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1.
Chinese Medical Journal ; (24): 963-970, 2021.
Article in English | WPRIM | ID: wpr-878129

ABSTRACT

BACKGROUND@#Histone deacetylase 4 (HDAC4) regulates chondrocyte hypertrophy and bone formation. The aim of the present study was to explore the effects of HDAC4 on Interleukin 1 beta (IL-1β)-induced chondrocyte extracellular matrix degradation and whether it is regulated through the WNT family member 3A (WNT3A)/β-catenin signaling pathway.@*METHODS@#Primary chondrocytes (CC) and human chondrosarcoma cells (SW1353 cells) were treated with IL-1β and the level of HDAC4 was assayed using Western blotting. Then, HDAC4 expression in the SW1353 cells was silenced using small interfering RNA to detect the effect of HDAC4 knockdown on the levels of matrix metalloproteinase 3 (MMP3) and MMP13 induced by IL-1β. After transfection with HDAC4 plasmids, the overexpression efficiency was examined using Real-time quantitative polymerase chain reaction (qRT-PCR) and the levels of MMP3 and MMP13 were assayed using Western blotting. After incubation with IL-1β, the translocation of β-catenin into the nucleus was observed using immunofluorescence staining in SW1353 cells to investigate the activation of the WNT3A/β-catenin signaling pathway. Finally, treatment with WNT3A and transfection with glycogen synthase kinase 3 beta (GSK3β) plasmids were assessed for their effects on HDAC4 levels using Western blotting.@*RESULTS@#IL-1β downregulated HDAC4 levels in chondrocytes and SW1353 cells. Furthermore, HDAC4 knockdown increased the levels of MMP3 and MMP13, which contributed to the degradation of the extracellular matrix. Overexpression of HDAC4 inhibited IL-1β-induced increases in MMP3 and MMP13. IL-1β upregulated the levels of WNT3A, and WNT3A reduced HDAC4 levels in SW1353 cells. GSK-3β rescued IL-1β-induced downregulation of HDAC4 in SW1353 cells.@*CONCLUSION@#HDAC4 exerted an inhibitory effect on IL-1β-induced extracellular matrix degradation and was regulated partially by the WNT3A/β-catenin signaling pathway.


Subject(s)
Cell Line, Tumor , Cells, Cultured , Chondrocytes/metabolism , Glycogen Synthase Kinase 3 beta/genetics , Histone Deacetylases/genetics , Humans , Interleukin-1beta/pharmacology , Matrix Metalloproteinase 13/metabolism , Matrix Metalloproteinase 3 , Repressor Proteins , Wnt Signaling Pathway , Wnt3A Protein/genetics , beta Catenin/metabolism
2.
Braz. j. med. biol. res ; 52(9): e8525, 2019. tab, graf
Article in English | LILACS | ID: biblio-1011614

ABSTRACT

Many compounds of ginsenosides show anti-inflammatory properties. However, their anti-inflammatory effects in intervertebral chondrocytes in the presence of inflammatory factors have never been shown. Increased levels of pro-inflammatory cytokines are generally associated with the degradation and death of chondrocytes; therefore, finding an effective and nontoxic substance that attenuates the inflammation is worthwhile. In this study, chondrocytes were isolated from the nucleus pulposus tissues, and the cells were treated with ginsenoside compounds and IL-1β, alone and in combination. Cell viability and death rate were assessed by CCK-8 and flow cytometry methods, respectively. PCR, western blot, and immunoprecipitation assays were performed to determine the mRNA and protein expression, and the interactions between proteins, respectively. Monomeric component of ginsenoside Rd had no toxicity at the tested range of concentrations. Furthermore, Rd suppressed the inflammatory response of chondrocytes to interleukin (IL)-1β by suppressing the increase in IL-1β, tumor necrosis factor (TNF)-α, IL-6, COX-2, and inducible nitric oxide synthase (iNOS) expression, and retarding IL-1β-induced degradation of chondrocytes by improving cell proliferation characteristics and expression of aggrecan and COL2A1. These protective effects of Rd were associated with ubiquitination of IL-1 receptor accessory protein (IL1RAP), blocking the stimulation of IL-1β to NF-κB. Bioinformatics analysis showed that NEDD4, CBL, CBLB, CBLC, and ITCH most likely target IL1RAP. Rd increased intracellular ITCH level and the amount of ITCH attaching to IL1RAP. Thus, IL1RAP ubiquitination promoted by Rd is likely to occur by up-regulation of ITCH. In summary, Rd inhibited IL-1β-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Chondrocytes/drug effects , Ginsenosides/pharmacology , Interleukin-1beta/drug effects , Interleukin-1 Receptor Accessory Protein/metabolism , Intervertebral Disc Degeneration/metabolism , Dinoprostone/metabolism , Cell Survival/drug effects , Tumor Necrosis Factor-alpha/metabolism , Low Back Pain/metabolism , Nitric Oxide Synthase/metabolism , Chondrocytes/cytology , Chondrocytes/metabolism , Ginsenosides/metabolism , Cyclooxygenase 2/metabolism , Aggrecans/metabolism , Interleukin-1beta/metabolism , Ubiquitination , Nucleus Pulposus/cytology , Nucleus Pulposus/drug effects , Nucleus Pulposus/metabolism , Inflammation/metabolism
3.
Acta cir. bras ; 31(2): 74-83, Feb. 2016. tab, graf
Article in English | LILACS | ID: lil-775567

ABSTRACT

PURPOSE: To investigated the effects of exposure to an 1800 MHz electromagnetic field (EMF) on bone development during the prenatal period in rats. METHODS: Pregnant rats in the experimental group were exposed to radiation for six, 12, and 24 hours daily for 20 days. No radiation was given to the pregnant rats in the control group. We distributed the newborn rats into four groups according to prenatal EMF exposure as follows: Group 1 was not exposed to EMF; groups 2, 3, and 4 were exposed to EMF for six, 12, and 24 hours a day, respectively. The rats were evaluated at the end of the 60th day following birth. RESULTS: Increasing the duration of EMF exposure during the prenatal period resulted in a significant reduction of resting cartilage levels and a significant increase in the number of apoptotic chondrocytes and myocytes. There was also a reduction in calcineurin activities in both bone and muscle tissues. We observed that the development of the femur, tibia, and ulna were negatively affected, especially with a daily EMF exposure of 24 hours. CONCLUSION: Bone and muscle tissue development was negatively affected due to prenatal exposure to 1800 MHz radiofrequency electromagnetic field.


Subject(s)
Humans , Animals , Male , Female , Infant, Newborn , Prenatal Exposure Delayed Effects/pathology , Bone Development/radiation effects , Calcineurin/metabolism , Electromagnetic Fields/adverse effects , Time Factors , Pregnancy , Cartilage/pathology , Rats, Sprague-Dawley , Apoptosis/radiation effects , Chondrocytes/metabolism , Chondrocytes/pathology , Models, Animal , Quadriceps Muscle/metabolism , Quadriceps Muscle/pathology , Femur Head/pathology
4.
Braz. j. med. biol. res ; 48(12): 1063-1070, Dec. 2015. tab, graf
Article in English | LILACS | ID: lil-762916

ABSTRACT

Damage to cartilage causes a loss of type II collagen (Col-II) and glycosaminoglycans (GAG). To restore the original cartilage architecture, cell factors that stimulate Col-II and GAG production are needed. Insulin-like growth factor I (IGF-I) and transcription factor SOX9are essential for the synthesis of cartilage matrix, chondrocyte proliferation, and phenotype maintenance. We evaluated the combined effect of IGF-I and SOX9 transgene expression on Col-II and GAG production by cultured human articular chondrocytes. Transient transfection and cotransfection were performed using two mammalian expression plasmids (pCMV-SPORT6), one for each transgene. At day 9 post-transfection, the chondrocytes that were over-expressing IGF-I/SOX9 showed 2-fold increased mRNA expression of the Col-II gene, as well as a 57% increase in Col-II protein, whereas type I collagen expression (Col-I) was decreased by 59.3% compared with controls. The production of GAG by these cells increased significantly compared with the controls at day 9 (3.3- vs 1.8-times, an increase of almost 83%). Thus, IGF-I/SOX9 cotransfected chondrocytes may be useful for cell-based articular cartilage therapies.


Subject(s)
Humans , Chondrocytes/metabolism , Collagen Type II/biosynthesis , Glycosaminoglycans/biosynthesis , Insulin-Like Growth Factor I/metabolism , Matrilin Proteins/biosynthesis , SOX9 Transcription Factor/metabolism , Transfection/methods , Cartilage, Articular/injuries , Cartilage, Articular/metabolism , Collagen Type II/analysis , Extracellular Matrix/chemistry , Gene Expression , Glycosaminoglycans/analysis , Insulin-Like Growth Factor I/genetics , Matrilin Proteins/genetics , Primary Cell Culture , Real-Time Polymerase Chain Reaction , RNA, Messenger/metabolism , SOX9 Transcription Factor/genetics , Spectrophotometry
5.
Biol. Res ; 48: 1-8, 2015. ilus, graf, tab
Article in English | LILACS | ID: biblio-950828

ABSTRACT

BACKGROUND: Osteoarthritis (OA) is a common arthritic disease and multifactorial whole-joint disease. Interactions of chemokines and OA is inadequately documented. RESULTS: In vivo and in vitro studies were conducted to investigate monocyte chemoattractant protein 1 (MCP-1) and receptor chemokine (C-C motif) receptor 2 (CCR2) in chondrocyte degradation and cartilage degeneration. Chondrocytes from 16 OA patients and 6 normal controls were involved in this study. After stimulation of MCP-1, the expression of MCP-1 and CCR2 increased significantly (P < 0.001) and the expression of MMP-13 also increased (P < 0.05). MCP-1 stimulation also induced (or enhanced) the apoptosis of OA chondrocytes (P < 0.05). Additionally, the degradation of cartilage matrix markers (metalloproteinase 3 and 13, MMP3 and MMP13) in the culture medium of normal chondrocytes was also assessed. Furthermore, intra-articular injection of MCP-1 in mouse knees induced cartilage degradation and the CCR2 antagonist did not impede cartilage destroy in rats knees of monosodium iodoacetate (MIA) model. CONCLUSIONS: The results of this study demonstrate that the MCP-1-CCR2 ligand-receptor axis plays a special role in the initiation and progression of OA pathology. Patients with ambiguous etiology can gain some insight from the MCP-1-CCR2 ligand-receptor axis.


Subject(s)
Humans , Animals , Male , Female , Adolescent , Middle Aged , Aged , Mice , Rats , Young Adult , Chemokine CCL2/metabolism , Chondrocytes/metabolism , Osteoarthritis, Knee/physiopathology , Receptors, CCR2/metabolism , Synovial Membrane/cytology , In Vitro Techniques , Enzyme-Linked Immunosorbent Assay , Rats, Sprague-Dawley , Apoptosis/physiology , Disease Progression , Chemokine CCL2/genetics , Matrix Metalloproteinase 3/metabolism , Chondrocytes/enzymology , Iodoacetic Acid , Reverse Transcriptase Polymerase Chain Reaction , Matrix Metalloproteinase 13/metabolism , Receptors, CCR2/antagonists & inhibitors , Receptors, CCR2/genetics , Fibroblasts/metabolism , Matrilin Proteins/metabolism , Mice, Inbred C57BL
6.
Article in English | WPRIM | ID: wpr-165767

ABSTRACT

IL-6 is an inflammatory cytokine and its overexpression plays an important role in osteoarthritis (OA) pathogenesis. Expression of IL-6 is regulated post-transcriptionally by MCPIP1. The 3' untranslated region (UTR) of MCPIP1 mRNA harbors a miR-139 'seed sequence', therefore we examined the post-transcriptional regulation of MCPIP1 by miR-139 and its impact on IL-6 expression in OA chondrocytes. Expression of miR-139 was found to be high in the damaged portion of the OA cartilage compared with unaffected cartilage from the same patient and was also induced by IL-1beta in OA chondrocytes. Inhibition of miR-139 decreased the expression of IL-6 mRNA by 38% and of secreted IL-6 protein by 40%. However, overexpression of miR-139 increased the expression of IL-6 mRNA by 36% and of secreted IL-6 protein by 56%. These data correlated with altered expression profile of MCPIP1 in transfected chondrocytes. Studies with a luciferase reporter construct confirmed the interactions of miR-139 with the 'seed sequence' located in the 3' UTR of MCPIP mRNA. Furthermore, miR-139 overexpression increased the catabolic gene expression but expression of anabolic markers remained unchanged. Overexpression of miR-139 also induced apoptosis in OA chondrocytes. Importantly, we also discovered that IL-6 is a potent inducer of miR-139 expression in OA chondrocytes. These findings indicate that miR-139 functions as a post-transcriptional regulator of MCPIP1 expression and enhances IL-6 expression, which further upregulates miR-139 expression in OA chondrocytes. These results support our hypothesis that miR-139-mediated downregulation of MCPIP1 promotes IL-6 expression in OA. Therefore, targeting miR-139 could be therapeutically beneficial in the management of OA.


Subject(s)
3' Untranslated Regions , Aged , Apoptosis , Chondrocytes/metabolism , Down-Regulation , Female , Gene Expression Regulation , Humans , Interleukin-6/genetics , Male , MicroRNAs/genetics , Middle Aged , Osteoarthritis/genetics , RNA, Messenger/genetics , Ribonucleases/genetics , Transcription Factors/genetics , Up-Regulation
7.
Int. j. morphol ; 32(4): 1347-1356, Dec. 2014. ilus
Article in English | LILACS | ID: lil-734682

ABSTRACT

Tissue engineering (TE) has become an alternative for auricular reconstruction based on the combination of cells, molecular signals and biomaterials. Scaffolds are biomaterials that provide structural support for cell attachment and subsequent tissue development. Ideally, a scaffold should have characteristics such as biocompatibility and bioactivity to adequate support cell functions. Our purpose was to evaluate biocompatibility of microtic auricular chondrocytes seeded onto a chitosan-polyvinyl alcohol-epichlorohydrin (CS-PVA-ECH) hydrogel to propose this material as a scaffold for tissue engineering application. After being cultured onto CS-PVA-ECH hydrogels, auricular chondrocytes viability was up to 81%. SEM analysis showed cell attachment and extracellular matrix formation that was confirmed by IF detection of type II collagen and elastin, the main constituents of elastic cartilage. Expression of elastic cartilage molecular markers during in vitro expansion and during culture onto hydrogels allowed confirming auricular chondrocyte phenotype. In vivo assay of tissue formation revealed generation of neotissues with similar physical characteristics and protein composition to those found in elastic cartilage. According to our results, biocompatibility of the CS-PVA-ECH hydrogel makes it a suitable scaffold for tissue engineering application aimed to elastic cartilage regeneration.


La ingeniería de tejidos (TE) es una alternativa para la reconstrucción auricular basada en la combinación de células, señales moleculares y biomateriales. Los andamios fabricados con biomateriales brindan un soporte estructural que favorece la adhesión cellular y el desarrollo del tejido. Un andamio debe poseer características como biocompatibilidad y bioactividad para soportar adecuadamente funciones celulares. Nuestro objetivo fue evaluar la biocompatibilidad de condrocitos auriculares de microtia cultivados sobre un hidrogel a base de quitosano-alcohol polivinílico-epiclorhidrina (CS-PVA-ECH) y proponerlo como andamio con aplicaciones en ingeniería de tejidos. La viabilidad de los condrocitos auriculares es superior al 81% después de ser cultivados sobre el hidrogel. El análisis por SEM reveló la unión celular y formación de matriz extracellular sobre el hidrogel; confirmada mediante detección por IF de colágena tipo II y elastina. La expresión de marcadores moleculares durante la expansión in vitro y el cultivo sobre los hidrogeles confirmaron el fenotipo condral. El ensayo de formación de tejido in vivo demostró la generación de neotejidos con características físicas y composición similar al cartílago elástico. Nuestros resultados indican que la biocompatibilidad del hidrogel de CS-PVA-ECH lo hace un andamio adecuado para aplicaciones en ingeniería de tejidos enfocadas a regeneración de cartílago elástico.


Subject(s)
Humans , Chondrocytes/cytology , Tissue Engineering/methods , Chitosan/chemistry , Ear Cartilage/cytology , Polyvinyls/chemistry , Biocompatible Materials , Immunohistochemistry , Cell Culture Techniques , Chondrocytes/metabolism , Hydrogels , Epichlorohydrin/chemistry
8.
Braz. j. med. biol. res ; 47(8): 637-645, 08/2014. tab, graf
Article in English | LILACS | ID: lil-716279

ABSTRACT

Tissue engineering encapsulated cells such as chondrocytes in the carrier matrix have been widely used to repair cartilage defects. However, chondrocyte phenotype is easily lost when chondrocytes are expanded in vitro by a process defined as “dedifferentiation”. To ensure successful therapy, an effective pro-chondrogenic agent is necessary to overcome the obstacle of limited cell numbers in the restoration process, and dedifferentiation is a prerequisite. Gallic acid (GA) has been used in the treatment of arthritis, but its biocompatibility is inferior to that of other compounds. In this study, we modified GA by incorporating sulfamonomethoxine sodium and synthesized a sulfonamido-based gallate, JJYMD-C, and evaluated its effect on chondrocyte metabolism. Our results showed that JJYMD-C could effectively increase the levels of the collagen II, Sox9, and aggrecan genes, promote chondrocyte growth, and enhance secretion and synthesis of cartilage extracellular matrix. On the other hand, expression of the collagen I gene was effectively down-regulated, demonstrating inhibition of chondrocyte dedifferentiation by JJYMD-C. Hypertrophy, as a characteristic of chondrocyte ossification, was undetectable in the JJYMD-C groups. We used JJYMD-C at doses of 0.125, 0.25, and 0.5 µg/mL, and the strongest response was observed with 0.25 µg/mL. This study provides a basis for further studies on a novel agent in the treatment of articular cartilage defects.


Subject(s)
Animals , Rabbits , Benzamides/chemical synthesis , Cell Dedifferentiation/drug effects , Cell Proliferation/drug effects , Chondrocytes/drug effects , Phenotype , Pyrimidines/chemical synthesis , Aggrecans/genetics , Aggrecans/metabolism , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Benzamides/pharmacology , Cell Survival , Cell Dedifferentiation/immunology , Chondrocytes/cytology , Chondrocytes/metabolism , Chondrogenesis/drug effects , Collagen Type I/genetics , Collagen Type I/metabolism , Collagen Type II/genetics , Collagen Type II/metabolism , Glycosaminoglycans/analysis , Immunohistochemistry , Laser Scanning Cytometry , Primary Cell Culture , Pyrimidines/pharmacology , Real-Time Polymerase Chain Reaction , SOX9 Transcription Factor/genetics , SOX9 Transcription Factor/metabolism , Tissue Engineering
9.
Braz. j. med. biol. res ; 47(6): 445-451, 06/2014. graf
Article in English | LILACS | ID: lil-709443

ABSTRACT

Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.


Subject(s)
Animals , Cattle , Apoptosis/physiology , /metabolism , Chondrocytes/metabolism , Lentivirus/genetics , RNA Interference/physiology , Starvation/metabolism , Blotting, Western , Cartilage/metabolism , Caspase 9/metabolism , /metabolism , Flow Cytometry , Genetic Vectors/metabolism , Microscopy, Fluorescence , Primary Cell Culture , Propidium , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Serum/physiology , Transfection
10.
Braz. j. med. biol. res ; 47(4): 279-286, 8/4/2014. tab, graf
Article in English | LILACS | ID: lil-705770

ABSTRACT

SRY-related high-mobility-group box 9 (Sox9) gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs). After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering.


Subject(s)
Humans , Cell Differentiation/genetics , Chondrogenesis/genetics , Fetal Blood/cytology , Mesenchymal Stem Cells/cytology , SOX9 Transcription Factor/genetics , Aggrecans/biosynthesis , Blotting, Western , Cartilage/metabolism , Cell Proliferation/genetics , Chondrocytes/metabolism , Collagen Type II/biosynthesis , Flow Cytometry , Green Fluorescent Proteins , Gene Expression Regulation/physiology , Human Umbilical Vein Endothelial Cells/cytology , Immunohistochemistry , Immunophenotyping , Primary Cell Culture , Reverse Transcriptase Polymerase Chain Reaction , Tissue Engineering , Transfection
11.
Article in English | WPRIM | ID: wpr-72398

ABSTRACT

Osteoarthritis is a common cause of functional deterioration in older adults and is an immense burden on the aging population. Altered chondrogenesis is the most important pathophysiological process involved in the development of osteoarthritis. However, the molecular mechanism underlying the regulation of chondrogenesis in patients with osteoarthritis requires further elucidation, particularly with respect to the role of microRNAs. MiR-21 expression in cartilage specimens was examined in 10 patients with knee osteoarthritis and 10 traumatic amputees. The effect of miR-21 on chondrogenesis was also investigated in a chondrocyte cell line. The effect of miR-21 on the expression of growth differentiation factor 5 (GDF-5) was further assessed by luciferase reporter assay and western blot. We found that endogenous miR-21 is upregulated in osteoarthritis patients, and overexpression of miR-21 could attenuate the process of chondrogenesis. Furthermore, we identified GDF-5 as the direct target of miR-21 during the regulation of chondrogenesis. Our data suggest that miR-21 has an important role in the pathogenesis of osteoarthritis and is a potential therapeutic target.


Subject(s)
Cartilage/metabolism , Case-Control Studies , Cell Line , Chondrocytes/metabolism , Growth Differentiation Factor 5/genetics , Humans , MicroRNAs/genetics , Osteoarthritis/metabolism , Up-Regulation
12.
Clinics ; 67(8): 939-944, Aug. 2012. ilus, graf, tab
Article in English | LILACS | ID: lil-647799

ABSTRACT

OBJECTIVES: The promotion of extracellular matrix synthesis by chondrocytes is a requisite part of an effective cartilage tissue engineering strategy. The aim of this in vitro study was to determine the effect of bi-axial cyclic mechanical loading on cell proliferation and the synthesis of glycosaminoglycans by chondrocytes in threedimensional cultures. METHOD: A strain comprising 10% direct compression and 1% compressive shear was applied to bovine chondrocytes seeded in an agarose gel during two 12-hour conditioning periods separated by a 12-hour resting period. RESULTS: The bi-axial-loaded chondrocytes demonstrated a significant increase in glycosaminoglycan synthesis compared with samples exposed to uni-axial or no loading over the same period (p<0.05). The use of a free-swelling recovery period prior to the loading regime resulted in additional glycosaminoglycan production and a significant increase in DNA content (p<0.05), indicating cell proliferation. CONCLUSIONS: These results demonstrate that the use of a bi-axial loading regime results in increased matrix production compared with uni-axial loading.


Subject(s)
Animals , Cattle , Chondrocytes/metabolism , Extracellular Matrix/metabolism , Glycosaminoglycans/biosynthesis , Up-Regulation/physiology , Cell Proliferation , Cells, Cultured , Compressive Strength , Chondrocytes/cytology , Extracellular Matrix/genetics , Sepharose , Stress, Mechanical , Time Factors , Tissue Engineering/methods
13.
Clinics ; 67(2): 99-106, 2012. ilus, graf, tab
Article in English | LILACS | ID: lil-614632

ABSTRACT

OBJECTIVES: Understanding the changes in chondrogenic gene expression that are involved in the differentiation of human adipose-derived stem cells to chondrogenic cells is important prior to using this approach for cartilage repair. The aims of the study were to characterize human adipose-derived stem cells and to examine chondrogenic gene expression after one, two, and three weeks of induction. MATERIALS AND METHODS: Human adipose-derived stem cells at passage 4 were evaluated by flow cytometry to examine the expression of surface markers. These adipose-derived stem cells were tested for adipogenic and osteogenic differentiation capacity. Ribonucleic acid was extracted from the cells for quantitative polymerase chain reaction analysis to determine the expression levels of chondrogenic genes after chondrogenic induction. RESULTS: Human adipose-derived stem cells were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen and successfully differentiated into adipogenic and osteogenic lineages. The human adipose-derived stem cells aggregated and formed a dense matrix after chondrogenic induction. The expression of chondrogenic genes (collagen type II, aggrecan core protein, collagen type XI, COMP, and ELASTIN) was significantly higher after the first week of induction. However, a significantly elevated expression of collagen type X was observed after three weeks of chondrogenic induction. CONCLUSION: Human adipose-derived stem cells retain stem cell characteristics after expansion in culture to passage 4 and serve as a feasible source of cells for cartilage regeneration. Chondrogenesis in human adiposederived stem cells was most prominent after one week of chondrogenic induction.


Subject(s)
Humans , Adipose Tissue/cytology , Cartilage, Articular/cytology , Cell Differentiation/genetics , Chondrocytes/metabolism , Chondrogenesis/genetics , Collagen/metabolism , Mesenchymal Stem Cells , Adipogenesis/genetics , Biomarkers/metabolism , Cells, Cultured , Chondrocytes/cytology , Collagen/genetics , Elastin/genetics , Elastin/metabolism , Flow Cytometry , Gene Expression Regulation , Mesenchymal Stem Cells , Osteogenesis/genetics , RNA, Messenger/genetics , SOX9 Transcription Factor/genetics , SOX9 Transcription Factor/metabolism , Time Factors
14.
Clinics ; 67(1): 35-40, 2012. ilus
Article in English | LILACS | ID: lil-610621

ABSTRACT

OBJECTIVE: Hypoxia-inducible factor 1 alpha regulates genes related to cellular survival under hypoxia. This factor is present in osteroarthritic chondrocytes, and cytokines, such as interleukin-1 beta, participate in the pathogenesis of osteoarthritis, thereby increasing the activities of proteolytic enzymes, such as matrix metalloproteinases, and accelerating cartilage destruction. We hypothesize that Hypoxia Inducible Factor-1 alpha (HIF-1α) can regulate cytokines (catabolic action) and/or growth factors (anabolic action) in osteoarthritis. The purpose of this study was to investigate the modulation of HIF-1α in human osteoarthritic chondrocytes by interleukin-1 beta (IL-1β) and insulin-like growth factors I (IGF-I) and II (IGF-II) and to determine the involvement of the phosphatidylinositol-3kinase (PI-3K) pathway in this process. METHODS: Human osteroarthritic chondrocytes were stimulated with IL-1β, IGF-I and IGF-II and LY294002, a specific inhibitor of PI-3K. Nuclear protein levels and gene expression were analyzed by western blot and quantitative reverse transcription-polymerase chain reaction analyses, respectively. RESULTS: HIF-1α expression was upregulated by IL-1β at the protein level but not at the gene level. IGF-I treatment resulted in increases in both the protein and mRNA levels of HIF-1α , whereas IGF-II had no effect on its expression. However, all of these stimuli exploited the PI-3K pathway. CONCLUSION: IL-1β upregulated the levels of HIF-1α protein post-transcriptionally, whereas IGF-I increased HIF-1α at the transcript level. In contrast, IGF-II did not affect the protein or gene expression levels of HIF-1α . Furthermore, all of the tested stimuli exploited the PI-3K pathway to some degree. Based on these findings, we are able to suggest that Hypoxia inducible Factor-1 exhibits protective activity in chondrocytes during osteoarthritis.


Subject(s)
Humans , Chondrocytes/drug effects , Gene Expression Regulation/drug effects , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Insulin-Like Growth Factor I/pharmacology , Insulin-Like Growth Factor II/pharmacology , Interleukin-1beta/pharmacology , Osteoarthritis/metabolism , Chondrocytes/metabolism , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Osteoarthritis/genetics , /antagonists & inhibitors , /metabolism , RNA, Messenger/analysis , Statistics, Nonparametric , Signal Transduction/drug effects , Signal Transduction/genetics
15.
Rev. chil. reumatol ; 27(2): 83-86, 2011. ilus
Article in Spanish | LILACS | ID: lil-609916

ABSTRACT

La osteoartritis es una enfermedad de alto impacto que produce importante morbilidad y discapacidad en adultos sobre 60 años. Su proceso patológico se caracteriza por degeneración del cartílago articular, asociado a inflamación secundaria, lo que produce cambios proliferativos con la formación de osteofitos y esclerosis del hueso subcondral. Actualmente existe evidencia de la importancia del proceso inflamatorio subyacente, destacando la participación de polimorfismos genéticos que fomentan el proceso inflamatorio. Asociado a esto, se ha evidenciado un cambio en el fenotipo del condrocito, el cual produce mayor cantidad de IL-1-beta y TNF-alfa, las que activan el catabolismo del cartílago. Además, se postula que la sinovial actuaría como un potenciador de la inflamación, pues los fibroblastos ahí presentes son capaces de producir diversas sustancias proinflamatorias, tales como citoquinas, prostaglandinas y óxido nítrico. Por otra parte, alteraciones mecánicas y la obesidad pueden activar al condrocito, fomentado su cambio de fenotipo, lo que aumenta la producción de citoquinas proinflamatorias que aumentan el daño cartilaginoso. Sin embargo, aún no existe claridad sobre los “gatillantes” exactos del proceso inflamatorio en OA.


Osteoarthritis (OA) is a disease of high impact that produces significant morbidity and disability in adults over 60 years. His disease process is characterized by degeneration of articular cartilage, associated with secondary inflammation that produces proliferative changes with osteophyte formation and subchondral bone sclerosis. There is evidence of the importance of the underlying inflammatory process, highlighting the involvement of genetic polymorphisms that promote the inflammatory process. Associated with this has shown a change in chondrocyte phenotype which produces more IL-1-beta and TNF-alpha, which activate the catabolism of cartilage. Furthermore, it is postulated that synovial act as an enhancer of inflammation present there as fibroblasts are capable of producing various proinflammatory substances such as cytokines, prostaglandins and nitric oxide. Moreover, mechanical alterations and obesity can activate the chondrocyte phenotype promoted the change of increasing the production of proinflammatory cytokines that promote cartilage damage. However, even there is no clarity on the exact “gatillantes” the inflammatory process in OA.


Subject(s)
Humans , Inflammation/metabolism , Osteoarthritis/metabolism , Biomechanical Phenomena , Cytokines/metabolism , Chondrocytes/metabolism , Inflammation/genetics , Obesity/metabolism , Osteoarthritis/genetics , Stress, Mechanical
16.
Article in English | WPRIM | ID: wpr-205261

ABSTRACT

Mesenchymal stem cells (MSCs) have the capacity to proliferate and differentiate into multiple connective tissue lineages, which include cartilage, bone, and fat. Cartilage differentiation and chondrocyte maturation are required for normal skeletal development, but the intracellular pathways regulating this process remain largely unclear. This study was designed to identify novel genes that might help clarify the molecular mechanisms of chondrogenesis. Chondrogenesis was induced by culturing human bone marrow (BM) derived MSCs in micromass pellets in the presence of defined medium for 3, 7, 14 or 21 days. Several genes regulated during chondrogenesis were then identified by reverse transcriptase-polymerase chain reaction (RT-PCR). Using an ABI microarray system, we determined the differential gene expression profiles of differentiated chondrocytes and BM-MSCs. Normalization of this data resulted in the identification of 1,486 differentially expressed genes. To verify gene expression profiles determined by microarray analysis, the expression levels of 10 genes with high fold changes were confirmed by RT-PCR. Gene expression patterns of 9 genes (Hrad6B, annexinA2, BMP-7, contactin-1, peroxiredoxin-1, heat shock transcription factor-2, synaptotagmin IV, serotonin receptor-7, Axl) in RT-PCR were similar to the microarray gene expression patterns. These findings provide novel information concerning genes involved in the chondrogenesis of human BM-MSCs.


Subject(s)
Bone Marrow Cells/cytology , Cell Differentiation , Chondrocytes/metabolism , Chondrogenesis/genetics , Gene Expression Profiling , Humans , Mesenchymal Stem Cells/cytology , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Time Factors
17.
Article in English | IMSEAR | ID: sea-135564

ABSTRACT

Background & objectives: Several in vitro studies have shown the importance of mechanical compression or hydrostatic pressure (HP) as a modulator of cartilage metabolism. The present study was undertaken to evaluate the in vitro effects of cyclical low HP (1-5 MPa) and continuous high HP (24 MPa) applied in the presence or absence of interleukin (IL)-1β on human osteoarthritis (OA) chondrocytes. Methods: Chondrocytes obtained from OA cartilage were cultivated for 48 h and then exposed to pressurization in the presence or absence of IL-1β. After pressurization, the culture medium was collected to detect the amount of proteoglycans (PG) and nitric oxide (NO) and the chondrocytes were immediately fixed for transmission electron microscopy (TEM) and processed for immunocytochemistry to localize the inducible nitric oxide synthase (iNOS). Results: A significant increase in the level of PG and a small, non-significant, decrease in NO production were observed upon exposure to cyclical low HP. On the other hand, exposure to continuous high HP resulted in a significant decrease in the PG levels and a significant increase in NO production. The presence of IL-1β led to a significant decrease in PG levels as well as a significant increase in NO production. The cyclical low HP did not increase the PG levels significantly but caused a statistically significant decrease in NO production in cultures damaged with IL-1β. The continuous high HP in chondrocyte cultures stimulated with IL-1β did not significantly decrease PG production, but significantly increased NO production. The results concerning metabolic production were further confirmed by morphological findings obtained by TEM and immunocytochemical studies. Interpretation & conclusion: The findings of this study confirmed that the response of chondrocytes varies with magnitude and frequency of HP. These findings are important to understand aetiopathogenetic mechanisms of OA and to find out which type of physical activity may be best suited for the prevention and therapy of OA.


Subject(s)
Cells, Cultured , Chondrocytes/metabolism , Chondrocytes/ultrastructure , Humans , Hydrostatic Pressure , Immunohistochemistry , Interleukin-1beta/metabolism , Microscopy, Electron, Transmission , Nitric Oxide/metabolism , Nitric Oxide Synthase/metabolism , Osteoarthritis/metabolism , Osteoarthritis/physiopathology , Proteoglycans/metabolism
19.
Acta ortop. bras ; 17(4): 242-246, 2009. ilus, tab
Article in English, Portuguese | LILACS | ID: lil-525657

ABSTRACT

OBJETIVOS: O presente estudo teve como objetivo cultivar condrócitos retirados da articulação do joelho de coelhos encapsulados em hidrogel de alginato (HA) e caracterizar a produção de matriz extracelular (ECM). MÉTODOS: A cartilagem articular foi removida do joelho de coelhos, com três a seis meses, fragmentada em pedaços de 1mm e submetida à digestão enzimática. Uma concentração de 1x106 céls/mL foram ressuspensas em uma solução de alginato de sódio a 1,5 por cento (w/v), em seguida fez-se o processo de gelatinização em CaCl2 (102 mM), permitindo a formação do HA e cultivo em meio DMEM-F12 durante quatro semanas. A distribuição das células e a ECM foram acessadas através das secções histológicas coradas com e azul de toluidina hematoxilina e eosina (HE). RESULTADOS: Houve um aumento no número e na viabilidade dos condrócitos durante as quatro semanas de cultura. Através das análises histológicas dos HAs corados com azul de toluidina e HE foi possível observar a distribuição definida dos condrócitos no hidrogel, assemelhando-se a grupos isógenos e formação de matriz territorial. CONCLUSÃO: Este estudo demonstrou a eficiência do HA como arcabouço para ser usado na cultura de condrócitos, constituindo uma alternativa no reparo de lesões na cartilagem articular.


OBJECTIVES: The aim of this study was to culture chondrocytes from knee joint cartilage of rabbits encapsulated in alginate hydrogel (HA) and to characterize the production of extracelular matrix (ECM). METHODS: Joint cartilage was obtained from rabbits' knees, three to six months old, fragmented into 1-mm pieces and submitted to enzymatic digestion. A concentration of 1x106 cells/mL were re-suspended into a 1.5 percent (w/v) sodium alginate solution, followed by gel formation process with CaCl2 (102 mM), allowing HA to build for culturing it into a DMEM-F12 medium for four weeks. The distribution of cells and ECM were assessed from histological slices stained toluidine blue and hematoxyline-eosin (HE). RESULTS: There was an increase of the number and viability of the chondrocytes during the four weeks of culture. By assessing the histological sections stained with toluidine blue and HE, we could note the definitive distribution of chondrocytes in the hydrogel, similarly to isogenous groups and territorial matrix formation. CONCLUSION: In this study, the alginate was shown to be an effective scaffold for use in chondrocytes culture, constituting an alternative for repairing joint cartilage defects.


Subject(s)
Animals , Rabbits , Bone Regeneration , Cartilage, Articular/physiopathology , Chondrocytes/metabolism , Hydrogels/pharmacokinetics , Alginates , Organ Culture Techniques
20.
Article in English | WPRIM | ID: wpr-223663

ABSTRACT

BACKGROUND: The mechanism by which mutant cartilage oligomeric matrix protein (COMP) induces a pseudoachondroplasia phenotype remains unknown, and the reason why a mutation of a minor protein of the growth plate cartilage causes total disruption of endochondral bone formation has not yet been determined. The current study was performed to investigate the effects of mutated COMP on the synthesis of the cartilage-specific major matrix proteins of Swarm rat chondrosarcoma chondrocytes. METHODS: The Swarm rat chondrosarcoma chondrocytes transfected with a chimeric construct, which consisted of a mutant gene of human COMP and an amino acid FLAG tag sequence, were cultured in agarose gel. Formation of extracellular proteoglycan and type-II collagen by the cells was evaluated by immunohistochemical staining and measuring the (35)S-sulfate incorporation. RESULTS: No difference was observed for the detection of type-II collagen among the cell lines expressing mutant COMP and the control cell lines. Histochemical staining of sulfated proteoglycans with safranin-O showed that lesser amounts of proteoglycans were incorporated into the extracellular matrix of the chondrocytes transfected with the mutant gene. (35)S-sulfate incorporation into the cell/matrix fractions demonstrated markedly lower radiolabel incorporation, as compared to that of the control cells. CONCLUSIONS: Mutation of COMP has an important impact on the processing of proteoglycans, rather than type-II collagen, in the three-dimensional culture of Swarm rat chondrosarcoma chondrocytes.


Subject(s)
Aggrecans/analysis , Animals , Cells, Cultured , Chondrocytes/metabolism , Chondrosarcoma/metabolism , Collagen Type II/biosynthesis , Extracellular Matrix/metabolism , Extracellular Matrix Proteins/genetics , Glycoproteins/genetics , Humans , Mutation , Rats , Transfection
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