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1.
Int J Nanomedicine ; 15: 7775-7789, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33116500

RESUMO

Purpose: Several scaffolds and cell sources are being investigated for cartilage regeneration. The aim of the study was to prepare nanocellulose-based thermosensitive injectable hydrogel scaffolds and assess their potential as 3D scaffolds allowing the chondrogenic differentiation of embedded human dental pulp stem and progenitor cells (hDPSCs). Materials and Methods: The hydrogel-forming solutions were prepared by adding ß-glycerophosphate (GP) to chitosan (CS) at different ratios. Nanocellulose (NC) suspension was produced from hemp hurd then added dropwise to the CS/GP mixture. In vitro characterization of the prepared hydrogels involved optimizing gelation and degradation time, mass-swelling ratio, and rheological properties. The hydrogel with optimal characteristics, NC-CS/GP-21, was selected for further investigation including assessment of biocompatibility. The chondrogenesis ability of hDPSCs embedded in NC-CS/GP-21 hydrogel was investigated in vitro and compared to that of bone marrow-derived mesenchymal stem cells (BMSCs), then was confirmed in vivo in 12 adult Sprague Dawley rats. Results: The selected hydrogel showed stability in culture media, had a gelation time of 2.8 minutes, showed a highly porous microstructure by scanning electron microscope, and was morphologically intact in vivo for 14 days after injection. Histological and immunohistochemical analyses and real-time PCR confirmed the chondrogenesis ability of hDPSCs embedded in NC-CS/GP-21 hydrogel. Conclusion: Our results suggest that nanocellulose-chitosan thermosensitive hydrogel is a biocompatible, injectable, mechanically stable and slowly degradable scaffold. hDPSCs embedded in NC-CS/GP-21 hydrogel is a promising, minimally invasive, stem cell-based strategy for cartilage regeneration.


Assuntos
Cartilagem/fisiologia , Diferenciação Celular/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Polpa Dentária/citologia , Hidrogéis/farmacologia , Regeneração/efeitos dos fármacos , Células-Tronco/citologia , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Cartilagem/citologia , Cartilagem/efeitos dos fármacos , Celulose/química , Quitosana/química , Humanos , Hidrogéis/química , Porosidade , Ratos , Ratos Sprague-Dawley , Células-Tronco/efeitos dos fármacos , Tecidos Suporte/química
2.
Int J Nanomedicine ; 15: 6247-6262, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32903812

RESUMO

Prosthesis-associated infections and aseptic loosening are major causes of implant failure. There is an urgent need to improve the antibacterial ability and osseointegration of orthopedic implants. Zinc oxide nanoparticles (ZnO-NPs) are a common type of zinc-containing metal oxide nanoparticles that have been widely studied in many fields, such as food packaging, pollution treatment, and biomedicine. The ZnO-NPs have low toxicity and good biological functions, as well as antibacterial, anticancer, and osteogenic capabilities. Furthermore, ZnO-NPs can be easily obtained through various methods. Among them, green preparation methods can improve the bioactivity of ZnO-NPs and strengthen their potential application in the biological field. This review discusses the antibacterial abilities of ZnO-NPs, including mechanisms and influencing factors. The toxicity and shortcomings of anticancer applications are summarized. Furthermore, osteogenic mechanisms and synergy with other materials are introduced. Green preparation methods are also briefly reviewed.


Assuntos
Antibacterianos/farmacologia , Nanopartículas Metálicas/química , Osteogênese/efeitos dos fármacos , Óxido de Zinco/farmacologia , Animais , Antibacterianos/química , Antineoplásicos/efeitos adversos , Antineoplásicos/química , Antineoplásicos/farmacologia , Condrogênese/efeitos dos fármacos , Química Verde , Humanos , Nanopartículas Metálicas/efeitos adversos , Nanopartículas Metálicas/uso terapêutico , Osteogênese/fisiologia , Próteses e Implantes , Óxido de Zinco/efeitos adversos , Óxido de Zinco/toxicidade
3.
Ecotoxicol Environ Saf ; 204: 111058, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32739676

RESUMO

Skeletal fluorosis causes growth plate impairment and growth retardation during bone development. However, the mechanism of how fluoride impairs chondrocyte is unclear. To explore the effect of fluoride on chondrocyte differentiation and the regulation of circadian clock signaling pathway during chondrogenesis, we treated ATDC5 cells with fluoride and carried out a series of experiments. 10-3 M fluoride inhibited cell viability and significantly decreased the expression of Sox9 and Col2a1 (P < 0.05). Fluoride inhibited proteoglycan synthesis and decreased significantly the expression of Aggrecan, Ihh and Col10a1 (P < 0.05). Meanwhile, fluoride significantly inhibited the expression of Bmal1 and disrupted circadian clock signaling pathway (P < 0.05). Furthermore, fluoride disrupted the time-dependent expression of circadian clock molecules and stage-specific differentiation markers. Overexpression of Bmal1 by lentivirus reversed the adverse effects of fluoride on chondrogenesis. These results suggested that fluoride inhibited chondrocyte viability and delayed chondrocyte differentiation. Fluoride delayed chondrogenesis partly via interfering with Bmal1 and circadian clock signaling pathway. Nevertheless, the specific mechanism of circadian clock in fluoride-induced cartilage damage needs to be further studied.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Relógios Circadianos , Poluentes Ambientais/toxicidade , Fluoretos/toxicidade , Animais , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/metabolismo , Condrogênese/fisiologia , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Lâmina de Crescimento/efeitos dos fármacos , Lâmina de Crescimento/metabolismo , Camundongos , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais
4.
Life Sci ; 253: 117636, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32251631

RESUMO

BMAL1 is a core component of the circadian clock loop, which directs the sophisticated circadian expression of clock-controlled genes. Skeletal Bone development is a complex biological process involving intramembranous ossification, endochondral ossification and bone remodeling, as well as specific cells, such as mesenchymal cells, osteoblasts, osteoclasts, chondrocytes, etc. Growing evidences suggest that BMAL1 is indispensable for hard tissue development, including bone, cartilage and teeth. Loss of BMAL1 in animals can inhibit bone and cartilage development, and result in abnormal bone mass. In mesenchymal cells, BMAL1 defect inhibits osteoblastic and chondrocytic differentiation. Inactivation of BMAL1 also can promote the differentiation and formation of osteoclasts and increase bone resorption. Specifically, preclinical data demonstrate that the abnormity of BMAL1 expression is associated with skeletal disorders such as skeletal mandibular hypoplasia, osteoarthritis, osteoporosis, etc. In this review, we systemically describe the impact of BMAL1 in skeletal development and homeostasis, and devote to searching new therapy strategies for bone disorders.


Assuntos
Fatores de Transcrição ARNTL/metabolismo , Desenvolvimento Ósseo/efeitos dos fármacos , Fatores de Transcrição ARNTL/genética , Animais , Densidade Óssea/efeitos dos fármacos , Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Cartilagem/metabolismo , Diferenciação Celular , Condrócitos/metabolismo , Condrogênese/efeitos dos fármacos , Relógios Circadianos/genética , Regulação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese , Dente/metabolismo
5.
Mar Drugs ; 18(2)2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32023805

RESUMO

Osteoarthritis is the most prevalent rheumatic disease. During disease progression, differences have been described in the prevalence of chondroitin sulfate (CS) isomers. Marine derived-CS present a higher proportion of the 6S isomer, offering therapeutic potential. Accordingly, we evaluated the effect of exogenous supplementation of CS, derived from the small spotted catshark (Scyliorhinus canicula), blue shark (Prionace glauca), thornback skate (Raja clavata) and bovine CS (reference), on the proliferation of osteochondral cell lines (MG-63 and T/C-28a2) and the chondrogenic differentiation of mesenchymal stromal cells (MSCs). MG-G3 proliferation was comparable between R. clavata (CS-6 intermediate ratio) and bovine CS (CS-4 enrichment), for concentrations below 0.5 mg/mL, defined as a toxicity threshold. T/C-28a2 proliferation was significantly improved by intermediate ratios of CS-6 and -4 isomers (S. canicula and R. clavata). A dose-dependent response was observed for S. canicula (200 µg/mL vs 50 and 10 µg/mL) and bovine CS (200 and 100 µg/mL vs 10 µg/mL). CS sulfation patterns discretely affected MSCs chondrogenesis; even though S. canicula and R. clavata CS up-regulated chondrogenic markers expression (aggrecan and collagen type II) these were not statistically significant. We demonstrate that intermediate values of CS-4 and -6 isomers improve cell proliferation and offer potential for chondrogenic promotion, although more studies are needed to elucidate its mechanism of action.


Assuntos
Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Sulfatos de Condroitina/farmacologia , Idoso , Idoso de 80 Anos ou mais , Animais , Bovinos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Condrócitos/metabolismo , Sulfatos de Condroitina/química , Sulfatos de Condroitina/isolamento & purificação , Feminino , Humanos , Isomerismo , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Tubarões , Rajidae
6.
Int J Mol Sci ; 21(4)2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-32093330

RESUMO

In vitro chondrogenically differentiated mesenchymal stem cells (MSCs) have a tendency to undergo hypertrophy, mirroring the fate of transient "chondrocytes" in the growth plate. As hypertrophy would result in ossification, this fact limits their use in cartilage tissue engineering applications. During limb development, retinoic acid receptor (RAR) signaling exerts an important influence on cell fate of mesenchymal progenitors. While retinoids foster hypertrophy, suppression of RAR signaling seems to be required for chondrogenic differentiation. Therefore, we hypothesized that treatment of chondrogenically differentiating hMSCs with the RAR inverse agonist, BMS204,493 (further named BMS), would attenuate hypertrophy. We induced hypertrophy in chondrogenic precultured MSC pellets by the addition of bone morphogenetic protein 4. Direct activation of the RAR pathway by application of the physiological RAR agonist retinoic acid (RA) further enhanced the hypertrophic phenotype. However, BMS treatment reduced hypertrophic conversion in hMSCs, shown by decreased cell size, number of hypertrophic cells, and collagen type X deposition in histological analyses. BMS effects were dependent on the time point of application and strongest after early treatment during chondrogenic precultivation. The possibility of modifing hypertrophic cartilage via attenuation of RAR signaling by BMS could be helpful in producing stable engineered tissue for cartilage regeneration.


Assuntos
Benzoatos/farmacologia , Células-Tronco Mesenquimais/metabolismo , Receptores do Ácido Retinoico/agonistas , Receptores do Ácido Retinoico/metabolismo , Estilbenos/farmacologia , Proteína Morfogenética Óssea 4/metabolismo , Condrogênese/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/patologia
7.
Int J Mol Sci ; 21(3)2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-32013232

RESUMO

Both inflammatory diseases like rheumatoid arthritis (RA) and anti-inflammatory treatment of RA with glucocorticoids (GCs) or non-steroidal anti-inflammatory drugs (NSAIDs) negatively influence bone metabolism and fracture healing. Janus kinase (JAK) inhibition with tofacitinib has been demonstrated to act as a potent anti-inflammatory therapeutic agent in the treatment of RA, but its impact on the fundamental processes of bone regeneration is currently controversially discussed and at least in part elusive. Therefore, in this study, we aimed to examine the effects of tofacitinib on processes of bone healing focusing on recruitment of human mesenchymal stromal cells (hMSCs) into the inflammatory microenvironment of the fracture gap, chondrogenesis, osteogenesis and osteoclastogenesis. We performed our analyses under conditions of reduced oxygen availability in order to mimic the in vivo situation of the fracture gap most optimal. We demonstrate that tofacitinib dose-dependently promotes the recruitment of hMSCs under hypoxia but inhibits recruitment of hMSCs under normoxia. With regard to the chondrogenic differentiation of hMSCs, we demonstrate that tofacitinib does not inhibit survival at therapeutically relevant doses of 10-100 nM. Moreover, tofacitinib dose-dependently enhances osteogenic differentiation of hMSCs and reduces osteoclast differentiation and activity. We conclude from our data that tofacitinib may influence bone healing by promotion of hMSC recruitment into the hypoxic microenvironment of the fracture gap but does not interfere with the cartilaginous phase of the soft callus phase of fracture healing process. We assume that tofacitinib may promote bone formation and reduce bone resorption, which could in part explain the positive impact of tofacitinib on bone erosions in RA. Thus, we hypothesize that it will be unnecessary to stop this medication in case of fracture and suggest that positive effects on osteoporosis are likely.


Assuntos
Inibidores de Janus Quinases/farmacologia , Janus Quinases/metabolismo , Osteogênese/efeitos dos fármacos , Piperidinas/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Hipóxia Celular , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condrogênese/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Janus Quinases/antagonistas & inibidores , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo
8.
J Biosci Bioeng ; 129(6): 756-764, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32107152

RESUMO

Cartilage defects resultant from trauma or degenerative diseases (e.g., osteoarthritis) can potentially be repaired using tissue engineering (TE) strategies combining progenitor cells, biomaterial scaffolds and bio-physical/chemical cues. This work examines promoting chondrogenic differentiation of human bone marrow mesenchymal stem/stromal cells (BM-MSCs) by combining the effects of modified poly (ε-caprolactone) (PCL) scaffolds hydrophilicity and chondroitin sulfate (CS) supplementation in a hypoxic 5% oxygen atmosphere. 3D-extruded PCL scaffolds, characterized by µCT, featured a 21 mm-1 surface area to volume ratio, 390 µm pore size and approximately 100% pore interconnectivity. Scaffold immersion in sodium hydroxide solutions for different periods of time had major effects in scaffold surface morphology, wettability and mechanical properties, but without improvements on cell adhesion. In-situ chondrogenic differentiation of BM-MSC seeded in 3D-extruded PCL scaffolds resulted in higher cell populations and ECM deposition along all scaffold structure, when chondrogenesis was preceded by an expansion phase. Additionally, CS supplementation during BM-MSC expansion was crucial to enhance aggrecan gene expression, known as a hallmark of chondrogenesis. Overall, this study presents an approach to tailor the wettability and mechanical properties of PCL scaffolds and supports the use of CS-supplementation as a biochemical cue in integrated TE strategies for cartilage regeneration.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Sulfatos de Condroitina/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Poliésteres/química , Adulto , Células Cultivadas , Humanos , Concentração de Íons de Hidrogênio , Masculino , Células-Tronco Mesenquimais/citologia , Porosidade
9.
Biochem Biophys Res Commun ; 523(2): 506-513, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31898972

RESUMO

In indirect co-culture system, chondrocytes can induce differentiation of bone marrow mesenchymal stem cells (BMSCs) to chondrocytes without additional inducer. The participation of microRNAs (miRNAs) may take part in the chondrogenic differentiation. Present study aimed to investigate the effect and mechanism of chondrocytes-derived exosomal miRNA in BMSCs chondrogenic differentiation. Our data showed that miR-8485 was the exosomal miRNA derived from chondrocytes and transmitted to BMSCs. Functionally, miR-8485 silence in chondrocytes impaired exosome-induced chondrogenic differentiation of BMSCs. Mechanistically, exosomal miR-8485 targeted GSK3B to repress GSK-3ß expression and targeted DACT1 to induce p-GSK-3ß (Ser9), activating Wnt/ß-catenin pathways. Our study firstly showed that chondrocytes-derived exosomal miR-8485 regulated the Wnt/ß-catenin pathways to promote chondrogenic differentiation of BMSCs, providing innovative thoughts for cartilage reconstruction.


Assuntos
Condrócitos/citologia , Exossomos/genética , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Condrogênese/efeitos dos fármacos , Condrogênese/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , MicroRNAs/isolamento & purificação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/genética
10.
Ann N Y Acad Sci ; 1460(1): 43-56, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31423598

RESUMO

Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by insulin deficiency, and patients with diabetes have an increased risk of bone fracture and significantly impaired fracture healing. Proinflammatory cytokine tumor necrosis factor-alpha is significantly upregulated in diabetic fractures and is believed to underlie delayed fracture healing commonly observed in diabetes. Our previous genetic screen for the binding partners of progranulin (PGRN), a growth factor-like molecule that induces chondrogenesis, led to the identification of tumor necrosis factor receptors (TNFRs) as the PGRN-binding receptors. In this study, we employed several in vivo models to ascertain whether PGRN has therapeutic effects in diabetic fracture healing. Here, we report that deletion of PGRN significantly delayed bone fracture healing and aggravated inflammation in the fracture models of mice with T1DM. In contrast, recombinant PGRN effectively promoted diabetic fracture healing by inhibiting inflammation and enhancing chondrogenesis. In addition, both TNFR1 proinflammatory and TNFR2 anti-inflammatory signaling pathways are involved in PGRN-stimulated diabetic fracture healing. Collectively, these findings illuminate a novel understanding concerning the role of PGRN in diabetic fracture healing and may have an application in the development of novel therapeutic intervention strategies for diabetic and other types of impaired fracture healing.


Assuntos
Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/patologia , Consolidação da Fratura/efeitos dos fármacos , Progranulinas/farmacologia , Animais , Condrogênese/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Deleção de Genes , Humanos , Inflamação/patologia , Camundongos , Progranulinas/deficiência , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
11.
Carbohydr Polym ; 229: 115551, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31826469

RESUMO

The field of cartilage tissue engineering has been evolved in the last decade and a myriad of scaffolding biomaterials and bioactive agents have been proposed. Controlled release of growth factors encapsulated in the polymeric nanomaterials has been of interest notably for the repair of damaged articular cartilage. Here, we proposed an on-chip hydrodynamic flow focusing microfluidic approach for synthesis of alginate nanogels loaded with the transforming growth factor beta 3 (TGF-ß3) through an ionic gelation method in order to achieve precise release profile of these bioactive agents during chondrogenic differentiation of mesenchymal stem cells (MSCs). Alginate nanogels with adjustable sizes were synthesized by fine-tuning the flow rate ratio (FRR) in the microfluidic device consisting of cross-junction microchannels. The result of present study showed that the proposed approach can be a promising tool to synthesize bioactive -loaded polymeric nanogels for applications in drug delivery and tissue engineering.


Assuntos
Alginatos/química , Microfluídica , Nanogéis/química , Fator de Crescimento Transformador beta3/química , Adulto , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Condrogênese/efeitos dos fármacos , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Nanogéis/toxicidade , Tamanho da Partícula , Fator de Crescimento Transformador beta3/metabolismo , Fator de Crescimento Transformador beta3/farmacologia
12.
Stem Cell Res Ther ; 10(1): 392, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31847882

RESUMO

BACKGROUND: Chondrogenesis represents a highly dynamic cellular process that leads to the establishment of various types of cartilage. However, when stress-related injuries occur, a rapid and efficient regeneration of the tissues is necessary to maintain cartilage integrity. Mesenchymal stem cells (MSCs) are known to exhibit high capacity for self-renewal and pluripotency effects, and thus play a pivotal role in the repair and regeneration of damaged cartilage. On the other hand, the influence of certain pathological conditions such as metabolic disorders on MSCs can seriously impair their regenerative properties and thus reduce their therapeutic potential. OBJECTIVES: In this investigation, we attempted to improve and potentiate the in vitro chondrogenic ability of adipose-derived mesenchymal stromal stem cells (ASCs) isolated from horses suffering from metabolic syndrome. METHODS: Cultured cells in chondrogenic-inductive medium supplemented with Cladophora glomerata methanolic extract were experimented for expression of the main genes and microRNAs involved in the differentiation process using RT-PCR, for their morphological changes through confocal and scanning electron microscopy and for their physiological homeostasis. RESULTS: The different added concentrations of C. glomerata extract to the basic chondrogenic inductive culture medium promoted the proliferation of equine metabolic syndrome ASCs (ASCsEMS) and resulted in chondrogenic phenotype differentiation and higher mRNA expression of collagen type II, aggrecan, cartilage oligomeric matrix protein, and Sox9 among others. The results reveal an obvious inhibitory effect of hypertrophy and a strong repression of miR-145-5p, miR-146-3p, and miR-34a and miR-449a largely involved in cartilage degradation. Treated cells additionally exhibited significant reduced apoptosis and oxidative stress, as well as promoted viability and mitochondrial potentiation. CONCLUSION: Chondrogenesis in EqASCsEMS was found to be prominent after chondrogenic induction in conditions containing C. glomerata extract, suggesting that the macroalgae could be considered for the enhancement of ASC cultures and their reparative properties.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Clorófitas/química , Condrogênese/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Síndrome Metabólica/patologia , Extratos Vegetais/farmacologia , Agrecanas/genética , Agrecanas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Clorófitas/metabolismo , Condrócitos/citologia , Condrócitos/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Cavalos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Síndrome Metabólica/metabolismo , MicroRNAs/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/fisiologia , Extratos Vegetais/química , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo
13.
Cell Commun Signal ; 17(1): 166, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31842918

RESUMO

BACKGROUND: In vitro chondrogenesis depends on the concerted action of numerous signalling pathways, many of which are sensitive to the changes of intracellular Ca2+ concentration. N-methyl-D-aspartate (NMDA) glutamate receptor is a cation channel with high permeability for Ca2+. Whilst there is now accumulating evidence for the expression and function of NMDA receptors in non-neural tissues including mature cartilage and bone, the contribution of glutamate signalling to the regulation of chondrogenesis is yet to be elucidated. METHODS: We studied the role of glutamatergic signalling during the course of in vitro chondrogenesis in high density chondrifying cell cultures using single cell fluorescent calcium imaging, patch clamp, transient gene silencing, and western blotting. RESULTS: Here we show that key components of the glutamatergic signalling pathways are functional during in vitro chondrogenesis in a primary chicken chondrogenic model system. We also present the full glutamate receptor subunit mRNA and protein expression profile of these cultures. This is the first study to report that NMDA-mediated signalling may act as a key factor in embryonic limb bud-derived chondrogenic cultures as it evokes intracellular Ca2+ transients, which are abolished by the GluN2B subunit-specific inhibitor ifenprodil. The function of NMDARs is essential for chondrogenesis as their functional knock-down using either ifenprodil or GRIN1 siRNA temporarily blocks the differentiation of chondroprogenitor cells. Cartilage formation was fully restored with the re-expression of the GluN1 protein. CONCLUSIONS: We propose a key role for NMDARs during the transition of chondroprogenitor cells to cartilage matrix-producing chondroblasts.


Assuntos
Condrogênese/genética , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Cálcio/análise , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Galinhas , Condrogênese/efeitos dos fármacos , Ácido Glutâmico/análise , N-Metilaspartato/farmacologia , Receptores de N-Metil-D-Aspartato/agonistas , Transdução de Sinais/efeitos dos fármacos
14.
Biomater Sci ; 7(12): 5388-5403, 2019 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-31626251

RESUMO

Human induced pluripotent stem cells (iPSCs) have emerged as a promising alternative to bone-marrow derived mesenchymal stem/stromal cells for cartilage tissue engineering. However, the effect of biochemical and mechanical cues on iPSC chondrogenesis remains understudied. This study evaluated chondrogenesis of induced pluripotent mesenchymal progenitor cells (iPS-MPs) encapsulated in a cartilage-mimetic hydrogel under different culture conditions: free swelling versus dynamic compressive loading and different growth factors (TGFß3 and/or BMP2). Human iPSCs were differentiated into iPS-MPs and chondrogenesis was evaluated by gene expression (qPCR) and protein expression (immunohistochemistry) after three weeks. In pellet culture, both TGFß3 and BMP2 were required to promote chondrogenesis. However, the hydrogel in growth factor-free conditions promoted chondrogenesis, but rapidly progressed to hypertrophy. Dynamic loading in growth factor-free conditions supported chondrogenesis, but delayed the transition to hypertrophy. Findings were similar with TGFß3, BMP2, and TGFß3 + BMP2. Dynamic loading with TGFß3, regardless of BMP2, was the only condition that promoted a stable chondrogenic phenotype (aggrecan + collagen II) accompanied by collagen X down-regulation. Positive TGFßRI expression with load-enhanced Smad2/3 signaling and low SMAD1/5/8 signaling was observed. In summary, this study reports a promising cartilage-mimetic hydrogel for iPS-MPs that when combined with appropriate biochemical and mechanical cues induces a stable chondrogenic phenotype.


Assuntos
Materiais Biomiméticos/farmacologia , Proteína Morfogenética Óssea 2/farmacologia , Condrogênese/efeitos dos fármacos , Hidrogéis/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Fenômenos Mecânicos , Fator de Crescimento Transformador beta/farmacologia , Fenômenos Biomecânicos , Materiais Biomiméticos/química , Cartilagem , Diferenciação Celular/efeitos dos fármacos , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Pessoa de Meia-Idade , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo
15.
Med Sci Monit ; 25: 7872-7881, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31631887

RESUMO

BACKGROUND Heterotopic ossification (HO) is a kind of abnormal mineralized bone which usually occurs in muscle, tendon, or ligament. There are currently no effective drugs for the treatment and prevention of HO. Developing effective drugs that can inhibit HO is of profound significance and would provide new strategies for clinical treatment of this disease. The present investigation evaluated the inhibitory effect of tamoxifen against HO. MATERIAL AND METHODS Using an Achilles tendon trauma-induced HO female mice model, we screened different doses of tamoxifen (1, 3, and 9 mg/kg) in mice to determine the optimal dosage on the inhibition of the HO formation. The curative effect of tamoxifen was also illustrated at different HO progression stages including inflammation, chondrogenesis, osteogenesis, and HO maturation. RESULTS Heterotopic bone was formed with typical endochondral ossification in Achilles tendons 6 weeks after surgery and continued to enlarge up to 12 weeks. The formation of HO was significantly inhibited with the treatment of tamoxifen at the dosage of 9 mg/kg, whereas 1 mg/kg and 3 mg/kg did not reduce HO bone volume remarkably. The progression of HO was both attenuated by tamoxifen from Day 1 and Week 4 post-surgery, whereas no inhibitory effect was shown at the osteogenesis and maturation stages treated with tamoxifen. CONCLUSIONS Tamoxifen exerts an inhibitory effect on the heterotopic bone progression at inflammation and chondrogenesis stages, with the TGF-ß signaling pathway suppressed following the increase in estrogen receptor alpha activity.


Assuntos
Tendão do Calcâneo/efeitos dos fármacos , Ossificação Heterotópica/tratamento farmacológico , Tamoxifeno/farmacologia , Animais , Osso e Ossos/metabolismo , China , Condrogênese/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Músculos/metabolismo , Ossificação Heterotópica/metabolismo , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/metabolismo , Traumatismos dos Tendões/tratamento farmacológico
16.
Int J Mol Sci ; 20(18)2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31514329

RESUMO

Cartilage repair using tissue engineering is the most advanced clinical application in regenerative medicine, yet available solutions remain unsuccessful in reconstructing native cartilage in its proprietary form and function. Previous investigations have suggested that the combination of specific bioactive elements combined with a natural polymer could generate carrier matrices that enhance activities of seeded stem cells and possibly induce the desired matrix formation. The present study sought to clarify this by assessing whether a chitosan-hyaluronic-acid-based biomimetic matrix in conjunction with adipose-derived stem cells could support articular hyaline cartilage formation in relation to a standard chitosan-based construct. By assessing cellular development, matrix formation, and key gene/protein expressions during in vitro cultivation utilizing quantitative gene and immunofluorescent assays, results showed that chitosan with hyaluronic acid provides a suitable environment that supports stem cell differentiation towards cartilage matrix producing chondrocytes. However, on the molecular gene expression level, it has become apparent that, without combinations of morphogens, in the chondrogenic medium, hyaluronic acid with chitosan has a very limited capacity to stimulate and maintain stem cells in an articular chondrogenic state, suggesting that cocktails of various growth factors are one of the key features to regenerate articular cartilage, clinically.


Assuntos
Tecido Adiposo/citologia , Materiais Biomiméticos/farmacologia , Cartilagem Articular/fisiologia , Quitosana/farmacologia , Condrogênese , Ácido Hialurônico/farmacologia , Células-Tronco/citologia , Cartilagem Articular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Condrogênese/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco/efeitos dos fármacos , Células-Tronco/ultraestrutura , Tecidos Suporte/química
17.
Indian J Med Res ; 149(5): 641-649, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31417032

RESUMO

Background & objectives: Seeding density is one of the major parameters affecting the quality of tissue-engineered cartilage. The objective of this study was to evaluate different seeding densities of osteoarthritis chondrocytes (OACs) to obtain the highest quality cartilage. Methods: The OACs were expanded from passage 0 (P0) to P3, and cells in each passage were analyzed for gross morphology, growth rate, RNA expression and immunochemistry (IHC). The harvested OACs were assigned into two groups: low (1×10[7] cells/ml) and high (3×10[7] cells/ml) cell density. Three-dimensional (3D) constructs for each group were created using polymerised fibrin and cultured for 7, 14 and 21 days in vitro using chondrocyte growth medium. OAC constructs were analyzed with gross assessments and microscopic evaluation using standard histology, IHC and immunofluorescence staining, in addition to gene expression and biochemical analyses to evaluate tissue development. Results: Constructs with a high seeding density of 3×10[7] cells/ml were associated with better quality cartilage-like tissue than those seeded with 1×10[7] cells/ml based on overall tissue formation, cell association and extracellular matrix distribution. The chondrogenic properties of the constructs were further confirmed by the expression of genes encoding aggrecan core protein and collagen type II. Interpretation & conclusions: Our results confirmed that cell density was a significant factor affecting cell behaviour and aggregate production, and this was important for establishing good quality cartilage.


Assuntos
Cartilagem/crescimento & desenvolvimento , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Osteoartrite/terapia , Cartilagem/efeitos dos fármacos , Cartilagem Articular , Técnicas de Cultura de Células/métodos , Condrócitos/metabolismo , Condrogênese/efeitos dos fármacos , Fibrina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Osteoartrite/patologia , Osteogênese/efeitos dos fármacos , RNA/genética
18.
Colloids Surf B Biointerfaces ; 183: 110403, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31400614

RESUMO

Efforts on bioengineering are directed towards the construction of biocompatible scaffolds and the determination of the most favorable microenvironment, which will better support cell proliferation and differentiation. Perfusion bioreactors are attracting growing attention as an effective, modern tool in tissue engineering. A natural biomaterial extensively used in regenerative medicine with outstanding biocompatibility, biodegradability and non-toxic characteristics, is collagen, a structural protein with undisputed beneficial characteristics. This is a study designed according to the above considerations. 3D printed polycaprolactone (PCL) scaffolds with rectangular pores were coated with collagen either as a coating on the scaffold's trabeculae, or as a gel-cell solution penetrating scaffolds' pores. We employed histological, molecular and imaging techniques to analyze colonization, proliferation and chondrogenic differentiation of Adipose Derived Mesenchymal Stem Cells (ADMSCs). Two different differentiation culture media were employed to test chondrogenic differentiation on gelated and non gelated PCL scaffolds in static and in perfusion bioreactors dynamic culture conditions. In dynamic culture, non gelated scaffolds combined with our in house TGF-ß2 based medium, augmented chondrogenic differentiation performance, which overall was significantly less favorable compared to StemPro™ propriety medium. The beneficial mechanical stimulus of dynamic culture, appears to outgrow the disadvantage of the "weaker" TGF-ß2 medium used for chondrogenic differentiation. Even though cells in static culture grew well on the scaffold, there was limited penetration inside the construct, so the purpose of the 3D culture was not fully served. In contrast dynamic culture achieved better penetration and uniform distribution of the cells within the scaffold.


Assuntos
Cartilagem/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Colágeno/farmacologia , Poliésteres/farmacologia , Engenharia Tecidual/métodos , Tecidos Suporte , Agrecanas/genética , Agrecanas/metabolismo , Materiais Biocompatíveis , Biomarcadores/metabolismo , Reatores Biológicos , Cartilagem/citologia , Cartilagem/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Condrogênese/genética , Colágeno/química , Meios de Cultura/química , Meios de Cultura/farmacologia , Expressão Gênica , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Poliésteres/química , Porosidade , Cultura Primária de Células , Impressão Tridimensional , Regeneração/genética , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Propriedades de Superfície
19.
Biomed Res Int ; 2019: 5141204, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31346519

RESUMO

Patients with bone and cartilage defects due to infection, tumors, and trauma are quite common. Repairing bone and cartilage defects is thus a major problem for clinicians. Autologous and artificial bone transplantations are associated with many challenges, such as limited materials and immune rejection. Bone and cartilage regeneration has become a popular research topic. Inorganic polyphosphate (polyP) is a widely occurring biopolymer with high-energy phosphoanhydride bonds that exists in organisms from bacteria to mammals. Much data indicate that polyP acts as a regulator of gene expression in bone and cartilage tissues and exerts morphogenetic effects on cells involved in bone and cartilage formation. Exposure of these cells to polyP leads to the increase of cytokines that promote the differentiation of mesenchymal stem cells into osteoblasts, accelerates the osteoblast mineralization process, and inhibits the differentiation of osteoclast precursors to functionally active osteoclasts. PolyP-based materials have been widely reported in in vivo and in vitro studies. This paper reviews the current cellular mechanisms and material applications of polyP in bone and cartilage regeneration.


Assuntos
Doenças Ósseas/tratamento farmacológico , Regeneração Óssea/efeitos dos fármacos , Cartilagem/efeitos dos fármacos , Polifosfatos/uso terapêutico , Doenças Ósseas/patologia , Cartilagem/crescimento & desenvolvimento , Cartilagem/patologia , Diferenciação Celular/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Humanos , Transplante de Células-Tronco Mesenquimais , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Tecidos Suporte/química
20.
Mater Sci Eng C Mater Biol Appl ; 103: 109833, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31349499

RESUMO

The different lineage-specific biological properties of articular cartilage and subchondral bone present a great challenge in the construction of bi-lineage scaffolds for simultaneous osteochondral regeneration. To overcome this challenge, strontium incorporated calcium silicate (Sr-CS) ceramic was prepared for bi-lineage formation of scaffolds in this study. The positive result of Sr-CS in the regeneration of osteochondral defects was first proven by its improved effect on the osteogenesis and chondrogenesis induction of mesenchymal stem cells (MSCs). After that, scaffold-mediated macrophage polarization between classically activated inflammatory macrophages (termed M1Ф) and alternatively activated inflammatory macrophages (termed M2Ф) was assayed to investigate whether the incorporation of Sr into calcium silicate could alter host-to-scaffold immune response. Furthermore, the interactions between Sr-CS pretreated macrophages and MSCs differentiation were performed to prove the enhancement effect of suppressed inflammatory response on osteogenesis and chondrogenesis. In vivo transplantation showed that the Sr-CS scaffolds distinctly improved the regeneration of cartilage and subchondral bone, as compared to the calcium silicate scaffolds. On the one hand, the mechanism attributes to enhancement of strontium on the osteogenic and chondrogenic differentiation of MSCs. On the other hand, the reason can partially be attributed to suppressed synovial inflammatory response, which has improved effects on enhancement of osteogenesis and chondrogenesis. These findings suggest that monophasic Sr-CS scaffolds with a bi-lineage conducive property and an inflammatory response regulatory property represents a viable strategy for simultaneous regeneration of osteochondral defects.


Assuntos
Condrogênese/efeitos dos fármacos , Fatores Imunológicos , Macrófagos/imunologia , Células-Tronco Mesenquimais/imunologia , Osteogênese/efeitos dos fármacos , Estrôncio , Tecidos Suporte/química , Animais , Compostos de Cálcio/química , Compostos de Cálcio/farmacocinética , Compostos de Cálcio/farmacologia , Células Cultivadas , Condrogênese/imunologia , Fatores Imunológicos/química , Fatores Imunológicos/farmacocinética , Fatores Imunológicos/farmacologia , Osteogênese/imunologia , Coelhos , Silicatos/química , Silicatos/farmacocinética , Silicatos/farmacologia , Estrôncio/química , Estrôncio/farmacocinética , Estrôncio/farmacologia
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