RESUMO
Mesenchymal stem cells (MSCs) can be isolated from dental tissues, such as pulp and periodontal ligament; the dental apical papilla (DAP) is a less-studied MSC source. These dental-derived MSCs are of great interest because of their potential as an accessible source for cell-based therapies and tissue-engineering (TE) approaches. Much of the interest regarding MSCs relies on the trophic-mediated repair and regenerative effects observed when they are implanted. TGFß3 is a key growth factor involved in tissue regeneration and scarless tissue repair. We hypothesized that human DAP-derived MSCs (hSCAPs) can produce and secrete TGFß3 in response to micro-environmental cues. For this, we encapsulated hSCAPs in different types of matrix and evaluated TGFß3 secretion. We found that dynamic changes of cell-matrix interactions and mechanical stress that cells sense during the transition from a monolayer culture (two-dimensional, 2D) towards a three-dimensional (3D) culture condition, rather than the different chemical composition of the scaffolds, may trigger the TGFß3 secretion, while monolayer cultures showed almost 10-fold less secretion of TGFß3. The study of these interactions is provided as a cornerstone in designing future strategies in TE and cell therapy that are more efficient and effective for repair/regeneration of damaged tissues. Copyright © 2015 John Wiley & Sons, Ltd.
Assuntos
Papila Dentária/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Fator de Crescimento Transformador beta3/metabolismo , Adolescente , Adulto , Antígenos CD/metabolismo , Separação Celular , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Modelos Biológicos , Análise de Componente Principal , Adulto JovemRESUMO
Gelatin-/chitosan-/hyaluronan-based biomaterials are used in tissue engineering as cell scaffolds. Three gamma radiation doses (1, 10 and 25 kGy) were applied to scaffolds for sterilization. Microstructural changes of the irradiated polymers were evaluated by using scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). A dose of 25 kGy produced a rough microstructure with a reduction of the porosity (from 99 to 96 %) and pore size (from 160 to 123 µm). Radiation also modified the glass transition temperature between 31.2 and 42.1 °C (1 and 25 kGy respectively). Human skin cells cultivated on scaffolds irradiated with 10 and 25 kGy proliferated at 48 h and secreted transforming growth factor ß3 (TGF-ß3). Doses of 0 kGy (non-irradiated) or 1 kGy did not stimulate TGF-ß3 secretion or cell proliferation. The specific growth rate and lactate production increased proportionally to radiation dose. The use of an appropriate radiation dose improves the cell scaffold properties of biomaterials.
Assuntos
Materiais Biocompatíveis/química , Pele/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Varredura Diferencial de Calorimetria , Proliferação de Células/efeitos da radiação , Quitosana/química , Relação Dose-Resposta à Radiação , Raios gama , Gelatina/química , Humanos , Ácido Hialurônico/química , Lactatos/metabolismo , Microscopia Eletrônica de Varredura , Porosidade , Temperatura , Fator de Crescimento Transformador beta3/metabolismoRESUMO
Radical scavenging activity (RSA), antioxidant content (TEAC), total phenolic compounds content (TPCC) and volatile profile (VOCs) were measured in 26 honeys obtained from the Valparaiso Region (Chile). Persea americana honey was the most interesting sample according to these evaluated parameters. A Projection to Latent Structures (PLS) based algorithm was used to model the possible relationship between antioxidant activity, total phenolic compounds content and volatile profile. Concerning the volatile profile, only nine volatile compounds, of a total of fifty, showed dependence on antioxidant activity and total phenolic compounds content.
Assuntos
Antioxidantes/química , Mel/análise , Algoritmos , ChileRESUMO
The inadequate treatments given to the served waste water which are disposal to the rivers and sea coast are the major sources of faecal Microorganisms and enteric bacterial pathogens. They are among the most serious effects of water pollution bringing risks on public health. None of the current methods for detection of pathogens offer real-time on site solutions, are capable of delivering a simple visual detection signal, or can be easily instrumented as an indicator of the presence of a pathogen in water. The use of lipid vesicles incorporating Polydiacetylenes (PDAs) for the development of biosensors for real-time detection of pathogens has become an alternative, due to its potential for simple colorimetric response against harmful environmental effectors. However, its actual application in the field has been complicated because lipid vesicles are unable to respond specifically to environmental changes. In this paper, we report several experimental trials leading to improved response in the detection of flagellated pathogens in drinking water. Chromatic biomimetic membranes of TRCDA/DMPC and TRCDA/DMPC/Tryptophan were used in agar and liquid media, which were challenged with different amounts of Escherichia coli and Salmonella typhimurium. In addition, the effect of some divalent cations on the interaction with vesicles TRCDA/DMPC was investigated. The results indicated an improvement in the response times, both visually and quantitatively, through the use of TRIS-EDTA and proper growing conditions for E. coli and Salmonella. With the application of both conditions, it was possible by incubation at 35ºC to promote bacterial growth, therefore avoiding a dramatic effect on the colour change over control samples which may invalidate the test. Our experiments indicated that the minimum bacterial concentration necessary to produce the transition from blue to red on the vesicles as biosensor approaches 10(8) CFU/ml within 4 hrs...
Assuntos
Água Potável/microbiologia , Técnicas Biossensoriais , Bactérias/isolamento & purificação , Membranas Artificiais , Polímero Poliacetilênico/química , Polímeros/química , Colorimetria , Microbiologia da ÁguaRESUMO
Gelatin, chitosan and hyaluronic acid are natural components used to prepare polymeric scaffold in tissue engineering. The physical properties of these materials confer an appropriate microenvironment for cells, which can be used as a regeneration system for skin and cartilage. In this work, we prepared and characterized a Gelatin/Chitosan/Hyaluronan lyophilized-polymer. Physical properties of lyophilized-polymer changed slightly with moisture, but when polymer was totally hydrated the elasticity changed significantly. Thermophysical characterisation indicated that temperatures higher than 30ºC could modify irreversibly the polymeric matrix probably due to protein denaturation. Besides, we used the polymer as scaffold to prepare a biosynthetic-skin, reporting biological behaviour and its mechanical properties.
Assuntos
Ácido Hialurônico/química , Gelatina/química , Quitosana/química , Varredura Diferencial de Calorimetria , Imuno-Histoquímica , Microscopia Eletrônica de Varredura , Materiais Biocompatíveis/química , Polímeros , Pele ArtificialRESUMO
BACKGROUND: This manuscript reports the production and preclinical studies to examine the tolerance and efficacy of an autologous cellular gel-matrix integrated implant system (IIS) aimed to treat full-thickness skin lesions. METHODS: The best concentration of fibrinogen and thrombin was experimentally determined by employing 28 formula ratios of thrombin and fibrinogen and checking clot formation and apparent stability. IIS was formed by integrating skin cells by means of the in situ gelification of fibrin into a porous crosslinked scaffold composed of chitosan, gelatin and hyaluronic acid. The in vitro cell proliferation within the IIS was examined by the MTT assay and PCNA expression. An experimental rabbit model consisting of six circular lesions was utilized to test each of the components of the IIS. Then, the IIS was utilized in an animal model to cover a 35% body surface full thickness lesion. RESULTS: The preclinical assays in rabbits demonstrated that the IIS was well tolerated and also that IIS-treated rabbit with lesions of 35% of their body surface, exhibited a better survival rate (p = 0,06). CONCLUSION: IIS should be further studied as a new wound dressing which shows promising properties, being the most remarkable its good biological tolerance and cell growth promotion properties.
Assuntos
Géis/farmacologia , Implantes Experimentais , Cicatrização/efeitos dos fármacos , Animais , Bioensaio , Coagulação Sanguínea/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fibrinogênio/metabolismo , Imuno-Histoquímica , Antígeno Nuclear de Célula em Proliferação/metabolismo , Coelhos , Pele/efeitos dos fármacos , Pele/patologia , Trombina/metabolismo , Resultado do TratamentoRESUMO
Biochemical oxygen demand (BOD) is a measure of the amount of dissolved oxygen that is required for the biochemical oxidation of the organic compounds in 5 days. New biosensor-based methods have been conducted for a faster determination of BOD. In this study, a mathematical model to evaluate the feasibility of using a BOD sensor, based on disposable alginate-entrapped bacteria, for monitoring BOD in situ was applied. The model considers the influences of alginate bead size and bacterial concentration. The disposable biosensor can be adapted according to specific requirements depending on the organic load contained in the wastewater. Using Klein and Washausen parameter in a Lineweaver-Burk plot, the glucose diffusivity was calculated in 6.4 × 10(-10) (m2/s) for beads of 1 mm in diameter and slight diffusion restrictions were observed (n = 0.85). Experimental results showed a correlation (p < 0.05) between the respirometric peak and the standard BOD test. The biosensor response was representative of BOD.
Assuntos
Alginatos/metabolismo , Reatores Biológicos , Enterobacter cloacae/metabolismo , Oxigênio/metabolismo , Técnicas Biossensoriais , Estudos de Viabilidade , Glucose/metabolismo , Ácido Glucurônico/metabolismo , Ácidos Hexurônicos/metabolismoRESUMO
Fibrin has been used extensively in cell encapsulation because it has important biological properties. Keratinocyte encapsulation in fibrin is a widely used technique in skin tissue engineering. The production of growth factors (EGF, TGF-beta1 and PDGF-BB) was evaluated when keratinocytes are encapsulated in fibrin. Secretions of TGF-beta1 and PDGF-BB increased more than five times compared to monolayer cultures. Encapsulated cells secreted about 80% active form of TGF-beta1 (monolayer cells only secreted inactive form). An enhanced secretion of TGF-beta1 and PDGF-BB was found in encapsulated cells, showing that fibrin capsules are favourable for the production of these growth factors.
Assuntos
Fibrina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Queratinócitos/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , HumanosRESUMO
It is known that skin releases volatile organic compounds to the environment, and also that its emission pattern changes with aging of the skin. It could be considered, that these compounds are intermediaries in cell metabolism, since many intermediaries of metabolic pathways have a volatile potential. In this work, a simple and non-destructive method consisting of SPME sampling and GC/MS analysis was developed to identify volatile organic emanations from cell cultures. This technique, applied to skin cells culture, indicates that the cells or cell metabolism produce several skin emissions. Chemometric analysis was performed in order to explore the relationship between a volatile profile and the senescence of cell cultures. Volatile profiles were different for cell cultures in different degrees of senescence, indicating that volatile compound patterns could be used to provide information about the age of skin cells.
Assuntos
Senescência Celular , Pele/citologia , Pele/metabolismo , Compostos Orgânicos Voláteis/análise , Células Cultivadas , Criança , Pré-Escolar , Biologia Computacional , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Análise dos Mínimos Quadrados , Redes e Vias Metabólicas , Análise de Componente Principal , Microextração em Fase Sólida , VolatilizaçãoRESUMO
One of the limitations in tissue engineering is the restricted ability to expand the number of cells, because somatic cells can duplicate a limited number of times before they lose the ability to divide, leading to a senescent state. Here we report that the interaction of senescent fibroblasts with fibrin polymer can modify the senescent phenotype and partially restore the ability of growth-arrested cells to continue replicating. Primary human dermal fibroblasts were grown to >90% SA/beta-Gal (senescence associated beta-galactosidase). The senescent cells were immobilized in fibrin-polymers by mixing fibrinogen and thrombin solutions. Immobilized senescent cell cultures grew, however, their growth arrested after 24 h of immobilization. The percentage of cells with a positive reaction at SA/beta-Gal did not decrease significantly after immobilization, but the intensity of the stain decreased. The glycolytic activity in immobilized senescent fibroblast was re-established at pre-senescent levels. In conclusion, fibrin induces changes in the phenotype of senescent human fibroblasts. This simple procedure could complement available tissue-engineering techniques to increase the amount of biomass seeded on a fibrin scaffold, which could be beyond senescence.