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Article in English | WPRIM | ID: wpr-1007920


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.

Animals , Mice , Cartilage/metabolism , Cell Differentiation , Cells, Cultured , Chondrocytes/metabolism , Chondrogenesis/physiology , Glycosaminoglycans/pharmacology
Article in English | WPRIM | ID: wpr-935065


@#End-stage ankle arthritis represents an “unmet medical need”, awaiting an appropriate time for joint arthroplasty or arthrodesis. We report three cases of end-stage ankle arthritis treated along the principles developed for chondrogenesis of the knee joint with autologous peripheral blood stem cells, resulting in reversal of the ankle arthritis. The improvement in clinical outcome measure scores (Ankle Osteoarthritis Scale total score) with a minimum two-year follow-up were comparable to total ankle replacement (TAR), arthroscopic ankle arthrodesis (AAA) and open ankle arthrodesis (OAA).

International Journal of Surgery ; (12): 273-278, 2022.
Article in Chinese | WPRIM | ID: wpr-930008


The normal ventilatory function is severely impaired by tracheal traumas, stenoses, tumors and some congenital diseases, which could result in tissue hypoxia and endangering the life of the patient. Resection and reconstruction of tracheal lesions is the most effective way to treat these diseases. At present, there is still no long-term safe and reliable method to achieve the reconstruction of long-segment trachea injury in clinical practice, and tissue-engineered trachea may be the solution to this situation. Cartilage, as one of the most important parts of tissue engineered trachea, plays a key role in providing mechanical support and maintaining the integrity of trachea. Tracheal tissue engineering cartilage regeneration process consists of several important parts, including the source of the cartilage cells, tissue engineering scaffold construction strategy and hydrogel composite scaffold material preparation, and the affecting factors of biological activity and application. This article reviews the new strategies of tissue engineered tracheal cartilage regeneration and the existing obstacles in order to provide reference for clinical practice.

Article in English | WPRIM | ID: wpr-962211


@#Introduction: Autologous matrix-induced chondrogenesis (AMIC) is a one-step surgical cartilage repair procedure involving the insertion of a scaffold into the chondral defect after microfracture. BST-CarGel [Smith and Nephew, Watford, England] is an injectable chitosan-based scaffold which can more easily fill defects with irregular shapes and be used to treat vertical or roof chondral lesions. The study aims to evaluate the clinical outcomes of knee cartilage repair with microfracture surgery and BST-CarGel using the AMIC technique for a minimum of two years. Materials and methods: A prospective study of patients undergoing cartilage repair with microfracture surgery and BST-CarGel at our institution from 2016 to 2019 was performed. Clinical outcomes were determined using the Lysholm Knee Scoring System and Knee Injury and Osteoarthritis Outcome Score (KOOS). These questionnaires were administered before the surgery and at a minimum of two years after surgery. Results: A total of 21 patients were identified and recruited into the study. 31 cartilage defects were seen and treated in 21 knees. These included horizontal lesions (e.g., trochlear, lateral tibial plateau), vertical lesions (e.g., medial femoral condyle, lateral femoral condyle) and inverted lesions (e.g., patella). No complications or reoperations were seen in our study population. For the average duration of follow-up of 42.5±8.55 months, there was an average improvement in Lysholm score of 25.8±18.6 and an average improvement in KOOS score of 22.5±15.0. Conclusion: BST-CarGel with microfracture surgery using the AMIC technique is a safe and effective treatment for cartilage defects in the short to medium term.

Rev. colomb. cienc. pecu ; 34(3): 212-223, July-Sept. 2021. tab, graf
Article in English | LILACS-Express | LILACS | ID: biblio-1408022


Abstract Background: Osteoarthritis is a complex degenerative disease with several factors contributing to joint damage. Objective: To compare the potential effect of hyaluronic acid (HA) and triamcinolone acetonide (TA), alone or combined, on the in vitro chondrogenic differentiation process of mesenchymal stem cells (MSCs). Methods: MSCs were divided into four groups: Control, HA, TA, and HA/TA combined. Each treatment group was cultured for 14 days in chondrogenic differentiation medium. The chondrogenic differentiation potential was assessed by histology and immunohistochemistry. Results: The HA and HA/TA-treated MSCs presented histological characteristics similar to native chondrocytes. The extracellular matrix (ECM) of TA-treated MSCs was compact and organized. Glycosaminoglycan staining was intense in Control, moderate in TA, slight in HA/TA, and undetectable in HA. Type II collagen immunoreactivity was high in the TA-treated ECM and MSCs. Conclusions: Histological analysis shows that HA influences morphological development similar to chondrocytes of the MSCs, but with low expression of specific cartilage molecules. The TA promotes formation of a compact and organized ECM.

Resumen Antecedentes: La osteoartritis es una enfermedad degenerativa compleja en la cual varios factores contribuyen al daño articular. Objetivo: Comparar el efecto del ácido hialurónico (HA) y acetónido de triamcinolona (TA), solos o en combinación, en el proceso de diferenciación condrogénica in vitro de células madre mesenquimales (MSCs). Métodos: Las MSCs fueron divididas en cuatro grupos: Control, HA, TA y HA/TA, y cultivadas por 14 días en medio de diferenciación condrogénica para cada tratamiento. El potencial de diferenciación condrogénica fue analizado por medio de histología e inmunohistoquímica. Resultados: Las MSCs tratadas con HA y HA/TA, presentaron características histológicas similares a los condrocitos nativos, y la matriz extracelular (ECM) de MSCs tratadas con TA fue más compacta y organizada. La tinción de glicosaminoglicanos fue intensa en el Control, moderada en TA, ligera en HA/TA, y sin tinción en HA. La inmunoreactividad para colágeno tipo II fue más alta en las MSCs y ECM tratadas con TA. Conclusión: El análisis histológico muestra que el HA influencia un desarrollo morfológico similar a los condrocitos de las MSCs, pero con baja expresión de moléculas específicas de cartílago. La TA promueve la formación de una ECM compacta y organizada.

Resumo Antecedentes: A osteoartrite é uma doença degenerativa complexa, na qual vários fatores contribuem ao dano articular. Objetivo: Comparar o efeito do ácido hialurônico (HA) e Triancinolona acetonida (TA), só ou combinado no processo de diferenciação condrogênica in vitro de células tronco mesenquimais (MSCs). Métodos: MSCs foram divididas em 4 grupos: Controle, HA, TA y HA/TA e cultivadas por 14 dias com meio de diferenciação condrogênica e seus respectivos tratamentos. O potencial de diferenciação condrogênica foi acessado por meio de histologia e imunohistoquímica. Resultados: Histologicamente, MSCs tratadas com HA e HA/TA apresentaram características semelhantes de condrócitos nativos, e a matriz extracelular de MSCs tratadas com TA foi mais compacta e organizada. A coloração para glicosaminoglicanos foi intensa no Controle, moderada no TA, leve no HA/TA e sem coloração com HA. Para os grupos tratamento, a imunoreatividade para colágeno tipo II foi maior nas células e matriz extracelular tratadas com TA. Conclusão: Mediante análise histológica, o HA influenciou o desenvolvimento morfológico semelhante a condrócitos das MSCs, mas com baixa expressão de moléculas específicas de cartilagem. A TA promoveu a formação de uma matriz extracelular compacta e organizada.

Rev. bras. ortop ; 56(3): 333-339, May-June 2021. tab, graf
Article in English | LILACS | ID: biblio-1288682


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.

Humans , Male , Female , Adolescent , Adult , Middle Aged , Postoperative Period , Magnetic Resonance Spectroscopy , Cartilage, Articular , Collagen , Chondrogenesis , Knee Injuries
Acta Anatomica Sinica ; (6): 453-459, 2021.
Article in Chinese | WPRIM | ID: wpr-1015458


Objective To investigate the effect of excessive caffeine intake on fetal cartilage ossification in female rats during pregnancy and its mechanism. Methods From gestational day(GD) 9 to GD 20, the pregnant Wistar rats in caffeine exposure group were intragastrically administered 120 mg/kg day caffeine, and the control group was administered the same volume of distilled water. The pregnant mice were sacrificed at day 20, and the body length of the fetal mice was measured. The distal femur of fetal rats was isolated, the length of distal femur cartilage was measured, and primary chondrocytes were prepared. The cells were treated with caffeine (0.1, 1 and 10 μmol/L), insulin-like growth factor 1 (IGF-1, 100 (μg/L) and extracellular regulated protein kinases(ERK) inhibitor (10 μmol/L), respectively. Then the cells were harvested for apoptosis, gene and protein analysis. Results Compared with the control group, the body length and femur length of the fetuses in the caffeine exposed group decreased significantly (P< 0.05), and the serum corticosterone levels increased significantly (P<0.05). Immunohistochemical analysis showed that the expressions of IGF-1, proliferating cell nuclear antigen (PCNA) and sex determining region Y box protein 9 (SOX9) in mast chondrocyte area of caffeine exposed group were significantly lower than those of control group (P<0.05). In vitro, caffeine treatment reduced the expression of IGF-1, PCNA, SOX9 mRNA and p-ERK protein in primary chondrocytes in a concentration-dependent manner, while exogenous IGF-1 could reversed these changes induced by caffeine, and the effect of exogenous IGF-1 was reduced by ERK inhibitors (all P<0.05). Conclusion Prenatal caffeine exposure leads to shortening of the long bones of the fetus and prolongation of the hypertrophy by inhibiting the proliferation of chondrocytes. The IGF-l/MAPK/ERK signaling pathway in chondrocytes may be partially involved in the adverse effects of caffeine on chondrocyte proliferation.

Article in Chinese | WPRIM | ID: wpr-867827


Growth plate,the developmental center of endochondral osteogenesis,can be divided morphologically and functionally into a resting zone,a proliferative zone,a prehypertrophic zone and a hypertrophic zone.Injuries to growth plate often lead to bone growth defects including limb length discrepancy and angulation deformity in children.Currently,their orthopedic corrective surgeries are invasive and limitedly effective and no effective biotherapy has been available.Previous studies on animal models of growth plate damage have investigated the related cellular and molecular events in the repair of damaged growth plates in the 4 distinct inflammatory,fibrogenic,osteogenic and remodeling phases.Related molecules involved in the regulation of the above processes,such as inflammatory cytokines tumor necrosis factor alpha,mitogenic platelet-derived growth factor and bone morphogenetic protein,are found to participate in the regulation of growth plate injury.Exploration of the mechanisms may provide new targets for biotherapy.In addition,development of cartilage tissue engineering,especially application of mesenchymal stem cells,also provides potential interventions for growth plate injury.

Rev. colomb. biotecnol ; 21(1): 71-81, ene.-jun. 2019. tab, graf
Article in Spanish | LILACS-Express | LILACS | ID: biblio-1013900


RESUMEN La fibroína de seda es una proteína que ha demostrado ser un biomaterial con gran potencial en medicina regenerativa, por sus características de biocompatibilidad y su amplia posibilidad de modificación estructural permite ser usada como andamio favoreciendo procesos de crecimiento, diferenciación celular y la regeneración del tejido afectado. En este estudio se utilizaron capullos de gusano de seda Bombyx mori L., para la fabricación de películas de fibroína, los capullos fueron desgomados utilizando Na2CO3 0,02M, la fibroína obtenida se disolvió con LiBr 9,3M, el cual fue eliminado mediante diálisis y finalmente la solución de fibroína fue concentrada mediante contradiálisis. La fibroína fue servida en cajas de poliestireno, secadas a 90°C/24 horas y esterilizadas con etanol al 70%. Células madre mesenquimales fueron sembradas sobre estas películas de fibroína e inducidas a diferenciación utilizando un medio condrogénico especifico. La diferenciación fue evaluada por triplicado a los 14 y 21 días mediante extracción de ARN total, síntesis de ADN copia y amplificación por PCR de un grupo de genes específicos de cartílago empleando cebadores específicos. Se fabricaron películas de fibroína estables y resistentes que permitieron el crecimiento y la multiplicación celular, así como la diferenciación condrogénica evidenciada por la expresión de genes condrogenicos, no se afectó la viabilidad ni el recuento celular, las células interactuaron con el andamio evidenciado por el área de tapizado formado sobre la superficie de la película de fibroína. Finalmente se concluye que la fibroína de seda es un biomaterial que puede servir de andamio potencial para la regeneración de lesiones articulares.

ABSTRACT Silk fibroin is a protein that has been shown to be a biomaterial with great potential in regenerative medicine, due to its biocompatibility characteristics and its wide possibility of structural modification can be used as scaffold, favoring growth processes, cell differentiation and the regeneration of affected tissue. Bombix mori L. silkworm cocoons were used to make fibroin films, the silk fibroin were degummed using 0.02M Na2CO3, the obtained fibroin was dissolved with 9.3M LiBr, which was eliminated by dialysis and finally the fibroin solution was concentrated to 17% by counterdialysis. The fibroin was served in polystyrene boxes, dried at 90°C/24 hours and sterilized with 70% ethanol. The mesenchymal stem cells were seeded on fibroin films and induced differentiation using a specific chondrogenic medium. Differentiation was assessed in triplicate at 14 and 21 days by total RNA extraction, DNA synthesis copy and PCR amplification of a group of cartilage-specific genes using specific primers. Stable and resistant fibroin films that allowed cell growth and multiplication were fabricated, as well as the chondrogenic differentiation evidenced by the expression of chondrogenic genes, the viability and the cell count were not affected, the cells interacted with the scaffolding evidenced by the area of upholstery formed on the surface of the fibroin film. Finally, it is concluded that silk fibroin is a biomaterial that can serve as a potential scaffold for the regeneration of joint injuries.

Arq. bras. med. vet. zootec. (Online) ; 71(2): 509-520, mar.-abr. 2019. graf, ilus
Article in English | VETINDEX, LILACS | ID: biblio-1011276


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)

Animals , Female , Rats , Caffeine , Cartilage, Articular/drug effects , Chondrocytes/drug effects , Chondrogenesis/drug effects
Article in English | WPRIM | ID: wpr-764053


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.

Humans , Blotting, Western , Bone Marrow , Cartilage , Chondrocytes , Chondrogenesis , Dexamethasone , Down-Regulation , Gene Expression , Growth Plate , Hypertrophy , In Vitro Techniques , Mesenchymal Stem Cells , Phosphorylation
Korean Journal of Medicine ; : 145-151, 2019.
Article in Korean | WPRIM | ID: wpr-759928


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.

Adult Stem Cells , Aging , Cartilage , Chondrogenesis , Induced Pluripotent Stem Cells , Joints , Musculoskeletal Diseases , Orthopedics , Osteoarthritis , Physicians' Offices , Stem Cells
Article in English | WPRIM | ID: wpr-761917


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.

Cartilage , Cartilage, Articular , Chondrogenesis , Cytokines , Immunity, Innate , Inflammation , Joints , Macrophages
Article | IMSEAR | ID: sea-187957


Mesenchymal stem cells (MSCs) are multipotent stem cells that are capable of self-renewal and can be committed into classical mesodermal tri-lineage differentiation (adipocytes, osteocytes and chondrocytes). During chondrogenic differentiation MSCs change their shape due to the reorganization of cytoskeletal components. This has been well documented for human and rodent models. Morphological changes of microtubule network and actin filaments that occur during the chondrogenic differentiation of MSCs from large animal models remain unknown. In this study we described the morphological changes of cell shape, area, actin structures and microtubule array that occur in bovine MSCs during the chondrogenic differentiation of bovine bone-marrow isolated MSCs. Chondrogenic differentiation of bMSCs occur more rapidly on glass substrate compared to the cells plated on vitronectin, and in 7 days after the commitment we observed clusters of small round-shaped cells that expressed glycosaminoglycans. During the differentiation microtubule (MT) array of MSCs became non-radial, and non-centrosomal MTs that grew transversely to the cell radius appeared in the inner cytoplasm and near the cell edges. At the end of differentiation process we observed the thick bundles of MTs that grew in parallel to the cell edge and basket-like structures of curved MTs around the nucleus. The main changes of actin structures in differentiating MSCs included the disappearance of thick transverse stress fibers and actin arches and reorganization of actin into chaotic network of thin cortical fibers. Our results imply the important role of both actin and MT cytoskeletal systems in chondrogenesis and reveals new perspectives for experimental regulation of these process in vitro systems.

Rev. bras. ortop ; 53(6): 733-739, Nov.-Dec. 2018. tab, graf
Article in English | LILACS | ID: biblio-977903


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.

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


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.

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
Article in Chinese | WPRIM | ID: wpr-777850


Objective@#This study investigated the effects of interleukin-1β (IL-1β) on human synovial fluid-derived mesenchymal stem cells (hSFMSCs) in the temporomandibular joint.@*Methods @#hSFMSCs from synovial fluid samples of temporomandibular disorder (TMD) patients were cultured in vitro. hSFMSCs were divided into three groups with different concentrations of rhIL-1β in complete medium (α-MEM cell culture medium + 10% FBS + 1× GlutaMAX): 0 ng/mL IL-1β group, 1 ng/mL IL-1β group and 10 ng/mL IL-1β group. Changes in the rate of colony formation, growth curve, cell cycle and apoptosis of hSFMSCs under IL-1β stimulation were evaluated. The osteogenic, adipogenic and chondrogenic potential of the cells were also determined using histochemical and real-time fluorescence quantitative PCR methods. @*Results @#No significant differences in growth or proliferation capacity were observed in any IL-1β-stimulated group in comparisons of the colony-formation rate (F = 0.665, P=0.548), growth curve (F=0.001, P=0.999), cell cycle (FG1=0.773, PG1=0.503; FS =0.636, PS =0.562) or apoptosis (F=0.196, P=0.827) of the cells. However, the multidifferentiation capacity of hSFMSCs was affected in the inflammatory environment. Mineralized nodule and lipid clusters decreased significantly, and the gene expression levels of runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), peroxisomal proliferative receptor G2 (PPARG2) and lipoprotein lipase (LPL) were suppressed significantly in IL-1β-mediated induction medium (P < 0.05). In general, cartilage pellets formed in all the IL-1β-mediated chondrogenic differentiation groups. The gene levels of sex-determining region Y-related high-mobility group box-9 (SOX9) and collagen II were decreased (P < 0.05), while that of matrix metalloproteinase 13 (MMP13) was increased significantly in the presence of IL-1β (P < 0.05). @*Conclusion@# IL-1β directly affects the multidifferentiation potential of hSFMSCs but not their cell growth or proliferation ability.

Journal of Medical Postgraduates ; (12): 1325-1328, 2018.
Article in Chinese | WPRIM | ID: wpr-818037


Long noncoding RNAs (lncRNAs) are important regulators for a variety of biological process. miRNAs appear to be important modulators of chondrogenesis and osteoarthritis (OA). LncRNAs are widely involved in the regulation of proliferation and apoptosis of chondrocytes as sponge of microRNAs. In this review, we discuss the role of lncRNA in cartilage repairation from the aspects of cartilage injury, chondrocyte proliferation and chondrocyte apoptosis. These findings will promote the development of novel molecular drugs which regulate the balance of cartilage injury and repairation.

Article in English | WPRIM | ID: wpr-713808


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.

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


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.

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