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Therapies for wound healing using the secretome and extracellular vesicles (EVs) of mesenchymal stem/stromal cells have been shown to be successful in preclinical studies. This study aimed to characterise the protein content of the secretome from stem cells from human exfoliated deciduous teeth (SHED) and analyse the in vitro effects of SHED-conditioned medium (SHED-CM) and SHED extracellular vesicles (SHED-EVs) on keratinocytes. EVs were isolated and characterised. The keratinocyte viability and migration of cells treated with SHED-EVs and conditioned medium (CM) were evaluated. An HaCaT apoptosis model induced by H2 O2 in vitro was performed with H2 O2 followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live/dead assays. Finally, the expression of vascular endothelial growth factor (VEGF) in keratinocytes treated with secretome and EVs was evaluated by immunofluorescence staining and confirmed with RT-qPCR. SHED-EVs revealed a cup-shaped morphology with expression of the classical markers for exosomes CD9 and CD63, and a diameter of 181 ± 87 nm. The internalisation of EVs by HaCaT cells was confirmed by fluorescence microscopy. Proteomic analysis identified that SHED-CM is enriched with proteins related to stress response and development, including cytokines (CXCL8, IL-6, CSF1, CCL2) and growth factors (IGF2, MYDGF, PDGF). The results also indicated that 50% CM and 0.4-0.6 µg/mL EVs were similarly efficient for improving keratinocyte viability, migration, and attenuation of H2 O2 -induced cytotoxicity. Additionally, expression of VEGF on keratinocytes increased when treated with SHED secretome and EVs. Furthermore, VEGF gene expression in keratinocytes increased significantly when treated with SHED secretome and EVs. Both SHED-CM and SHED-EVs may therefore be promising therapeutic tools for accelerating re-epithelialization in wound healing.
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Vesículas Extracelulares , Cicatrização , Humanos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Meios de Cultivo Condicionados/farmacologia , Meios de Cultivo Condicionados/metabolismo , Proteômica , Secretoma , Células-Tronco/metabolismo , Queratinócitos , Vesículas Extracelulares/metabolismo , Dente DecíduoRESUMO
The process of electrohydrodynamic living cell microencapsulation inside a scaffold during the electrospinning (ES) process is called cell electrospinning (CE). Several studies demonstrate the feasibility of using cell electrospinning for biomedical applications, allowing for the direct biofabrication of living cells to be encapsulated in fibers for the formation of active biological scaffolds. In this review, a comprehensive overview of the materials and methodologies used in cell electrospinning, as well as their biomedical application in tissue engineering, is provided. Cell ES represents an innovative technique for automated application in regenerative medicine.
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Decellularization of organs creates an acellular scaffold, ideal for being repopulated by cells. In this work, a low-cost perfusion system was created to be used in the process of liver decellularization and as a bioreactor after recellularization. It consists of a glass chamber to house the organ coupled to a peristaltic pump to promote liquid flow through the organ vascular tree. The rats' liver decellularization was made with a solution of sodium dodecyl sulfate. The recellularization was made with 108 mesenchymal stromal/stem cells and cultivated for seven days. The decellularized matrices showed an absence of DNA while preserving the collagen and glycosaminoglycans quantities, confirming the efficiency of the process. The functional analyses showed a rise in lactate dehydrogenase levels occurring in the first days of the cultivation, suggesting that there is cell death in this period, which stabilized on the seventh day. Histological analysis showed conservation of the collagen web and some groups of cells next to the vessels. It was possible to establish a system for decellularization and a bioreactor to use for the recellularization method. It is easy to assemble, can be ready to use in little time and be easily sterilized.
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Hybrid scaffolds from natural and synthetic polymers have been widely used due to the complementary nature of their physical and biological properties. The aim of the present study, therefore, has been to analyzein vivoa bilayer scaffold of poly(lactide-co-glycolide)/fibrin electrospun membrane and fibrin hydrogel layer on a rat skin model. Fibroblasts were cultivated in the fibrin hydrogel layer and keratinocytes on the electrospun membrane to generate a skin substitute. The scaffolds without and with cells were tested in a full-thickness wound model in Wistar Kyoto rats. The histological results demonstrated that the scaffolds induced granulation tissue growth, collagen deposition and epithelial tissue remodeling. The wound-healing markers showed no difference in scaffolds when compared with the positive control. Activities of antioxidant enzymes were decreased concerning the positive and negative control. The findings suggest that the scaffolds contributed to the granulation tissue formation and the early collagen deposition, maintaining an anti-inflammatory microenvironment.
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Hidrogéis , Alicerces Teciduais , Ratos , Animais , Fibrina , Colágeno/farmacologia , Polímeros , Engenharia Tecidual/métodosRESUMO
A oleorresina obtida de copaíferas é amplamente utilizada na medicina tradicional brasileira. Este estudo avaliou a composição química por Cromatografia Gasosa (CG) e o efeito da oleorresina de Copaifera officinalis em células-tronco. Para isso as células foram tratadas com a oleorresina nas concentrações de 0,5, 20, 110, 140, 170 ou 200 µg/ml por 24h. A avaliação por CG identificou os sesquiterpenos ß-cariofileno, trans-α-bergamoteno e óxido de cariofileno II como os compostos majoritários da oleorresina. As menores concentrações de oleorresina utilizadas apresentaram resultados semelhantes ao grupo controle e as maiores concentrações diminuíram significativamente a viabilidade celular e apresentaram maior citotoxicidade. Como conclusão, os principais componentes encontrados na oleorresina de copaíba foram os sesquiterpenos e as baixas concentrações testadas não foram citotóxicas. O aumento das concentrações de oleorresina de copaíba promoveu diminuição da viabilidade celular e aumento dos efeitos citotóxicos nas células-tronco. Embora a oleorresina de copaíba tenha uso etnofarmacológico na cicatrização, este estudo demonstrou efeito citotóxico em células-tronco, as quais estão relacionadas ao processo de regeneração corpóreo. Portanto, deve-se ter cuidado com a dosagem de oleorresina a ser utilizada, uma vez que este estudo in vitro mostrou citotoxicidade e um impacto negativo na viabilidade das células-tronco nas mais altas concentrações testadas. [au]
Oleoresinobtained from Copaifera trees is extensively used in Brazilian traditional medicine. This study hasevaluated the chemical composition and effect of Copaifera officinalisoleoresin on stem cells. The oleoresin was analyzed by Gas Chromatography (GC) and the cells were treated with the oleoresin at concentrations of 0.5, 20, 110, 140, 170 or 200 µg/ml for 24h for cellular tests. GC identified the sesquiterpenes beta-caryophyllene, trans-alpha-bergamotene, and caryophyllene oxide II as the main compounds in oleoresin. The cell viability and cytotoxicity assays showed the lowest concentrations of oleoresin used presented similar results to the control group and the higher concentrations tested significantly decreased cell viability and increased cytotoxicity. In a conclusion, the main components found in copaiba oleoresin were sesquiterpenes and the low tested concentrations were not cytotoxic. The increased concentrations of copaiba oleoresin promoted a decrease in cell viability and an increase of cytotoxicity in the stem cells. Although copaiba oleoresin has ethnopharmacology use in healing, this study showed toxicity in stem cells, which are related to the corporeal regeneration process. Therefore, caution must be taken with the dosage of the oleoresin to be used since this in vitro study showed cytotoxicity and a negative impact on stem cell viability at the higher tested concentrations. [au]
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Glass and bioactive glass-ceramic can be used in several applications. In bone growth where good bone/biomaterial adhesion was required, bioactive coatings for implants can improve bone formation. The glass and glass-ceramics of the LZS (Li2 O-ZrO2 -SiO2 ) system are very interesting because of their mechanical, electrical, and thermal properties. Very recently, their biological response in contact with human osteoblast has been evaluated. However, despite several initiatives, there are still no studies that systematically assess this system's bioactivity, dissolution, and cytotoxicity in vitro. This work aims to investigate the dissolution, bioactivity behavior, and cytotoxicity of LZS glass-ceramic. LZS glass-ceramics were produced from SiO2 , Li2 CO3, and ZrSiO4 by melting followed by quenching. The obtained glass frits were milled and uniaxially pressed and heat-treated at 800 and 900°C and submitted to physical-chemical, structural and mechanical characterization. Their dissolution behavior was studied in Tris-HCl, while bioactivity was performed in simulated solution body fluid (SBF). The cytotoxicity test was performed using glass-ceramic in direct contact with mesenchymal stem/stromal cells (SC) isolated from human exfoliated deciduous teeth. Structural and microstructural analyzes confirmed bioactivity. The results show that it was possible to produce bioactive glass-ceramic from LZS, proven by the formation of new calcium phosphate structures such as hydroxyapatite on the surface of the samples after exposure to SBF. The SC viability test performed indicated that the materials were not cytotoxic at 0.25, 0.5, and 1.0 mg/ml. The glass-ceramic system under study is very promising for a medicinal application that requires bioactivity and/or biocompatibility for bone regeneration.
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Cerâmica , Dióxido de Silício , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Cerâmica/química , Cerâmica/farmacologia , Vidro/química , Humanos , Dióxido de Silício/química , SolubilidadeRESUMO
Exosomes are extracellular vesicles secreted by various cell types, which play important roles in physiological processes. In particular, stem cell-derived exosomes have been shown to play crucial functions in intercellular communication during the tissue healing process. This review summarizes the effects of exosomes derived from different stem cell sources on the repair of cutaneous and bone tissue, focusing on the different pathways that could be involved in the regeneration process. The biogenesis, isolation, and content of exosomes have also been discussed. The effectiveness of exosomes is broadly demonstrated for skin and bone regeneration in animal models, supporting the basis for clinical translation of exosomes as a ready-to-use cell-free therapeutic for skin and bone regeneration.
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Regeneração Óssea , Osso e Ossos , Exossomos , Pele , Células-Tronco/metabolismo , Animais , Osso e Ossos/lesões , Osso e Ossos/metabolismo , Exossomos/metabolismo , Exossomos/transplante , Humanos , Pele/lesões , Pele/metabolismoRESUMO
The genus Hypericum (Hypericaceae) is a recognized source of therapeutic agents, being some species widely used due to their wound healing properties. In a previous study, south Brazilian species H. caprifoliatum, H. carinatum, H. connatum, H. myrianthum and H. polyanthemum demonstrated potential to induce proliferation of keratinocytes. In the present study, the effect of phloroglucinol derivatives isolated from Hypericum on cell proliferation of human keratinocytes, fibroblasts and stem cells was investigated. The best results, determined by the MTT assay, were achieved with cariphenone B at concentrations of 0.01 and 0.1 µM (122.3% and 114%, respectively) on HaCaT cells. Uliginosin B was able to induce the proliferation of mesenchymal stem cells (129% at 10 µM) and MRC5 fibroblasts (152.5% at 5 µM). These findings confirm the capacity of phloroglucinol derivatives to induce the in vitro cellular proliferation and reinforce the importance of Hypericum species as potential sources of wound healing compounds.
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Hypericum , Proliferação de Células , Humanos , Floroglucinol/farmacologia , Extratos Vegetais/farmacologia , CicatrizaçãoRESUMO
The aim of this study has been to fabricate a hybrid electrospun nanofibrous scaffold composed of poly(lactic-co-glycolic) acid (PLGA)/fibrin polymers to be used as a skin substitute and analyze its physical and biological properties. Fibrin was obtained from rat blood plasma, characterized and solubilized in formic acid. The final electrospinning solution concentration was 40% PLGA (w/v) and 1% fibrin (w/v). To improve spinnability, 3% PEG (w/v) was added. The scaffolds were characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Water contact angle, maximum elongation, thermal stability, degree of swelling, blood compatibility, cytotoxicity and cell viability were analyzed. The characterization by SEM showed randomly oriented nanofibers with a mean diameter of 639.8 ± 241.8 nm for the PLGA/fibrin and 1051.0 ± 290.2 nm for the PLGA. FTIR spectra confirmed the presence of fibrin in the mats. Fibrin incorporation reduced the water contact angle from 118.9 ± 2.9 to 111.1 ± 2.8. The fibrin increased tensile strength and decreased elongation at break. The scaffolds demonstrated blood compatibility and fibrin incorporation improved cell adhesion and viability when direct and indirect MTT analyses were carried out. Thus, it can be concluded that the PLGA/fibrin mat is a promising material for use as a skin substitute.
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Fibrina/química , Ácido Poliglicólico/química , Pele Artificial , Animais , Adesão Celular , Sobrevivência Celular , Células Cultivadas , Formiatos/química , Ácido Láctico/química , Masculino , Microscopia Eletrônica de Varredura , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros/química , Ratos , Ratos Wistar , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície , Resistência à Tração , Sais de Tetrazólio/química , Tiazóis/química , Engenharia Tecidual/métodos , Alicerces Teciduais/químicaRESUMO
The transplantation of stem cells from human exfoliated deciduous teeth (SHED) has been studied as a possible treatment strategy for spinal cord injuries (SCIs) due to its potential for promoting tissue protection and functional recovery. The aim of the present study was to investigate the effects of the early transplantation of SHED on glial scar formation and astrocytic reaction after an experimental model of SCI. Wistar rats were spinalized using the NYU Impactor. Animals were randomly distributed into three groups: control (naive) (animal with no manipulation); SCI (receiving laminectomy followed by SCI and treated with vehicle), and SHED (SCI rat treated with intraspinal SHED transplantation, 1 h after SCI). In vitro investigation demonstrated that SHED were able to express mesenchymal stem cells, vimentin and S100B markers, related with neural progenitor and glial cells, respectively. The acute SHED transplantation promoted functional recovery, measured as from the first week after spinal cord contusion by Basso, Beattie, and Bresnahan scale. Twenty-four and 48 h after lesion, flow cytometry revealed a spinal cord vimentin+ cells increment in the SHED group. The increase of vimentin+ cells was confirmed by immunofluorescence. Moreover, the bioavailability of astrocytic proteins such as S100B and Kir4.1 shown to be increased in the spinal cord of SHED group, whereas there was a glial scar reduction, as indicated by ELISA and Western blot techniques. The presented results support that SHED act as a neuroprotector agent after transplantation, probably through paracrine signaling to reduce glial scar formation, inducing tissue plasticity and functional recovery.
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Astrócitos/patologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/terapia , Esfoliação de Dente/patologia , Dente Decíduo/citologia , Animais , Aquaporina 4/metabolismo , Astrócitos/metabolismo , Células Cultivadas , Proteína Glial Fibrilar Ácida/metabolismo , Membro Posterior/fisiopatologia , Humanos , Masculino , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Ratos Wistar , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Traumatismos da Medula Espinal/metabolismo , Vimentina/metabolismoRESUMO
The authors hereby declare that the Figure 4 in page eight of the paper "Stem cells from human exfoliated deciduous teeth modulate early astrocyte response after spinal cord contusion" authored by Fabrício Nicola and colleagues (DOI: 10.1007/s12035-018-1127-4) was mistakenly included.
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Peripheral nerve injury is an important cause of incapability and has limited available treatment. Autologous donor nerve implant is the golden standard treatment, however, may cause secondary deficits. Stem cells show positive results in preclinical settings, preserving tissue and function. We tested the efficacy of stem cells derived from human exfoliated deciduous teeth seeded in poly (lactide-co-glycolide) scaffolds in sciatic nerve transection model. Seventy-two adult male Wistar rats had 7-mm nerve gap bridge using scaffolds with (or without) stem cells. Animals were randomly divided into: sham-operated; sham-operated without scaffold; sham-operatedâ¯+â¯scaffoldâ¯+â¯stem cells; sciatic transectionâ¯+â¯no treatment; sciatic transectionâ¯+â¯acellular scaffolds; sciatic transectionâ¯+â¯scaffoldâ¯+â¯stem cells. Sciatic Functional Index and Ladder Rung Walking tests were performed before (-1), 14 and 28â¯days after surgery. Morphometric nerve measurement and muscle weights were assessed. Scaffolds with stem cells improved function in Sciatic Functional Index. Acellular scaffold was effective, promoting functional recovery and nerve regeneration following nerve injury. Scaffolds provide better nerve regeneration and functional recovery after sciatic transection. Despite cell therapy promoting faster recovery after sciatic transection in the Sciatic Index Score, stem cells did not improve functional and morphological recovery after nerve injury. This is the first study testing the potential use of scaffolds combined with stem cells in the early stages after injury. Scaffolds with stem cells could accelerate nerve recovery and favor adjuvant therapies, evidencing the need for further studies to increase the knowledge about stem cells' mechanisms.
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Regeneração Nervosa/efeitos dos fármacos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/uso terapêutico , Recuperação de Função Fisiológica/efeitos dos fármacos , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/lesões , Alicerces Teciduais , Animais , Humanos , Masculino , Ratos , Recuperação de Função Fisiológica/fisiologia , Nervo Isquiático/citologia , Nervo Isquiático/fisiologia , Transplante de Células-Tronco , Caminhada/fisiologiaRESUMO
Electrospinning and electrospraying technologies provide an accessible and universal synthesis method for the continuous preparation of nanostructured materials. This chapter introduces recent uses of electrospun and electrosprayed scaffolds for tissue regeneration applications. More recent in vitro and in vivo of electrospun fibers are also discussed in relation to soft and hard tissue engineering applications. The focus is made on the bone, vascular, skin, neural and soft tissue regeneration. An introduction is presented regarding the production of biomaterials made by synthetic and natural polymers and inorganic and metallic materials for use in the production of scaffolds for regenerative medicine. For this proposal, the following techniques are discussed: electrospraying, co-axial and emulsion electrospinning and bio-electrospraying. Tissue engineering is an exciting and rapidly developing field for the understanding of how to regenerate the human body.
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Nanofibras , Medicina Regenerativa/tendências , Engenharia Tecidual , Alicerces Teciduais , Materiais Biocompatíveis , HumanosRESUMO
Regenerative medicine involves the study of cells, signaling cues and biomatrices to restore normal function of tissues and organs. To develop the matrices for use in tissue engineering there are three main groups of biomaterials: (i) naturally derived materials; (ii) synthetic polymers; and (iii) decellularized organ or tissue scaffolds. These biomaterials, in various forms such as hydrogels, nanofibers and 3D scaffolds, among others, have been employed for different tissue regeneration purposes, with several techniques involved in their production, including rapid prototyping, tissue decellularization and electrospinning. In this review, the main topics of hydrogels, 3D printing and electrospun scaffolds, other biomaterials and decellularization and recellularization will be discussed.
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Materiais Biocompatíveis , Matriz Extracelular/fisiologia , Polímeros/química , Medicina Regenerativa/métodos , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Matriz Extracelular/transplante , Humanos , Hidrogéis , Impressão TridimensionalRESUMO
In this study, an evaluation was performed to determine the in vitro bioactivity, viability of stem cells, and antibiofilm effect against Streptococcus mutans of two bioactive gel-glass 60SiO2-36CaO-4P2O5 (BG-A) and 80SiO2-15CaO-5P2O5 (BG-B) compositions. Both materials were bioactive and undergo the formation of hydroxycarbonate apatite (HCA) on their surfaces when immersed in simulated body fluid (SBF) after 12h, but the BG-A composition showed a more significant formation rate. The pH variation of the samples during the test in SBF indicated that an abrupt change had occurred for the BG-A composition within the first few hours, and the pH was subsequently maintained over time, supporting its stronger antibacterial effects against S. mutans. For the in vitro viability test using mesenchymal stem cells (MSCs), the BG-B showed significantly higher cell viability compared to the BG-A composition at concentrations of 0.125, 1.25 and 12.50mg/mL for 2days. These results indicated that the higher solubility of the BG-A glass favors bioactivity and antibacterial effects. However, as a result of rapid degradation, the increase in the concentration of ions in the cell culture medium was not favorable for cell proliferation. Thus, by varying the composition of glasses, and consequently their dissolution rate, it is possible to favor bioactivity, antimicrobial activity or stem cell proliferation for a particular application of interest.
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Streptococcus mutans , Materiais Biocompatíveis , Biofilmes , Líquidos Corporais , Sobrevivência Celular , Géis , Vidro , Células-TroncoRESUMO
Stem cells from human exfoliated deciduous teeth (SHED) transplants have been investigated as a possible treatment strategy for spinal cord injuries (SCI) due to their potential for promoting functional recovery. The aim of present study was to investigate the effects of SHED on neuronal death after an experimental model of SCI. METHODS: Wistar rats were spinalized using NYU impactor®. Animals were randomly distributed into 4 groups: Control (Naive) or Surgical control, Sham (laminectomy with no SCI); SCI (laminectomy followed by SCI, treated with vehicle); SHED (SCI treated with intraspinal transplantation of 3×105 SHED, 1h after SCI). Functional evaluations and morphological analysis were performed to confirm the spinal injury and the benefit of SHED transplantation on behavior, tissue protection and motor neuron survival. Flow cytometry of neurons, astrocytes, macrophages/microglia and T cells of spinal cord tissue were run at six, twenty-four, forty-eight and seventy-two hours after lesion. Six hours after SCI, ELISA and Western Blot were run to assess pro- and anti-apoptotic factors. The SHED group showed a significant functional improvement in comparison to the SCI animals, as from the first week until the end of the experiment. This behavioral protection was associated with less tissue impairment and greater motor neuron preservation. SHED reduced neuronal loss over time, as well as the overexpression of pro-apoptotic factor TNF-α, while maintained basal levels of the anti-apoptotic BCL-XL six hours after lesion. Data here presented show that SHED transplantation one hour after SCI interferes with the balance between pro- and anti-apoptotic factors and reduces early neuronal apoptosis, what contributes to tissue and motor neuron preservation and hind limbs functional recovery.
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Células-Tronco Adultas/transplante , Traumatismos da Medula Espinal/terapia , Dente Decíduo/transplante , Células-Tronco Adultas/patologia , Animais , Apoptose , Astrócitos/patologia , Sobrevivência Celular , Células Cultivadas , Humanos , Masculino , Neurônios/fisiologia , Fármacos Neuroprotetores/metabolismo , Ratos , Ratos Wistar , Recuperação de Função Fisiológica/fisiologia , Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Células-Tronco/patologia , Dente Decíduo/metabolismoRESUMO
OBJECTIVES: The hexane (HEX) and dichloromethane (DCM) fractions from Blechnum binervatum, Blechnum brasiliense and Blechnum occidentale were studied about phytochemicals and biological properties using multitarget approach. METHODS: The chemical composition was performed by gas chromatography coupled with mass spectrometry detector (GC-MS) analysis. Antioxidant capacity was evaluated against free radicals and on lipid peroxidation. Monoamine oxidases (MAO) and cholinesterases enzymatic modulation, as well as effects on rat and human cells, were assessed. KEY FINDINGS: The CG-MS analysis allowed the identification of a non-polar compound series, being neophytadiene the major constituent in all DCM fractions and in HEX fractions from B. binervatum and B. occidentale. In B. brasiliense HEX fraction, ß-sitosterol was the main compound. In general, B. brasiliense DCM fraction presented the highest antioxidant activity, with IC50 values around 9, 2 and 1.2 times lower than those found for the other species, against HOË, NOË and on lipid peroxidation, respectively. Regarding enzyme modulations, B. brasiliense DCM fraction presented higher MAO-A inhibition (IC50 : 31.83 µg/ml), with a better selectivity index (SI MAO-A/MAO-B: 6.77). The lack of harmful effects was observed in rat cells, also highlighted in the stem cells for all Blechnum samples. CONCLUSION: These findings encourage the search for multibinding natural products, mainly from B. brasiliense DCM fraction.
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Antioxidantes/farmacologia , Gleiquênias/química , Inibidores da Monoaminoxidase/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Sitosteroides/farmacologia , Terpenos/análise , Animais , Antioxidantes/análise , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Monoaminoxidase/metabolismo , Inibidores da Monoaminoxidase/análise , Extratos Vegetais/química , Ratos Wistar , Sitosteroides/análise , Especificidade da EspécieRESUMO
BACKGROUND: Investigation of selected plant extracts on multi-targets related to neurodegeneration, such as monoamine oxidases (MAO), cholinesterase enzymes, and antioxidant activities (AOA) is a useful tool for identification of new scaffolds. OBJECTIVE: This work investigated biological effects of three Blechnum methanol extracts from Brazil and chemical profile of the most active sample. MATERIALS AND METHODS: AOA included scavenging of hydroxyl and nitric oxide radicals, also lipid peroxidation inhibition. Enzymatic modulation of Blechnum binervatum, Blechnum brasiliense, and Blechnum occidentale extracts on MAO and cholinesterases was conducted. Moreover, total phenol content was performed with all samples, and high-performance liquid chromatography-diode array detection mass spectrometry HPLC-DAD-MS analysis was carried out with B. brasiliense. Possible toxic effects were evaluated on Wistar rats polymorphonuclear cells (PMN) and human stem cells. RESULTS: B. brasiliense extract presented the highest phenolic amount (9.25 g gallic acid equivalent/100 g extract) and lowest IC50 values (112.3 ± 2.61 and 176.1 ± 1.19 µg/mL) against hydroxyl radicals and on lipid peroxidation, respectively, showing strong AO effects. On nitric oxide assay and cholinesterase inhibition, all extracts were considered inactive. MAO-A selective action was evidenced, being B. brasiliense powerful against this enzyme (IC50: 72.7 µg/mL), followed by B. occidentale and B. binervatum (IC50: 130.85 and 165.2 µg/mL). No cytotoxic effects were observed on PMN and human stem cells treated with Blechnum extracts. HPLC-DAD-MS analysis of B. brasiliense allowed the identification of chlorogenic and rosmarinic acids. CONCLUSION: Our results especially highlight B. brasiliense, with pronounced phenols content and strong effects on selected targets related to neurodegeneration, being characterized as a natural safe source of bioactive hydroxycinnamic acids. SUMMARY: Blechnum crude extracts showed high phenolic amounts and valuable IC50 values on targets related with neurodegenerative disordersBlechnum brasiliense was the most active sample, with strong radical scavenging and lipid peroxidation inhibition, also with monoamine oxidases: A selective modulationNo cytotoxic effects were observed on polymorphonuclear cells rat cells and human stem cells treated with Blechnum extractsHigh-performance liquid chromatography-diode array detection-mass spectrometry analysis of Blechnum brasiliense allowed the identification of hydroxycinnamic derivatives: Chlorogenic and rosmarinic acids. Abbreviations used: IC50: half maximal inhibitory concentration; MAO: monoamine oxidase; MAO-A: monoamine oxidase isoform A; MAO-B: monoamine oxidase isoform B; HOâ¢: hydroxyl radical.
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For many bioengineered tissues to have practical clinical application, cryopreservation for use on demand is essential. This study examined different thermal histories on warming and short holding periods at different subzero temperatures on subsequent functional recoveries of alginate encapsulated liver spheroids (ELS) for use in a bioartificial liver device. This mimicked transport at liquid nitrogen (-196°C) or dry ice (â¼-80°C) temperatures. Holding at -80°C on warming after -196°C storage resulted in ELS expressing significant (p < 0.001) damage compared with direct thaw from liquid nitrogen, with viable cell number falling from 74.0 ± 8.4 million viable cells/mL without -80°C storage to 1.9 ± 0.6 million viable cells/mL 72 h post-thaw after 8 days storage at -80°C. Even 1 day at -80°C after -196°C storage resulted in lower viability (down 21% 24 h post-thaw), viable cell count (down 29% 24 h post-thaw), glucose, and alpha-1-fetoprotein production (reduced by 59% and 95% 24 h from 1 day post-thaw, respectively). Storage at -80°C was determined to be harmful only during the warming cycle. Chemical measurements of the alginate component of ELS were unchanged by cryogenic exposure in either condition.
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Established fear-related memories can undergo phenomena such as extinction or reconsolidation when recalled. Extinction probably involves the creation of a new, competing memory trace that decreases fear expression, whereas reconsolidation can mediate memory maintenance, updating, or strengthening. The factors determining whether retrieval will initiate extinction, reconsolidation, or neither of these two processes include training intensity, duration of the retrieval session, and age of the memory. However, previous studies have not shown that the same behavioral protocol can be used to induce either extinction or reconsolidation and strengthening, depending on the pharmacological intervention used. Here we show that, within an experiment that leads to extinction in control rats, memory can be strengthened if rolipram, a selective inhibitor of phosphodiesterase type 4 (PDE4), is administered into the dorsal hippocampus immediately after retrieval. The memory-enhancing effect of rolipram lasted for at least 1 week, was blocked by the protein synthesis inhibitor anisomycin, and did not occur when drug administration was not paired with retrieval. These findings indicate that the behavioral outcome of memory retrieval can be pharmacologically switched from extinction to strengthening. The cAMP/protein kinase A (PKA) signaling pathway might be a crucial mechanism determining the fate of memories after recall.