar to that of the extract. Additionally, KP extract significantly suppressed NF-κB and MAPK signaling pathways. The suppressing expression of necroptosis genes and promoted anti-apoptosis were also found. Collectively, these results provided supportive evidence of the anti-arthritis properties of KP extract, which are associated with its three major components.


Assuntos
Artrite/tratamento farmacológico , Extratos Vegetais/farmacologia , Zingiberaceae/metabolismo , Animais , Apoptose/efeitos dos fármacos , Artrite/genética , Artrite/imunologia , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Modelos Animais de Doenças , Expressão Gênica/efeitos dos fármacos , Glicosaminoglicanos/metabolismo , Humanos , Inflamação/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , NF-kappa B/metabolismo , Cultura Primária de Células , Proteoglicanas/metabolismo , Ratos , Ratos Sprague-Dawley , Rizoma/metabolismo , Suínos , Fator de Transcrição RelA/metabolismo
17.
Development ; 148(7)2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33741714

RESUMO

During craniofacial development, different populations of cartilage- and bone-forming cells develop in precise locations in the head. Most of these cells are derived from pluripotent cranial neural crest cells and differentiate with distinct developmental timing and cellular morphologies. The mechanisms that divide neural crest cells into discrete populations are not fully understood. Here, we use single-cell RNA sequencing to transcriptomically define different populations of cranial neural crest cells. We discovered that the gene family encoding the Alx transcription factors is enriched in the frontonasal population of neural crest cells. Genetic mutant analyses indicate that alx3 functions to regulate the distinct differentiation timing and cellular morphologies among frontonasal neural crest cell subpopulations. This study furthers our understanding of how genes controlling developmental timing shape craniofacial skeletal elements.


Assuntos
Diferenciação Celular/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Crista Neural/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais , Cartilagem/metabolismo , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Cabeça , Camundongos Transgênicos , Morfogênese , Crista Neural/citologia , Organogênese , Crânio/metabolismo , Fatores de Transcrição/genética , Transcriptoma , Peixe-Zebra/embriologia
18.
Adv Clin Chem ; 101: 95-120, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33706891

RESUMO

Osteoarthritis (OA) is a multifactorial disease with huge phenotypic heterogeneity. The disease affects all tissues in the joint, and the loss of articular cartilage is its hallmark. The main biochemical components of the articular cartilage are type II collagen, aggrecan, and water. Transforming growth factor-beta (TGF-ß) signaling is one of the signaling pathways that maintains the healthy cartilage. However, the two subpathways of the TGF-ß signaling-TGF-ß and bone morphogenetic proteins (BMP) subpathways, lose their balance in OA, resulting an increased expression of cartilage degradation enzymes including matrix metallopeptidase 13 (MMP13), cathepsin B (CTSB), and cathepsin K (CTSK) and a decreased expression of aggrecan (ACAN). Thus, restoring the balance of two subpathways might provide a new avenue for treating OA patients. Further, metabolic changes are seen in OA and can be used to distinguish different subtypes of OA patients. Metabolomics studies showed that at least three endotypes of OA can be distinguished: 11% of OA patients are characterized by an elevated blood butyryl carnitine, 33% of OA patients have significant reduced arginine concentration, and 56% with metabolic alteration in phospholipid metabolism. While these findings need to be confirmed, they are promising personalized medicine tools for OA management.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Biomarcadores/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Cartilagem/metabolismo , Cartilagem/patologia , Humanos , Sinais de Exportação Nuclear , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
19.
Ned Tijdschr Geneeskd ; 1652021 01 27.
Artigo em Holandês | MEDLINE | ID: mdl-33651519

RESUMO

A 52-year-old men suffered from osteoarthritis of the knee. During knee replacement surgery, the remaining cartilage appeared black. This discoloration and early degeneration of the cartilage is characteristic for the metabolic disorder alkaptonuria in which homogentisic acid accumulates in the body.


Assuntos
Alcaptonúria , Artroplastia do Joelho , Cartilagem/patologia , Articulação do Joelho/patologia , Joelho/patologia , Ocronose , Alcaptonúria/complicações , Alcaptonúria/metabolismo , Alcaptonúria/cirurgia , Cartilagem/metabolismo , Cartilagem/cirurgia , Cor , Ácido Homogentísico/metabolismo , Humanos , Achados Incidentais , Joelho/cirurgia , Articulação do Joelho/metabolismo , Articulação do Joelho/cirurgia , Masculino , Pessoa de Meia-Idade , Ocronose/etiologia , Ocronose/metabolismo , Ocronose/cirurgia , Osteoartrite/complicações , Osteoartrite/cirurgia
20.
Biochem Biophys Res Commun ; 553: 119-125, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33765556

RESUMO

Excessive activation of inflammation in chondrocyte has been considered to be a major reason cause of cellular death and degeneration in osteoarthritis (OA) development. The NLRP3 inflammasome-mediated pyroptosis pathway is closely related to inflammation regulation. This research was conducted to confirm whether NLRP3 expression and activity are impacted in the development of OA and to detect the role of CY-09, a selective and direct inhibitor of NLRP3 in the in vitro and in vivo models of OA. Our findings corroborated that the expression of NLRP3 is stimulated in OA cartilage. CY-09 can maintain extracellular matrix (ECM) homeostasis and regulate inflammation in TNF-α treated chondrocytes via inhibition of NLRP3 inflammasome-mediated pyroptosis. Moreover, the chondrocyte protective effects of CY-09 were further confirmed in vivo in a DMM-induced OA model. In conclusion, our research indicates that experimental OA activated the NLRP3 activity, and pharmacological inhibition of NLRP3 inflammasome activation by CY-09 protects chondrocytes against inflammation and attenuates OA development.


Assuntos
Inflamassomos/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Osteoartrite/tratamento farmacológico , Substâncias Protetoras/farmacologia , Piroptose/efeitos dos fármacos , Tiazolidinas/farmacologia , Tionas/farmacologia , Animais , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Cartilagem/patologia , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Modelos Animais de Doenças , Progressão da Doença , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/patologia , Feminino , Homeostase/efeitos dos fármacos , Inflamassomos/metabolismo , Inflamação/induzido quimicamente , Inflamação/patologia , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Osteoartrite/patologia , Osteoartrite/prevenção & controle , Fator de Necrose Tumoral alfa/farmacologia
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