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BACKGROUND: The innate immune response (IMR) is critical for the oral mucosa due to their continuous exposure to various oral pathogens. Keratinocytes play important role in IMR. Therefore, to date, keratinocytes from different sources have been used as in vitro research model for the study of IMR. However, current keratinocyte research models are hampered by the limited supply, patients' dependency and batch to batch variation. Therefore, in this study, we demonstrated the use of human embryonic stem cells (hESCs) derived keratinocytes (H9-Kert) as an alternative research model for the study of IMR. METHODS: The expression kinetics of toll-like receptor (TLR) 2, TLR 4, interleukin (IL) -6, IL-8, inducible nitric oxide synthase (iNOS) and tumour necrosis factor-alpha (TNF-α), in H9-Kert and immortalized human keratinocyte cell line (HaCaT) were analysed at mRNA levels by both reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. The activation of the inflammatory transcription factor nuclear factor kappa-b (NFĸB) was assayed in these cells by transiently transfecting the cells with NFĸB reporter plasmid. Activation of NFĸB following treatment with heat-killed Porphyromonas gingivalis (P. gingivalis), an oral pathogen, was determined by assaying for the reporter, secreted alkaline phosphatase activity. RESULTS: The expression of TLRs, cytokines and activation of NFĸB following bacterial stimulation showed in both H9-Kert and the widely used HaCaT keratinocyte cell line was similar. CONCLUSION: Overall, our results support the potential application of hESCs as an alternative limitless cell source for primary keratinocytes which can be used as consistent and dependable research tool with minimum variations and no donor's dependency.
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Células-Tronco Embrionárias/fisiologia , Imunidade Inata/imunologia , Queratinócitos/imunologia , Fosfatase Alcalina/análise , Linhagem Celular , Linhagem da Célula , Meios de Cultura , Fibroblastos/citologia , Vetores Genéticos/genética , Humanos , Interleucina-6/análise , Interleucina-8/análise , Queratinócitos/citologia , NF-kappa B/análise , Óxido Nítrico Sintase Tipo II/análise , Porphyromonas gingivalis/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 2 Toll-Like/análise , Receptor 4 Toll-Like/análise , Fator de Necrose Tumoral alfa/análiseRESUMO
To understand, in greater detail, the molecular mechanisms regulating the complex relationship between mechanical strain and alveolar bone metabolism during orthodontic treatment, passive cross-arch palatal springs were bonded to the maxillary molars of 6-wk-old rats, which were killed after 4 and 8 d. Outcome measures included serum assays for markers of bone formation and resorption and for the multifunctional hormone leptin, and histomorphometry of the inter-radicular bone. The concentration of the bone-formation marker alkaline phosphatase (ALP) was significantly reduced at both time points in the appliance group, accompanied by a 50% reduction in inter-radicular bone volume; however, osteocalcin (bone Gla protein) levels remained unaffected. Bone collagen deoxypyridinoline (DPD) crosslinks increased 2.3-fold at 4 d only, indicating a transient increase in bone resorption; in contrast, the level of the osteoclast-specific marker, tartrate-resistant acid phosphatase 5b (TRACP 5b), was unchanged. Leptin levels closely paralleled ALP reductions at both time points, suggesting an important role in the mechanostat negative-feedback loop required to normalize bone mass. These data suggest that an orthodontic appliance, in addition to remodeling the periodontal ligament (PDL)-bone interface, may exert unexpected side-effects on the tooth-supporting alveolar bone, and highlights the importance of recognizing that bone strains can have negative, as well as positive, effects on bone mass.
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Fosfatase Alcalina/sangue , Processo Alveolar/metabolismo , Doenças Ósseas Metabólicas/metabolismo , Reabsorção Óssea/metabolismo , Leptina/sangue , Aparelhos Ortodônticos/efeitos adversos , Fosfatase Ácida/sangue , Processo Alveolar/patologia , Aminoácidos/sangue , Animais , Biomarcadores/sangue , Doenças Ósseas Metabólicas/etiologia , Doenças Ósseas Metabólicas/patologia , Ensaio de Imunoadsorção Enzimática , Isoenzimas/sangue , Masculino , Osteocalcina/sangue , Ratos , Ratos Wistar , Fosfatase Ácida Resistente a TartaratoRESUMO
The rapid development of minimally invasive surgery technology requires higher flexibility of surgical treatment and small volume of medical instrument. This paper proposed a new type of minimally invasive surgery wrist institution actuated by TiNi shape memory alloy (SMA) wire. The wrist institution has some advantages such as compact structure, flexible function, light weight, big movement space, and high output position precision. The paper briefly introduces the properties of TiNi SMA and describes the configuration of wrist institution. We also carried out mechanism simulation analysis to the mechanics model and set up kinematics equations, and finally presented the workspace of the institution.
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Ligas , Procedimentos Cirúrgicos Minimamente Invasivos/instrumentação , Níquel , Robótica/instrumentação , Titânio , Fenômenos Biomecânicos/fisiologia , Desenho de EquipamentoRESUMO
OBJECTIVE: To investigate the cytotoxicity of calcium hydroxide (CH) and composite resin on fibroblasts derived from human embryo body fibroblasts-H9 (EBf-H9), human dental pulp cells (hDPCs) and immortalized fibroblasts L929; and to evaluate the use of EBf-H9 as a cellular model for cytotoxicity screening of dental materials. METHODS: The EBf-H9 cells were derived from human embryonic stem cells (H9) via outgrowth of embryonic body (EB); hDPCs were isolated from healthy dental pulp, and identified by immunochemical staining. Cell Counting Kit-8 (CCK-8) assay was applied to analyze the cytotoxicity of CH and composite resin with serial concentrations on the 3 kinds of cells. RESULTS: Following 24 h and 48 h (or 72 h) post-treatment of CH and composite resin, the viability of L929 cells was significantly lower than that of EBf-H9 and hDPCs (P<0.05), and there was no significant difference between the last two groups (P>0.05). CONCLUSION: Immortalized fibroblasts L929 cells exhibited different response to CH and composite resin compared with EBf-H9 and hDPCs, and the last two cell types were similar to each other. This study indicated that fibroblasts derived from human embryonic stem cells were a potential cellular model instead of traditional immortalized murine cell line for cytotoxicity screening assay.
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Hidróxido de Cálcio/toxicidade , Resinas Compostas/toxicidade , Materiais Dentários/toxicidade , Células-Tronco Embrionárias/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Animais , Células Cultivadas , Polpa Dentária/citologia , Células-Tronco Embrionárias/citologia , Fibroblastos/citologia , Humanos , Teste de Materiais , Camundongos , Testes de ToxicidadeRESUMO
Articular cartilage injuries are one of the most challenging problems in musculoskeletal medicine due to the poor intrinsic regenerative capacity of this tissue. The lack of efficient treatment modalities motivates research into tissue engineering: combining cells, biomaterials mimicking extracellular matrix (scaffolds) and microenvironmental signaling cues. The aim of this review is to focus on the use of biomaterials as delivery systems for microenvironmental cues in relation to their applications for treatment of cartilage defects. The latest advances in cartilage tissue engineering and regeneration are critically reviewed to demonstrate an outline of challenges toward biomaterial-based approaches of cartilage regeneration.
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Materiais Biocompatíveis/química , Cartilagem Articular/citologia , Engenharia Tecidual/métodos , Cartilagem Articular/lesões , Humanos , Medicina Regenerativa/métodosRESUMO
Tofacitinib citrate (TC) has recently gained interest in treating skin disorders such as psoriasis, atopic dermatitis and baldness. Unfortunately, the oral administration shows side effects, such as decreased neutrophil counts. To this end, the topical delivery of TC can be used to reduce the risk associated with systemic exposure. However, TC shows minimal absorption via skin. Hence, the objective of this study is to enhance the skin delivery of TC using a non-invasive approach. The liposomes based on propylene glycol, named as proposomes, carrying TC, were studied. The vesicle characteristics and in vitro skin permeation were assessed. The proposomes enhanced the skin permeability of TC by 4-11 folds. The composition of proposomes was found to affect the skin permeation and deposition of TC. The proposomes were stable for at least 6 months. Overall, proposomes were effective for targeted topical drug delivery.
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Sistemas de Liberação de Medicamentos/métodos , Lipossomos/química , Piperidinas/farmacocinética , Inibidores de Proteínas Quinases/farmacocinética , Pirimidinas/farmacocinética , Absorção Cutânea/fisiologia , Administração Cutânea , Cadáver , Química Farmacêutica/métodos , Estabilidade de Medicamentos , Humanos , Masculino , Pessoa de Meia-Idade , Tamanho da Partícula , Piperidinas/administração & dosagem , Propilenoglicol/química , Inibidores de Proteínas Quinases/administração & dosagem , Pirimidinas/administração & dosagemRESUMO
Dental pulp stem cells (DPSCs), one type of mesenchymal stem cells, are considered to be a type of tool cells for regenerative medicine and tissue engineering. Our previous studies found that the stimulation with lipopolysaccharide (LPS) might introduce senescence of DPSCs, and this senescence would have a positive correlation with the concentration of LPS. The ß-galactosidase (SA-ß-gal) staining was used to evaluate the senescence of DPSCs and immunofluorescence to show the morphology of DPSCs. Our findings suggested that the activity of SA-ß-gal has increased after repeated stimulation with LPS and the morphology of DPSCs has changed with the stimulation with LPS. We also found that LPS bound to the Toll-like receptor 4 (TLR4)/myeloid differentiation factor (MyD) 88 signaling pathway. Protein and mRNA expression of TLR4, MyD88 were enhanced in DPSCs with LPS stimulation, resulting in the activation of nuclear factor-κB (NF-κB) signaling, which exhibited the expression of p65 improved in the nucleus while the decreasing of IκB-α. Simultaneously, the expression of p53 and p21, the downstream proteins of the NF-κB signaling, has increased. In summary, DPSCs tend to undergo senescence after repeated stimulation in an inflammatory microenvironment. Ultimately, these findings may lead to a new direction for cell-based therapy in oral diseases and other regenerative medicines.
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OBJECTIVE: Graphene and its derivatives, graphene oxide (GO) and reduced graphene oxide (rGO), are 2D carbon-based materials with remarkable physical, chemical and biological properties. Graphene sheets have high specific surface area and mechanical strength. Moreover, they have been shown to influence the differentiation of stem cells and to improve properties of biomaterials. METHODS: Here, we present the recent achievements on the use of graphene and its derivatives to improve properties and enhance bioactivity of biomaterials. We also discuss the biosafety constraints to be solved to translate these carbonaceous materials to the clinic. RESULTS: Graphene and its derivatives can be functionalized and further modified with several bioactive molecules. They can be combined with several biomaterials used in regenerative and reconstructive dentistry and medicine. The resultant graphene-modified composites often present improved physico-mechanical properties and enhanced bioactivity. Moreover, graphene-modified composites are promising candidates to deliver growth factors, drugs and others bioactive compounds. SIGNIFICANCE: Graphene can improve the physical, chemical and mechanical properties of biomaterials. As it can be functionalized and combined with several biomolecules, graphene holds enormous potential to be used as drug carriers or substrates and scaffolds for cell-based tissue engineering strategies.
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Grafite , Engenharia Tecidual , Materiais Biocompatíveis , Diferenciação Celular , Teste de Materiais , ÓxidosRESUMO
OBJECTIVE: The objective was to investigate the potential of graphene (Gp) to induce odontogenic and osteogenic differentiation in dental pulp stem cells (DPSC). METHODS: Gp was produced by chemical vapor deposition. DPSC were seeded on Gp or glass (Gl). Cells were maintained in culture medium for 28 days. Every two days, culture medium from Gp was used to treat cells on Gl and vice versa. Mineralization and differentiation of DPSC on all substrates were evaluated after 14 and 28 days by alizarin red S staining, qPCR, immunofluorescence and FACS. Statistics were performed with two-way ANOVA and multiple comparisons were performed using Tukey's post hoc test at a pre-set significance level of 5%. RESULTS: After 14 and 28 days, Gp induced higher levels of mineralization as compared to Gl. Odontoblastic genes (MSX-1, PAX and DMP) were down-regulated and osteogenic genes and proteins (RUNX2, COL and OCN) were significantly upregulated on Gp comparing to Gl (p<0.05 for all cases). Medium from Gp induced downregulation of odontoblastic genes and increased bone-related gene and protein on Gl. SIGNIFICANCE: Graphene induced osteogenic and not odontoblastic differentiation of DPSC without the use of chemical inducers for osteogenesis. Graphene has the potential to be used as a substrate for craniofacial bone tissue engineering research.
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Diferenciação Celular , Polpa Dentária/citologia , Grafite/farmacologia , Osteogênese/efeitos dos fármacos , Células Cultivadas , Polpa Dentária/efeitos dos fármacos , Humanos , Células-TroncoRESUMO
Neural crest stem cells (NCSCs) represent a transient and multipotent cell population that contributes to numerous anatomical structures such as peripheral nervous system, teeth, and cornea. NCSC maldevelopment is related to various human diseases including pigmentation abnormalities, disorders affecting autonomic nervous system, and malformations of teeth, eyes, and hearts. As human pluripotent stem cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can serve as an unlimited cell source to generate NCSCs, hESC/hiPSC-derived NCSCs can be a valuable tool to study the underlying mechanisms of NCSC-associated diseases, which paves the way for future therapies for these abnormalities. In addition, hESC/hiPSC-derived NCSCs with the capability of differentiating to various cell types are highly promising for clinical organ repair and regeneration. In this review, we first discuss NCSC generation methods from human pluripotent stem cells and differentiation mechanism of NCSCs. Then we focus on the clinical application potential of hESC/hiPSC-derived NCSCs on peripheral nerve injuries, corneal blindness, tooth regeneration, pathological melanogenesis, Hirschsprung disease, and cardiac repair and regeneration.
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Biological function of adherent cells depends on the cell-cell and cell-matrix interactions in three-dimensional space. To understand the behavior of cells in 3D environment and their interactions with neighboring cells and matrix requires 3D culture systems. Here, we present a novel 3D cell carrier scaffold that provides an environment for routine 3D cell growth in vitro We have developed thin, mechanically stable electrohydrodynamic jet (E-jet) 3D printed polycaprolactone and polycaprolactone/Chitosan macroporous scaffolds with precise fiber orientation for basic 3D cell culture application. We have evaluated the application of this technology by growing human embryonic stem cell-derived fibroblasts within these 3D scaffolds. Assessment of cell viability and proliferation of cells seeded on polycaprolactone and polycaprolactone/Chitosan 3D-scaffolds show that the human embryonic stem cell-derived fibroblasts could adhere and proliferate on the scaffolds over time. Further, using confocal microscopy we demonstrate the ability to use fluorescence-labelled cells that could be microscopically monitored in real-time. Hence, these 3D printed polycaprolactone and polycaprolactone/Chitosan scaffolds could be used as a cell carrier for in vitro 3D cell culture-, bioreactor- and tissue engineering-related applications in the future.
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Quitosana/química , Fibroblastos/citologia , Células-Tronco Embrionárias Humanas/citologia , Poliésteres/química , Alicerces Teciduais/química , Adesão Celular , Técnicas de Cultura de Células , Diferenciação Celular , Humanos , Teste de Materiais , Microscopia Confocal , Impressão Tridimensional , Espectroscopia de Infravermelho com Transformada de Fourier , Resistência à Tração , Engenharia TecidualRESUMO
Periodontitis involves complex interplay of bacteria and host immune response resulting in destruction of supporting tissues of the tooth. Toll-like receptors (TLRs) play a role in recognizing microbial pathogens and eliciting an innate immune response. Recently, the potential application of multipotent stem cells and pluripotent stem cells including human embryonic stem cells (hESCs) in periodontal regenerative therapy has been proposed. However, little is known about the impact of periodontopathogens on hESC-derived progenies. This study investigates the effects of heat-killed periodontopathogens, namely, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, on TLR and cytokine expression profile of hESC-derived progenies, namely, fibroblasts (hESC-Fib) and mesenchymal stem cells (hESC-MSCs). Additionally, the serotype-dependent effect of A. actinomycetemcomitans on hESC-derived progenies was explored. Both hESC-Fib and hESC-MSCs constitutively expressed TLR-2 and TLR-4. hESC-Fib upon exposure to periodontopathogens displayed upregulation of TLRs and release of cytokines (IL-1ß, IL-6, and IL-8). In contrast, hESC-MSCs were largely nonresponsive to bacterial challenge, especially in terms of cytokine production. Further, exposure of hESC-Fib to A. actinomycetemcomitans serotype c was associated with higher IL-8 production than serotype b. In contrast, the hESC-MSCs displayed no serotype-dependent response. Differential response of the two hESC progenies implies a phenotype-dependent response to periodontopathogens and supports the concept of immunomodulatory properties of MSCs.
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The aim was to compare the use of different cell-material contact test methods with two different biological systems (cell line and tooth slice cultures) for cytotoxicity assessment of dental materials. Cytotoxicity of composites polymerized with two halogen-based and two light-emitting diode (LED) light-curing units (LCUs) served as the basis for comparison. Disk shaped specimens (7 x 2 mm) were fabricated using the four light sources. Composites were tested using L-929 cell line using direct/indirect/extract tests in accordance to standard protocols. Cytotoxicity was assessed using neutral red uptake. Tooth slice organ cultures were also employed to test the dental materials using direct/indirect test methods. Histomorphometric cell counting of intact odontoblasts and pulp fibroblasts and the use of tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were applied for cytotoxicity evaluation. Discrepancy in result presentation was observed in the different tests used with L-929. Sensitivity levels of the L-929 tests ranked as follows: extract test < direct contact test < indirect contact test. Tooth slice tests confirmed that L-929 direct contact test proved to be the most reliable test among the three. In conclusion, this study highlights the risk involved when relying on a single test method for cytotoxicity assessment. It would be advisable to test different culture models and then proceed using more clinically relevant biological system that stimulate the in vivo situation for confirmation.
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Linhagem Celular , Resinas Compostas/toxicidade , Materiais Dentários/toxicidade , Polpa Dentária/efeitos dos fármacos , Técnicas de Cultura de Órgãos/métodos , Testes de Toxicidade/métodos , Animais , Contagem de Células , Resinas Compostas/química , Materiais Dentários/química , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Incisivo , Masculino , Camundongos , Vermelho Neutro/metabolismo , Odontoblastos/citologia , Odontoblastos/efeitos dos fármacos , Odontoblastos/patologia , Ratos , Ratos Wistar , Sensibilidade e EspecificidadeRESUMO
A major challenge in the therapeutic application of stem cells in regenerative medicine is the lineage-specific selection of their committed/differentiated progenies for transplantation. This is necessary to avoid engraftment of undesired lineages at the transplantation site, i.e. fibroblastic scar tissue, as well as to enhance the efficacy of transplantation therapy. Commonly used techniques for lineage-specific selection of committed/differentiated stem cell progenies include marker gene transfection, fluorescence-activated (FACS) and magnetic-affinity (MACS) cell-sorting. Nevertheless, these have their disadvantages for therapeutic applications. Marker gene transfection invariably leads to permanent genetic modification of stem cells, which in turn limits their use in human clinical therapy due to overwhelming ethical and safety concerns. FACS requires expensive instrumentation and highly-skilled personnel, and is unsuited for handling bulk quantities of cells that would almost certainly be required for transplantation therapy. MACS is a cheaper alternative, but the level of purity attained is also reduced. A possible novel approach that has yet to be investigated is immunoliposome-mediated delivery of neomycin phosphotranferase (NPT) for lineage-specific selection of stem cell progenies. This would avoid permanent genetic modification to the cell, unlike recombinant NPT expression linked to activation of specific promoter sequences. Moreover, it could potentially provide a much more practical and cost-effective alternative for handling bulk quantities of cells that would be required for transplantation therapy, as compared to FACS or MACS. As such, this alternative approach needs to be rigorously investigated, in view of its potentially useful applications in stem cell therapeutics.
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Citometria de Fluxo/métodos , Técnicas de Transferência de Genes , Lipossomos/metabolismo , Células-Tronco/citologia , Diferenciação Celular , Linhagem da Célula , DNA/química , Canamicina Quinase/metabolismo , Magnetismo , TransfecçãoRESUMO
To engineer constructs of the periodontal ligament (PDL), human PDL cells were incorporated into a matrix of hyaluronan, gelatin, and type I collagen (COLI) in sample holders (13×1 mm) of six-well Biopress culture plates. The loading dynamics of the PDL were mimicked by applying a cyclic compressive strain of 33.4 kPa (340.6 gm/cm(2)) to the constructs for 1.0 s every 60 s, for 6, 12, and 24 h in a Flexercell FX-4000C Strain Unit. Compression significantly increased the number of nonviable cells and increased the expression of several apoptosis-related genes, including initiator and executioner caspases. Of the 15 extracellular matrix genes screened, most were upregulated at some point after 6-12 h deformation, but all were downregulated at 24 h, except for MMPs1-3 and CTGF. In culture supernatants, matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinases-1 (TIMP-1) protein levels were upregulated at 24 h; receptor activator of nuclear kappa factor B (RANKL), osteoprotegerin (OPG) and fibroblast growth factor-2 (FGF-2) were unchanged; and connective tissue growth factor (CTGF) not detected. The low modulus of elasticity of the constructs was a disadvantage-future mechanobiology studies and tissue engineering applications will require constructs with much higher stiffness. Since the major structural protein of the PDL is COLI, a more rational approach would be to permeabilize preformed COLI scaffolds with PDL-populated matrices.
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Apoptose/fisiologia , Colágeno Tipo I/química , Regeneração Tecidual Guiada Periodontal/instrumentação , Ácido Hialurônico/química , Ligamento Periodontal/citologia , Ligamento Periodontal/fisiologia , Materiais Biocompatíveis/síntese química , Células Cultivadas , Força Compressiva/fisiologia , Módulo de Elasticidade/fisiologia , Desenho de Equipamento , Análise de Falha de Equipamento , Gelatina/química , Regulação da Expressão Gênica/fisiologia , Regeneração Tecidual Guiada Periodontal/métodos , Humanos , Teste de Materiais , Mecanotransdução Celular/fisiologia , Estresse Mecânico , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodosRESUMO
OBJECTIVE: To investigate the physical (setting time, hardness, flowability, microstructure) and chemical (pH change, calcium release, crystallinity) properties and the biological outcomes (cell survival and differentiation) of mineral trioxide aggregate (MTA) mixed using different proportions of propylene glycol (PG) and water. MATERIAL AND METHODS: White MTA was mixed with different water/PG ratios (100/0, 80/20 and 50/50). Composition (XRD), microstructure (SEM), setting time (ASTM C266-13), flowability (ANSI/ADA 57-2000), Knoop hardness (100 g/10 s) and chemical characteristics (pH change and Ca2+ release for 7 days) were evaluated. Cell proliferation, osteo/odontoblastic gene expression and mineralization induced by MTA mixed with PG were evaluated. MTA discs (5 mm in diameter, 2 mm thick) were prepared and soaked in culture medium for 7 days. Next, the discs were removed and the medium used to culture dental pulp stem cells (DPSC) for 28 days. Cells survival was evaluated using MTS assay (24, 72 and 120 h) and differentiation with RT-PCR (ALP, OCN, Runx2, DSPP and MEPE) and alizarin red staining (7 and 14 days). Data were analysed using one-way ANOVA and Tukey's post-hoc analysis (a=0.05). RESULTS: The addition of PG significantly increased setting time, flowability and Ca2+ release, but it compromised the hardness of the material. SEM showed that 50/50 group resulted porous material after setting due to the incomplete setting reaction, as shown by XRD analysis. The addition of PG (80/20 and 50/50) was not capable to improve cell proliferation or to enhance gene expression, and mineralized deposition of DPSC after 7 and 14 days as compared to the 100/0. CONCLUSION: Except for flowability, the addition of PG did not promote further improvements on the chemical and physical properties evaluated, and it was not capable of enhancing the bioactivity of the MTA.
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Compostos de Alumínio/química , Compostos de Cálcio/química , Óxidos/química , Propilenoglicol/química , Silicatos/química , Análise de Variância , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Polpa Dentária , Combinação de Medicamentos , Expressão Gênica , Testes de Dureza , Humanos , Teste de Materiais , Microscopia Eletrônica de Varredura , Reação em Cadeia da Polimerase em Tempo Real , Reologia , Células-Tronco/efeitos dos fármacos , Fatores de TempoRESUMO
Tissue engineering offers an alternative method that can overcome some of the existing drawbacks of current articular defect repair methods because articular cartilage has a limited capacity to respond to injury. The solution may lie in the design of a three-dimensional load-bearing scaffold. Here we describe the tissue engineering of an osteochondral construct by coculturing osteogenic cells and chondrogenic cells on a three-dimensional load-bearing bioresorbable polymer scaffold. Porous polycaprolactone scaffolds were designed and fabricated via fused deposition modeling. Osteogenic cells were seeded and precultured in one-half of the partitioned scaffolds. Chondrogenic cells were later seeded into the other half. The cell-seeded scaffolds were cultured in a coculture medium. Both cell types proliferated, migrated, linked in their scaffold compartments, and integrated at the interface. Osteoblasts and chondrocytes produced different extracellular matrices in each scaffold compartment. Mineralized nodules deposited in the osteogenic cell seeded compartment. High osteocalcin was detected in precultured osteogenic cell supernatant and high alkaline phosphatase was detected in the coculture supernatant of osteochondral constructs. This study suggests that a tissue-engineered osteochondral construct with a three-dimensional polycaprolactone scaffold has the potential for osteochondral defect repair.
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Substitutos Ósseos , Cartilagem/metabolismo , Poliésteres , Engenharia Tecidual , Fosfatase Alcalina/metabolismo , Animais , Técnicas de Cocultura , Matriz Extracelular , Humanos , Microscopia Eletrônica de Varredura , Microscopia de Contraste de Fase , Osteocalcina/metabolismoRESUMO
Molybdenum(V) thiocyanate complex extracted by Tween-80, a non-ionic surface active agent, in chloroform has been studied. When the concentration of Tween-80 is 0.1%, extractive ratio can reach 94%, while tungsten(III) thiocyanate can not extracted in the same condition. Therefore, the two kinds of complexes can be separated very well. It enjoys a better extraction efficiency compared with petroleum ether or dimethyl sulfoxide and the advantage of low cost, no toxicity and no pollution. The extraction mechanism has been studied and may be a kind of extraction belonging to the category of supermolecular, which has not been reported publicly.
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Molibdênio/química , Tiocianatos/química , Quelantes/química , Clorofórmio , Polissorbatos , Espectrofotometria Ultravioleta/métodos , Tungstênio/químicaRESUMO
Human embryonic stem cells (hESCs) have the ability to self-renew and differentiate into any cell lineage of the three germ layers, therefore holding great promise for regenerative applications in dentistry and medicine. We previously described a micromass culture system as a model system to induce and study the chondrogenic commitment of hESCs. Using this system, chondrogenic cells can be further isolated and expanded under specific growth factor conditions. When encapsulated in hyaluronic acid (HA)-based hydrogels and cultured under appropriate growth factor and medium conditions, these chondrogenic cells synthesized and deposited extracellular matrix (ECM) characteristic of neocartilage. Here, we describe the micromass culture of hESCs, the isolation and expansion of hESC-derived chondrogenic cells, and the three-dimensional (3-D) culture of the chondrogenic cells in hydrogels for cartilage tissue engineering. We will also describe the various tools and techniques used for characterizing the tissue-engineered cartilage.
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INTRODUCTION: The induced pluripotent stem cells (iPSCs) have characteristics similar to embryonic stem cells, including the capability of self-renewal and large-scale expansion and the ability to differentiate into all types of cells including germ cells, which defines pluripotency. Using iPSC avoids problems of immunological rejection and ethical controversy. The possible future uses of iPSC are diverse and go beyond the differentiation into somatic cells for regeneration of damaged tissues. AREAS COVERED: A unique feature of iPSC is the potential to generate patient disease-specific tissues. Thus, cells from patients can be differentiated into relevant cells of interest for drug screening, characterization of drug effects and cytotoxic assays. This review presents key aspects related to iPSC, such as their generation, potential for disease modeling, treatment, drug development and future contributions to the craniofacial complex. EXPERT OPINION: It is undisputable that the evolution in iPSC knowledge will improve the approaches for drug screening and development, help to understand and treat disease origins and mechanisms and provide new strategies to clinical treatment. However, it is necessary to fine-tune protocols to establish iPSCs that are cost-effective and safe for clinical use.