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1.
Article in English | WPRIM | ID: wpr-1007920

ABSTRACT

OBJECTIVES@#This study aims to investigate the effects and mechanisms of chondroitin sulfate (CS), dermatan sulfate (DS), and heparin (HEP) on chondrogenesis of murine chondrogenic cell line (ATDC5) cells and the maintenance of murine articular cartilage in vitro.@*METHODS@#ATDC5 and articular cartilage tissue explant were cultured in the medium containing different sulfated glycosaminoglycans. Cell proliferation, differentiation, cartilage formation, and mechanism were observed using cell proliferation assay, Alcian blue staining, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blot, respectively.@*RESULTS@#Results showed that HEP and DS primarily activated the bone morphogenetic protein (BMP) signal pathway, while CS primarily activated the protein kinase B (AKT) signal pathway, further promoted ATDC5 cell proliferation and matrix production, and increased Sox9, Col2a1, and Aggrecan expression.@*CONCLUSIONS@#This study investigated the differences and mechanisms of different sulfated glycosaminoglycans in chondrogenesis and cartilage homeostasis maintenance. HEP promotes cartilage formation and maintains the normal state of cartilage tissue in vitro, while CS plays a more effective role in the regeneration of damaged cartilage tissue.


Subject(s)
Animals , Mice , Cartilage/metabolism , Cell Differentiation , Cells, Cultured , Chondrocytes/metabolism , Chondrogenesis/physiology , Glycosaminoglycans/pharmacology
2.
Rev. Bras. Ortop. (Online) ; 56(3): 333-339, May-June 2021. tab, graf
Article in English | LILACS | ID: biblio-1288682

ABSTRACT

Abstract Objective To evaluate clinically and radiologically the results of the treatment of chondral lesions using collagen membrane - autologous matrix-induced chondrogenesis (AMIC). Methods This is a series of observational cases, in which 15 patients undergoing AMIC were analyzed. The clinical evaluation was made by comparing the Lysholm and International Knee Document Commitee (IKDC) scores in the pre- and postoperative period of 12 months, and radiological evaluation using the Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score in the same postoperative period. Results The mean age of the patients was 39.2 years old, and the mean size of the chondral lesions was 1.55cm2. There was a significant improvement in clinical scores, with a mean increase of 24.6 points on Lysholm and of 24.3 on IKDC after 12 months. In the radiological evaluation, MOCART had a mean of 65 points. It was observed that the larger the size of the lesion, the greater the improvement in scores. Conclusion Evaluating subjective clinical scores, the treatment of chondral lesions with the collagen membrane showed good results, as well as the evaluation of MOCART, with greater benefit in larger lesions.


Resumo Objetivo Avaliar clínica e radiologicamente os resultados do tratamento das lesões condrais com a membrana de colágeno - condrogênese autóloga induzida por matriz. Métodos Trata-se de uma série de casos observacional, na qual foram analisados 15 pacientes submetidos a condrogênese autóloga induzida por matriz. A avaliação clínica foi feita comparando os escores de Lysholm e International Knee Document Commitee (IKDC, na sigla em inglês) no pré- e pós-operatório de 12 meses, e avaliação radiológica através do escore de Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART, na sigla em inglês) no mesmo período de pós-operatório. Resultados A média de idade dos pacientes foi 39,2 anos, e a média do tamanho das lesões condrais foi de 1,55cm2. Houve uma melhora significativa nos escores clínicos, com média de aumento de 24,6 pontos no Lysholm e de 24,3 no IKDC, após 12 meses. Na avaliação radiológica, o MOCART teve média de 65 pontos. Observou-se que quanto maior o tamanho da lesão, maior foi a melhora nos escores. Conclusão Avaliando escores clínicos subjetivos, o tratamento das lesões condrais com a membrana de colágeno mostrou bons resultados, assim como a avaliação de MOCART, com maior benefício em lesões maiores.


Subject(s)
Humans , Male , Female , Adolescent , Adult , Middle Aged , Postoperative Period , Magnetic Resonance Spectroscopy , Cartilage, Articular , Collagen , Chondrogenesis , Knee Injuries
3.
Article in English | WPRIM | ID: wpr-881038

ABSTRACT

Due to the poor repair ability of cartilage tissue, regenerative medicine still faces great challenges in the repair of large articular cartilage defects. Quercetin is widely applied as a traditional Chinese medicine in tissue regeneration including liver, bone and skin tissues. However, the evidence for its effects and internal mechanisms for cartilage regeneration are limited. In the present study, the effects of quercetin on chondrocyte function were systematically evaluated by CCK8 assay, PCR assay, cartilaginous matrix staining assays, immunofluorescence assay, and western blotting. The results showed that quercetin significantly up-regulated the expression of chondrogenesis genes and stimulated the secretion of GAG (glycosaminoglycan) through activating the ERK, P38 and AKT signalling pathways in a dose-dependent manner. Furthermore, in vivo experiments revealed that quercetin-loaded silk protein scaffolds dramatically stimulated the formation of new cartilage-like tissue with higher histological scores in rat femoral cartilage defects. These data suggest that quercetin can effectively stimulate chondrogenesis in vitro and in vivo, demonstrating the potential application of quercetin in the regeneration of cartilage defects.


Subject(s)
Animals , Rats , Cartilage/cytology , Chondrocytes/drug effects , Chondrogenesis/drug effects , Extracellular Matrix/metabolism , Quercetin/pharmacology , Signal Transduction/drug effects , Tissue Scaffolds
4.
Arq. bras. med. vet. zootec. (Online) ; 71(2): 509-520, mar.-abr. 2019. graf, ilus
Article in English | VETINDEX, LILACS | ID: biblio-1011276

ABSTRACT

The aim of this study was to evaluate the effect of concentrations of caffeine on the viability, synthesis activity and gene expression in cultures of chondrocytes. Extracted articular cartilage from the femurs and tibias of 15 Wistar rats at three days old to isolate chondrocytes. Chondrocytes were cultured in chondrogenic medium (control) or supplemented with caffeine (0.5, 1.0, 2.0mM). Cell viability, alkaline phosphatase activity and collagen synthesis were assessed using colorimetric assays at 7, 14, 21 days. The chondrocyte cultures of all groups grown under coverslips were stained with hematoxylin-eosin to determine the percentage of cells/field and with PAS, safranin O, alcian blue to determine the percentage of matrix chondrogenic/field at 21 days. The expressions of gene transcripts for aggrecan, collagen-II, Sox-9, Runx-2 and alkaline phosphatase were also evaluated by RT-PCR at 21 days. The means were compared using Student-Newman-Keuls. Caffeine significantly reduced the conversion of MTT to formazan, percentage of cells/field, collagen synthesis, alkaline phosphatase activity, synthesis of PAS+, safranin O+ and alcian blue+ chondrogenic matrix, and the expression of aggrecan, Sox-9 and II collagen. It is concluded that caffeine at concentrations of 0.5, 1.0, 2.0mM has a direct inhibitory effect on chondrogenesis in cultures of chondrocytes from rats.(AU)


O objetivo deste estudo foi avaliar o efeito direto de concentrações de cafeína sobre a viabilidade, atividade de síntese e expressão gênica em culturas de condrócitos de ratos. As cartilagens dos fêmures e tíbias de 15 ratos Wistar com três dias foram extraídas para isolamento de condrócitos. Os condrócitos foram cultivados em meio condrogênico (controle) ou em meio acrescido de diferentes concentrações de cafeína (0,5, 1,0, 2,0mM). Foram avaliadas a viabilidade celular, a atividade da fosfatase alcalina e a síntese de colágeno por ensaios colorimétricos aos sete, 14 e 21 dias. Condrócitos cultivados sob lamínulas foram corados pela hematoxilina e eosina, para se determinar a porcentagem de células/campo, e pelo PAS, safranina O, alcian Blue, para se determinar a porcentagem de matriz condrogênica/campo aos 21 dias. Foi avaliada a expressão de transcriptos gênicos para Sox-9, Runx-2, agrecano, colágeno-II e fosfatase alcalina por qRT-PCR, aos 21 dias. As médias foram comparadas pelo Student-Newman-Keuls. A cafeína reduziu significativamente o MTT em cristais de formazan, a porcentagem de células/campo, a síntese de colágeno, a atividade da fosfatase alcalina e a síntese de matriz condrogênica PAS+, safranina O+, alcian blue+ e expressão de Sox-9 e colágeno-II. Conclui-se que a cafeína, nas concentrações de 0,5, 1,0, 2,0mM, apresenta efeito inibidor direto sobre a condrogênese em culturas de condrócitos de ratos.(AU)


Subject(s)
Animals , Female , Rats , Caffeine , Cartilage, Articular/drug effects , Chondrocytes/drug effects , Chondrogenesis/drug effects
5.
Article in English | WPRIM | ID: wpr-764053

ABSTRACT

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSCs) become hypertrophic in long term despite chondrogenic differentiation following the pathway of growth plate chondrocytes. This terminal differentiation leads to phenotypically unstable cartilage and was mirrored in vitro by addition of hypertrophy inducing medium. We investigated how intrinsic TGF-β signaling is altered in pro-hypertrophic conditions. METHODS AND RESULTS: Human bone marrow derived MSC were chondrogenically differentiated in 3D culture. At day 14 medium conditions were changed to 1. pro-hypertrophic by addition of T3 and withdrawal of TGF-β and dexamethasone 2. pro-hypertrophic by addition of BMP 4 and withdrawal of TGF-β and dexamethasone and 3. kept in prochondrogenic medium conditions. All groups were treated with and without TGFβ-type-1-receptor inhibitor SB431542 from day 14 on. Aggregates were harvested for histo- and immunohistological analysis at d14 and d28, for gene expression analysis (rt-PCR) on d1, d3, d7, d14, d17, d21 and d28 and for Western blot analysis on d21 and d28. Induction of hypertrophy was achieved in the pro-hypertrophic groups while expression of TGFβ-type-1- and 2-receptor and Sox 9 were significantly downregulated compared to pro-chondrogenic conditions. Western blotting showed reduced phosphorylation of Smad 2 and 3 in hypertrophic samples, reduced TGF-β-1 receptor proteins and reduced SOX 9. Addition of SB431542 did not initiate hypertrophy under pro-chondrogenic conditions, but was capable of enhancing hypertrophy when applied simultaneously with BMP-4. CONCLUSIONS: Our results suggest that the enhancement of hypertrophy in this model is a result of both activation of pro-hypertrophic BMP signaling and reduction of anti-hypertrophic TGFβ signaling.


Subject(s)
Humans , Blotting, Western , Bone Marrow , Cartilage , Chondrocytes , Chondrogenesis , Dexamethasone , Down-Regulation , Gene Expression , Growth Plate , Hypertrophy , In Vitro Techniques , Mesenchymal Stem Cells , Phosphorylation
6.
Article in Chinese | WPRIM | ID: wpr-813090

ABSTRACT

Articular cartilage lesions due to injury or other pathology are often difficult to heal, and the outcomes of the clinical treatment widely used today are far from satisfaction. Adipose-derived stem cells (ADSCs) are multipotent stem cells from adipose tissue. Tissue engineering based on the ability of ADSCs to differentiate into chondrocytes provides a new idea for the repair and regeneration of articular cartilage defects. The method for inducing the differentiation of ADSCs into chondrocytes in vitro who have been quite well established, which mainly include the use of growth factors and scaffolds to mimic the in vivo microenvironment, thereby promoting the differentiation of ADSCs into chondrocytes.


Subject(s)
Adipocytes , Adipose Tissue , Cartilage, Articular , Cell Differentiation , Cells, Cultured , Chondrocytes , Chondrogenesis , Stem Cells , Tissue Engineering
7.
Article in English | WPRIM | ID: wpr-761917

ABSTRACT

BACKGROUND: Articular cartilage lesions occur frequently but unfortunately damaged cartilage has a very limited intrinsic repair capacity. Therefore, there is a high need to develop technology that makes cartilage repair possible. Since joint damage will lead to (sterile) inflammation, development of this technology has to take into account the effects of inflammation on cartilage repair. METHODS: A literature search has been performed including combinations of the following keywords; cartilage repair, fracture repair, chondrogenesis, (sterile) inflammation, inflammatory factors, macrophage, innate immunity, and a number of individual cytokines. Papers were selected that described how inflammation or inflammatory factors affect chondrogenesis and tissue repair. A narrative review is written based on these papers focusing on the role of inflammation in cartilage repair and what we can learn from findings in other organs, especially fracture repair. RESULTS: The relationship between inflammation and tissue repair is not straightforward. Acute, local inflammation stimulates fracture repair but appears to be deleterious for chondrogenesis and cartilage repair. Systemic inflammation has a negative effect on all sorts of tissue repair. CONCLUSION: Findings on the role of inflammation in fracture repair and cartilage repair are not in line. The currently widely used models of chondrogenesis, using high differentiation factor concentrations and corticosteroid levels, are not optimal. To make it possible to draw more valid conclusions about the role of inflammation and inflammatory factors on cartilage repair, model systems must be developed that better mimic the real conditions in a joint with damaged cartilage.


Subject(s)
Cartilage , Cartilage, Articular , Chondrogenesis , Cytokines , Immunity, Innate , Inflammation , Joints , Macrophages
8.
Korean Journal of Medicine ; : 145-151, 2019.
Article in Korean | WPRIM | ID: wpr-759928

ABSTRACT

Osteoarthritis is a musculoskeletal disease representative of an aging society. As medical conditions are usually complicated in an aging population, osteoarthritis becomes more frequently encountered in the physician's office. There is a growing need, therefore, for physicians to pay attention to this common orthopedic condition. Cartilage degeneration, arthritic pain, and joint dysfunction are major manifestations of osteoarthritis, and degenerated cartilage is difficult to repair with conventional treatment modalities. Scientists and physicians have developed various therapeutic strategies, including the use of stem cells. Here, we discuss previous and current progress in cartilage regenerative therapy against osteoarthritis.


Subject(s)
Adult Stem Cells , Aging , Cartilage , Chondrogenesis , Induced Pluripotent Stem Cells , Joints , Musculoskeletal Diseases , Orthopedics , Osteoarthritis , Physicians' Offices , Stem Cells
9.
Rev. Bras. Ortop. (Online) ; 53(6): 733-739, Nov.-Dec. 2018. tab, graf
Article in English | LILACS | ID: biblio-977903

ABSTRACT

ABSTRACT Objectives: To evaluate the clinical and functional results of patients diagnosed with full-thickness chondral defects on symptomatic knees who underwent a biological repair technique using autologous matrix-induced chondrogenesis. Methods: Seven patients who underwent surgical treatment due to chondral lesions in the knee by autologous matrix-induced chondrogenesis were evaluated. The Lysholm, Kujala and visual analog scale of pain questionnaires were applied before and 12 months after the surgery. Nuclear magnetic resonance images were evaluated 12 months after surgery according to MOCART (magnetic resonance observation of cartilage repair tissue) cartilage repair tissue score. Results: Of the seven patients evaluated, three presented defects classified as grade III and four as grade IV according to the International Cartilage Repair Society classification. Chondral defects were located in the medial femoral condyle (n = 2), patella (n = 2), and trochlea (n = 3). The mean age of the patients (six men and one woman) was 37.2 years (24-54 years). The mean chondral defect size was 2.11 cm2 (1.0-4.6 cm2). After 12 months, post-operative nuclear magnetic resonance showed resurfacing of the lesion site with scar tissue less thick than normal cartilage in all patients. The mean MOCART score was 66.42 points. A significant decrease in pain and an improvement in the Lysholm and Kujala scores were observed. Conclusion: The use of the collagen I/III porcine membrane was favorable for the treatment of chondral and osteochondral lesions of the knee when assessing the results using the VAS, Lysholm, and Kujala scores 1 year after surgery, as well as when assessing the magnetic resonance image of the lesion 6 months after surgery.


RESUMO Objetivos: Avaliar os resultados clínicos e funcionais dos pacientes com diagnóstico de lesões condrais de espessura total em joelhos sintomáticos submetidos a um método de reparação biológica por meio da técnica de condrogênese autóloga induzida por matriz. Métodos: Foram avaliados sete pacientes submetidos a tratamento cirúrgico devido a lesões condrais no joelho pela técnica de condrogênese autóloga induzida por matriz. Foram usados os questionários Lysholm e Kujala e a escala visual analógica da dor antes e após um ano de cirurgia. As imagens de ressonância nuclear magnética foram avaliadas após 12 meses de acordo com os critérios de reparo cartilaginoso de Mocart (magnetic resonance observation of cartilage repair tissue). Resultados: Dos sete pacientes avaliados, três apresentavam defeitos classificados como grau III e quatro como grau IV, de acordo com a classificação da International Cartilage Repair Society. Os defeitos condrais estavam no côndilo femoral medial (n = 2), na patela (n = 2) e na tróclea (n = 3). A média de idade dos sete pacientes (seis homens e uma mulher) foi de 37,2 anos (24 a 54). O tamanho médio dos defeitos condrais foi de 2,11 cm2 (1,0 a 4,6 cm2). Após 12 meses, a ressonância nuclear magnética pós-operatória mostrou preenchimento do local da lesão com tecido cicatricial menos espesso do que a cartilagem normal em todos os pacientes. O valor médio do questionário de Mocart após 12 meses foi de 66,42 pontos. Observou-se diminuição importante na dor e melhoria da avaliação dos questionários de Lysholm e Kujala. Conclusão: O uso da membrana de colágeno I/III de origem porcina se mostrou favorável no tratamento de lesões condrais e osteocondrais do joelho quando se avaliaram os resultados obtidos com a escala visual analógica da dor e o questionário de Lysholme Kujala um ano após a cirurgia, bem como quando se avaliou a imagem da lesão na ressonância magnética seis meses após a cirurgia.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Cartilage, Articular , Collagen , Chondrogenesis , Arthroplasty, Subchondral , Knee Injuries
10.
Article in Korean | WPRIM | ID: wpr-741518

ABSTRACT

Mesenchymal stem cells (MSCs) are useful candidates for tissue engineering and cell therapy. Physiological cell environment not only connects cells to each other, but also connects cells to the extracellular matrix that provide mechanical support, thus exposing the entire cell surface and activating signaling pathways. Hydrogel is a polymeric material that swells in water and maintains a distinct 3-dimensional (3D) network structure by cross linking. In this study, we investigated the optimized cellular function for canine adipose tissue-derived MSCs (cAD-MSCs) using hydrogel. We observed that the expression levels of Ki67 and proliferating cell nuclear antigen, which are involved in cell proliferation and stemness, were increased in transwell-hydrogel (3D-TN) compared to the transwell-normal (TN). Also, transforming growth factor-β1 and SOX9, which are typical bone morphogenesis-inducing factors, were increased in 3D-TN compared to the TN. Collagen type II alpha 1, which is a chondrocyte-specific marker, was increased in 3D-TN compared to the TN. Osteocalcin, which is a osteocyte-specific marker, was increased in 3DTN compared to the TN. Collectively, preconditioning cAD-MSCs via 3D culture systems can enhance inherent secretory properties that may improve the potency and efficacy of MSCs-based therapies for bone regeneration process.


Subject(s)
Bone Regeneration , Cell Proliferation , Cell- and Tissue-Based Therapy , Chondrogenesis , Collagen Type II , Extracellular Matrix , Hydrogels , Hydrogels , Mesenchymal Stem Cells , Osteocalcin , Osteogenesis , Polymers , Proliferating Cell Nuclear Antigen , Tissue Engineering , Water
11.
Article in English | WPRIM | ID: wpr-742376

ABSTRACT

Coculture between mesenchymal stem cells (MSCs) and chondrocytes has significant implications in cartilage regeneration. However, a conclusive understanding remains elusive. Previously, we reported that rabbit bone marrow-derived MSCs (rbBMSCs) could downregulate the differentiated phenotype of rabbit articular chondrocytes (rbACs) in a non-contact coculture system for the first time. In the present study, a systemic investigation was performed to understand the biological characteristics of chondrocytes in coculture with MSCs. Firstly, cells (MSCs and chondrocytes) from different origins were cocultured in transwell system. Different chondrocytes, when cocultured with different MSCs respectively, consistently demonstrated stimulated proliferation, transformed morphology and declined glycosaminoglycan secretion of chondrocytes. Next, cell surface molecules and the global gene expression of rbACs were characterized. It was found that cocultured rbACs showed a distinct surface molecule profile and global gene expression compared to both dedifferentiated rbACs and rbBMSCs. In the end, cocultured rbACs were passaged and induced to undergo the chondrogenic redifferentiation. Better growth and chondrogenesis ability were confirmed compared with control cells without coculture. Together, chondrocytes display comprehensive changes in coculture with MSCs and the cocultured rbACs are beneficial for cartilage repair.


Subject(s)
Cartilage , Chondrocytes , Chondrogenesis , Coculture Techniques , Gene Expression , Mesenchymal Stem Cells , Phenotype , Population Characteristics , Regeneration
12.
Kosin Medical Journal ; : 200-207, 2018.
Article in English | WPRIM | ID: wpr-718466

ABSTRACT

Tracheobronchopathia osteoplastica (TO) is a rare benign disease in which the anterior inner wall of the tracheobronchus changes because of abnormal chondrogenesis or ossification, while the posterior wall of the trachea is spared. The etiology is not clearly understood, but may relate with chronic infection, inflammation, and trauma. In some case studies, it has also been reported to be accompanied by other chronic diseases such as atrophic rhinitis and amyloidosis. However, Coexistence of TO and tuberculosis has rarely been reported, and has never been reported in Korea. Here, we report a case of a 70-year-old male patient who complained of hemoptysis and whose case was diagnosed as TO and pulmonary tuberculosis through bronchoscopy with bronchial washing and biopsy.


Subject(s)
Aged , Humans , Male , Amyloidosis , Biopsy , Bronchoscopy , Chondrogenesis , Chronic Disease , Hemoptysis , Inflammation , Korea , Rhinitis, Atrophic , Trachea , Tuberculosis , Tuberculosis, Pulmonary
13.
Article in English | WPRIM | ID: wpr-713808

ABSTRACT

For the cartilage repair, the cell sources currently adopted are primarily chondrocytes or mesenchymal stem cells (MSCs). Due to the fact that chondrocytes dedifferentiate during 2-dimensional (2D) expansion, MSCs are generally more studied and considered to have higher potential for cartilage repair purposes. Here we question if the dedifferentiated chondrocytes can regain the chondrogenic potential, to find potential applications in cartilage repair. For this we chose chondrocytes at passage 12 (considered to have sufficiently dedifferentiated) and the expression of chondrogenic phenotypes and matrix syntheses were examined over 14 days. In particular, the chondrogenic potential of MSCs was also compared. Results showed that the dedifferentiated chondrocytes proliferated actively over 14 days with almost 2.5-fold increase relative to MSCs. Moreover, the chondrogenic ability of chondrocytes was significantly higher than that of MSCs, as confirmed by the expression of a series of mRNA levels and the production of cartilage extracellular matrix molecules in 2D-monolayer and 3-dimensional (3D)-spheroid cultures. Of note, the significance was higher in 3D-culture than in 2D-culture. Although more studies are needed such as the use of different cell passages and human cell source, and the chondrogenic confirmation under in vivo conditions, this study showing that the dedifferentiated chondrocytes can also be a suitable cell source for the cell-based cartilage repair, as a counterpart of MSCs, will encourage further studies regarding this issue.


Subject(s)
Animals , Humans , Rats , Cartilage , Chondrocytes , Chondrogenesis , Extracellular Matrix , Mesenchymal Stem Cells , Phenotype , RNA, Messenger
14.
Article in English | WPRIM | ID: wpr-715003

ABSTRACT

The aim of this study was to prepare inclusion nanocomplexes of hyaluronic acid-β-cyclodextrin and simvastatin (HA-β-CD/SIM) and evaluate in vitro anti-inflammation effects on lipopolysaccharide (LPS)-activated synoviocytes and chondrogenic differentiation effects on rat adipose-derived stem cells (rADSCs). The β-CD moieties in HA-β-CD could incorporate SIM to form HA-β-CD/SIM nanocomplexes with diameters of 297–350 nm. HA-β-CD/SIM resulted in long-term release of SIM from the nanocomplexes for up to 63 days in a sustained manner. In vitro studies revealed that HA-β-CD/SIM nanocomplexes were able to effectively and dose-dependently suppress the mRNA expression levels of proinflammatory markers such as matrix metallopeptidase-3 (MMP-3), MMP-13, cyclooxygenase-2 (COX-2), a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), interleukin-6 (IL-6), and tumor necrosis factor (TNF-α) in LPS-stimulated synoviocytes. HA-β-CD/SIM-treated rADSCs significantly and dose-dependently enhanced mRNA expressions of aggrecan, collagen type II (COL2A1), and collagen type X (COL10A1), implying that HA-β-CD/SIM greatly induced the chondrogenic differentiation of rADSCs. Conclusively, HA-β-CD/SIM nanocomplexes will be a promising therapeutic material to alleviate inflammation as well as promote chondrogenesis.


Subject(s)
Animals , Rats , Aggrecans , Chondrogenesis , Collagen Type II , Collagen Type X , Cyclooxygenase 2 , In Vitro Techniques , Inflammation , Interleukin-6 , RNA, Messenger , Simvastatin , Stem Cells , Thrombospondins , Tumor Necrosis Factor-alpha
15.
Clinics ; Clinics;73: e268, 2018. tab, graf
Article in English | LILACS | ID: biblio-890754

ABSTRACT

OBJECTIVES: Articular cartilage is vulnerable to injuries and undergoes an irreversible degenerative process. The use of amniotic fluid mesenchymal stromal stem cells for the reconstruction of articular cartilage is a promising therapeutic alternative. The aim of this study was to investigate the chondrogenic potential of amniotic fluid mesenchymal stromal stem cells from human amniotic fluid from second trimester pregnant women in a micromass system (high-density cell culture) with TGF-β3 for 21 days. METHODS: Micromass was performed using amniotic fluid mesenchymal stromal stem cells previously cultured in a monolayer. Chondrocytes from adult human normal cartilage were used as controls. After 21 days, chondrogenic potential was determined by measuring the expression of genes, such as SOX-9, type II collagen and aggrecan, in newly differentiated cells by real-time PCR (qRT-PCR). The production of type II collagen protein was observed by western blotting. Immunohistochemistry analysis was also performed to detect collagen type II and aggrecan. This study was approved by the local ethics committee. RESULTS: SOX-9, aggrecan and type II collagen were expressed in newly differentiated chondrocytes. The expression of SOX-9 was significantly higher in newly differentiated chondrocytes than in adult cartilage. Collagen type II protein was also detected. CONCLUSION: We demonstrate that stem cells from human amniotic fluid are a suitable source for chondrogenesis when cultured in a micromass system. amniotic fluid mesenchymal stromal stem cells are an extremely viable source for clinical applications, and our results suggest the possibility of using human amniotic fluid as a source of mesenchymal stem cells.


Subject(s)
Humans , Pregnancy , Cell Culture Techniques/methods , Chondrocytes/cytology , Chondrogenesis , Mesenchymal Stem Cells/cytology , Gene Expression , Cell Differentiation , Collagen Type II/analysis , Aggrecans/metabolism , Transforming Growth Factor beta3/metabolism , SOX9 Transcription Factor/metabolism , Amniotic Fluid
16.
Braz. j. microbiol ; Braz. j. microbiol;48(1): 125-131, Jan.-Mar. 2017. graf
Article in English | LILACS | ID: biblio-839349

ABSTRACT

Abstract Small ruminant lentiviruses isolated from peripheral blood leukocytes and target organs can be propagated in vitro in fibroblasts derived from goat synovial membrane cells. These cells are obtained from tissues collected from embryos or fetuses and are necessary for the establishment of the fibroblast primary culture. A new alternative type of host cells, derived from goat umbilical cord, was isolated and characterized phenotypically with its main purpose being to obtain cell monolayers that could be used for the diagnosis and isolation of small ruminant lentiviruses in cell culture. To accomplish this goal, cells were isolated from umbilical cords; characterized phenotypically by flow cytometry analysis; differentiate into osteogenic, chondrogenic and adipogenic lineage; and submitted to viral challenge. The proliferation of goat umbilical cord cells was fast and cell monolayers formed after 15 days. These cells exhibited morphology, immunophenotype, growth characteristics, and lineage differentiation potential similar to mesenchymal stem cells of other origins. The goat umbilical cord derived cells stained positive for vimentin and CD90, but negative for cytokeratin, CD34 and CD105 markers. Syncytia and cell lysis were observed in cell monolayers infected by CAEV-Cork and MVV-K1514, showing that the cells are permissive to small ruminant lentivirus infection in vitro. These data demonstrate the proliferative competence of cells derived from goat umbilical cords and provide a sound basis for future research to standardize this cell lineage.


Subject(s)
Animals , Umbilical Cord/cytology , Lentivirus/physiology , Mesenchymal Stem Cells/virology , Osteogenesis , Virus Replication , In Vitro Techniques , Goats , Biomarkers , Cell Differentiation , Cells, Cultured , Immunophenotyping , Cell Culture Techniques , Chondrogenesis , Cytopathogenic Effect, Viral , Adipogenesis , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/pathology
17.
Article in English | WPRIM | ID: wpr-96452

ABSTRACT

Endochondral ossification is the fundamental process of skeletal development in vertebrates. Chondrocytes undergo sequential steps of differentiation, including mesenchymal condensation, proliferation, hypertrophy, and mineralization. These steps, which are required for the morphological and functional changes in differentiating chondrocytes, are strictly regulated by a complex transcriptional network. Biochemical and mice genetic studies identified chondrogenic transcription factors critical for endochondral ossification. The transcription factor sex-determining region Y (SRY)-box 9 (Sox9) is essential for early chondrogenesis, and impaired Sox9 function causes severe chondrodysplasia in humans and mice. In addition, recent genome-wide chromatin immunoprecipitation-sequencing studies revealed the precise regulatory mechanism of Sox9 during early chondrogenesis. Runt-related transcription factor 2 promotes chondrocyte hypertrophy and terminal differentiation. Interestingly, endoplasmic reticulum (ER) stress-related transcription factors have recently emerged as novel regulators of chondrocyte differentiation. Here we review the transcriptional mechanisms that regulate endochondral ossification, with a focus on Sox9.


Subject(s)
Animals , Humans , Mice , Chondrocytes , Chondrogenesis , Chromatin , Endoplasmic Reticulum , Gene Regulatory Networks , Hypertrophy , Miners , Osteogenesis , SOX9 Transcription Factor , Transcription Factors , Vertebrates
18.
Article in English | WPRIM | ID: wpr-655773

ABSTRACT

Histone deacetylase inhibitors (HDACi) are a class of compounds that suppress the function of histone deacetylases (HDACs). This study was performed to examine the effects of Trichostatin A (TSA), a typical HDACi, on chondrogenesis of human bone marrow mesenchymal stem cells (hBMMSCs) and related molecular pathways. After evaluating the concentration for cytotoxicity and HDAC activity, hBMMSCs underwent chondrogenic differentiation in pellet culture with or without TSA for 21 days. The weight of TSA-treated pellets was 25% lower than that of untreated pellets. DNA level was not significantly different, but glycosaminoglycan content per DNA level was lower in TSA-treated pellets than that of untreated pellets. Gene expression of the chondrogenic markers (SOX9, Aggrecan, and Col2A1) decreased by by 12.9-fold, 8.9-fold, and 7.6-fold respectively in TSA-treated pellets compared with that in TSA-untreated pellets. TSA-treated pellets had lower cell density and lower proteoglycan staining content compared with those of TSA-untreated pellets. A microarray analysis from TSA-treated pellets showed that 1,467 chondrogenic-related genes were downregulated and 1,524 were upregulated by more than 2-fold compared with TSA-untreated pellets. Col10A1, TGF-β3, and SOX9 decreased significantly by 10-fold, 2.1-fold, and 3.2-fold respectively in TSA-treated pellets compared with those in untreated pellets, whereas expression of BMP4 and FGFR3 increased significantly by 2.1-fold and 5.4-fold respectively. It is concluded that TSAinhibits chondrogenesis and does not seem to be useful for cartilage tissue engineering of hBMMSCs.


Subject(s)
Humans , Aggrecans , Bone Marrow , Cartilage , Cell Count , Chondrogenesis , DNA , Gene Expression , Histone Deacetylase Inhibitors , Histone Deacetylases , Mesenchymal Stem Cells , Microarray Analysis , Proteoglycans , Tissue Engineering
19.
Protein & Cell ; (12): 439-445, 2017.
Article in English | WPRIM | ID: wpr-757009

ABSTRACT

Mesenchymal stem cells (MSCs) are progenitors of connective tissues, which have emerged as important tools for tissue engineering due to their differentiation potential along various cell types. In recent years, accumulating evidence has suggested that the regulation of mitochondria dynamics and function is essential for successful differentiation of MSCs. In this paper, we review and provide an integrated view on the role of mitochondria in MSC differentiation. The mitochondria are maintained at a relatively low activity level in MSCs, and upon induction, mtDNA copy number, protein levels of respiratory enzymes, the oxygen consumption rate, mRNA levels of mitochondrial biogenesis-associated genes, and intracellular ATP content are increased. The regulated level of mitochondrial ROS is found not only to influence differentiation but also to contribute to the direction determination of differentiation. Understanding the roles of mitochondrial dynamics during MSC differentiation will facilitate the optimization of differentiation protocols by adjusting biochemical properties, such as energy production or the redox status of stem cells, and ultimately, benefit the development of new pharmacologic strategies in regenerative medicine.


Subject(s)
Animals , Humans , Adipogenesis , Physiology , Cell Differentiation , Physiology , Chondrogenesis , Physiology , Mesenchymal Stem Cells , Cell Biology , Metabolism , Mitochondria , Genetics , Metabolism , Mitochondrial Proteins , Genetics , Metabolism , Osteogenesis , Physiology , RNA , Genetics , Metabolism , RNA, Messenger , Genetics , Metabolism , RNA, Mitochondrial , Reactive Oxygen Species , Metabolism
20.
Rev. bras. pesqui. méd. biol ; Braz. j. med. biol. res;50(2): e5988, 2017. graf
Article in English | LILACS | ID: biblio-839254

ABSTRACT

This study was undertaken to clarify the role and mechanism of pyruvate dehydrogenase kinase isoform 2 (PDK2) in chondrogenic differentiation of mesenchymal stem cells (MSCs). MSCs were isolated from femurs and tibias of Sprague-Dawley rats, weighing 300-400 g (5 females and 5 males). Overexpression and knockdown of PDK2 were transfected into MSCs and then cell viability, adhesion and migration were assessed. Additionally, the roles of aberrant PDK2 in chondrogenesis markers SRY-related high mobility group-box 6 (Sox6), type ΙΙ procollagen gene (COL2A1), cartilage oligomeric matrix protein (COMP), aggrecan (AGC1), type ΙX procollagen gene (COL9A2) and collagen type 1 alpha 1 (COL1A1) were measured by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The expressions of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) and extracellular regulated protein kinase (ERK) were measured. Overexpressing PDK2 promoted cell viability, adhesion and inhibited cell migration in MSCs (all P<0.05). qRT-PCR assay showed a potent increase in the mRNA expressions of all chondrogenesis markers in response to overexpressing PDK2 (P<0.01 or P<0.05). PDK2 overexpression also induced a significant accumulation in mRNA and protein expressions of JNK, p38MAPK and ERK in MSCs compared to the control (P<0.01 or P<0.05). Meanwhile, silencing PDK2 exerted the opposite effects on MSCs. This study shows a preliminary positive role and potential mechanisms of PDK2 in chondrogenic differentiation of MSCs. It lays the theoretical groundwork for uncovering the functions of PDK2 and provides a promising basis for repairing cartilage lesions in osteoarthritis.


Subject(s)
Animals , Male , Female , Rats , Chondrogenesis/physiology , JNK Mitogen-Activated Protein Kinases/physiology , MAP Kinase Signaling System/physiology , Mesenchymal Stem Cells/physiology , Protein Serine-Threonine Kinases/physiology , SOXE Transcription Factors/physiology , Cell Differentiation , Rats, Sprague-Dawley , Transcriptional Activation , Up-Regulation
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