RESUMO
Aneurysms pose life-threatening risks due to the dilatation of the arteries and carry a high risk of rupture. Despite continuous research efforts, there are still no satisfactory or clinically effective pharmaceutical treatments for this condition. Accelerated inflammatory processes during aneurysm development lead to increased levels of matrix metalloproteinases (MMPs) and destabilization of the vessel wall through the degradation of the structural components of the extracellular matrix (ECM), mainly collagen and elastin. Tissue inhibitors of metalloproteinases (TIMPs) directly regulate MMP activity and consequently inhibit ECM proteolysis. In this work, the synthesis of TIMP-1 protein was increased by the exogenous delivery of synthetic TIMP-1 encoding mRNA into aortic vessel tissue in an attempt to inhibit MMP-9. In vitro, TIMP-1 mRNA transfection resulted in significantly increased TIMP-1 protein expression in various cells. The functionality of the expressed protein was evaluated in an appropriate ex vivo aortic vessel model. Decreased MMP-9 activity was detected using in situ zymography 24 h and 48 h post microinjection of 5 µg TIMP-1 mRNA into the aortic vessel wall. These results suggest that TIMP-1 mRNA administration is a promising approach for the treatment of aneurysms.
Assuntos
Metaloproteinase 9 da Matriz , RNA Mensageiro , Inibidor Tecidual de Metaloproteinase-1 , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Humanos , Ratos , Aneurisma/terapia , Aneurisma/genética , Aorta/metabolismo , Masculino , Artérias/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologiaRESUMO
It has long been known that containers for sample analysis or storage can play a role in endotoxin recovery and have to be taken into account when determining endotoxin concentrations. However, there is little data on the effects of containers regarding (1â3)-ß-D-glucan, which plays a role as a contaminant in endotoxin measurements. To determine the effect of the container on (1â3)-ß-D-glucan measurements, four different types of containers were investigated at different temperatures and stored for up to 28 days. For short-term storage for 3 h at room temperature, no effect of the container on the (1â3)-ß-D-glucan recovery could be observed, but for storage at -20 °C, the results indicate that the storage time and temperature influences (1â3)-ß-D-glucan detection. All containers showed a trend of lower recoveries over time, but the polyethylene container showed a significantly lower recovery compared to the other containers. We also showed that freeze/thaw cycles had a strong influence on the recovery of (1â3)-ß-D-glucan in polyethylene containers. Our study showed that the container can affect not only the detection of endotoxins but also the detection of (1â3)-ß-D-glucans.
Assuntos
Glucanos , beta-Glucanas , Glucanos/análise , beta-Glucanas/análise , Endotoxinas , Temperatura , PolietilenosRESUMO
The enzyme poly-ADP-ribose-polymerase (PARP) has important roles for many forms of DNA repair and it also participates in transcription, chromatin remodeling and cell death signaling. Currently, some PARP inhibitors are approved for cancer therapy, by means of canceling DNA repair processes and cell division. Drug repurposing is a new and attractive aspect of therapy development that could offer low-cost and accelerated establishment of new treatment options. Excessive PARP activity is also involved in neurodegenerative diseases including the currently untreatable and blinding retinitis pigmentosa group of inherited retinal photoreceptor degenerations. Hence, repurposing of known PARP inhibitors for patients with non-oncological diseases might provide a facilitated route for a novel retinitis pigmentosa therapy. Here, we demonstrate and compare the efficacy of two different PARP inhibitors, BMN-673 and 3-aminobenzamide, by using a well-established retinitis pigmentosa model, the rd1 mouse. Moreover, the mechanistic aspects of the PARP inhibitor-induced protection were also investigated in the present study. Our results showed that rd1 rod photoreceptor cell death was decreased by about 25-40% together with the application of these two PARP inhibitors. The wealth of human clinical data available for BMN-673 highlights a strong potential for a rapid clinical translation into novel retinitis pigmentosa treatments. Remarkably, we have found that the efficacy of 3 aminobenzamide was able to decrease PARylation at the nanomolar level. Our data also provide a link between PARP activity with the Wnt/ß-catenin pathway and the major intracellular antioxidant concentrations behind the PARP-dependent retinal degeneration. In addition, molecular modeling studies were integrated with experimental studies for better understanding of the role of PARP1 inhibitors in retinal degeneration.
Assuntos
Benzamidas/uso terapêutico , Ftalazinas/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Degeneração Retiniana/tratamento farmacológico , Retinose Pigmentar/tratamento farmacológico , Animais , Reposicionamento de Medicamentos/métodos , Humanos , Camundongos , Poli(ADP-Ribose) Polimerases/metabolismo , Retina/efeitos dos fármacos , Retina/metabolismo , Retina/patologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologiaRESUMO
Traumatic injuries, tumor resections, and degenerative diseases can damage skeletal muscle and lead to functional impairment and severe disability. Skeletal muscle regeneration is a complex process that depends on various cell types, signaling molecules, architectural cues, and physicochemical properties to be successful. To promote muscle repair and regeneration, various strategies for skeletal muscle tissue engineering have been developed in the last decades. However, there is still a high demand for the development of new methods and materials that promote skeletal muscle repair and functional regeneration to bring approaches closer to therapies in the clinic that structurally and functionally repair muscle. The combination of stem cells, biomaterials, and biomolecules is used to induce skeletal muscle regeneration. In this review, we provide an overview of different cell types used to treat skeletal muscle injury, highlight current strategies in biomaterial-based approaches, the importance of topography for the successful creation of functional striated muscle fibers, and discuss novel methods for muscle regeneration and challenges for their future clinical implementation.
Assuntos
Materiais Biocompatíveis , Músculo Esquelético , Doenças Musculares/terapia , Regeneração , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Linhagem Celular , Humanos , Músculo Esquelético/lesões , Músculo Esquelético/fisiologiaRESUMO
Tissue engineering offers auspicious opportunities in oral and maxillofacial surgery to heal bone defects. For this purpose, the combination of cells with stability-providing scaffolds is required. Jaw periosteal cells (JPCs) are well suited for regenerative therapies, as they are easily accessible and show strong osteogenic potential. In this study, we analyzed the influence of uncoated and polylactic-co-glycolic acid (PLGA)-coated ß-tricalcium phosphate (ß-TCP) scaffolds on JPC colonization and subsequent osteogenic differentiation. Furthermore, interaction with the human blood was investigated. This study demonstrated that PLGA-coated and uncoated ß-TCP scaffolds can be colonized with JPCs and further differentiated into osteogenic cells. On day 15, after cell seeding, JPCs with and without osteogenic differentiation were incubated with fresh human whole blood under dynamic conditions. The activation of coagulation, complement system, inflammation, and blood cells were analyzed using ELISA and scanning electron microscopy (SEM). JPC-seeded scaffolds showed a dense cell layer and osteogenic differentiation capacity on both PLGA-coated and uncoated ß-TCP scaffolds. SEM analyses showed no relevant blood cell attachment and ELISA results revealed no significant increase in most of the analyzed cell activation markers (ß-thromboglobulin, Sc5B-9, polymorphonuclear (PMN)-elastase). However, a notable increase in thrombin-antithrombin III (TAT) complex levels, as well as fibrin fiber accumulation on JPC-seeded ß-TCP scaffolds, was detected compared to the scaffolds without JPCs. Thus, this study demonstrated that besides the scaffold material the cells colonizing the scaffolds can also influence hemostasis, which can influence the regeneration of bone tissue.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Fosfatos de Cálcio/farmacologia , Arcada Osseodentária/citologia , Periósteo/citologia , Alicerces Teciduais/química , Contagem de Células Sanguíneas , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proteínas do Sistema Complemento/metabolismo , Humanos , Osteogênese/efeitos dos fármacos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologiaRESUMO
Induced pluripotent stem cell-derived mesenchymal stem cell-like cells (iMSCs) are considered to be a promising source of progenitor cells for approaches in the field of bone regeneration. In a previous study, we described the generation of footprint-free induced pluripotent stem cells (iPSCs) from human jaw periosteal cells (JPCs) by transfection of a self-replicating RNA (srRNA) and subsequent differentiation into functional osteogenic progenitor cells. In order to facilitate the prospective transfer into clinical practice, xeno-free reprogramming and differentiation methods were established. In this study, we compared the properties and stem cell potential of the iMSCs produced from JPC-derived iPSCs with the parental primary JPCs they were generated from. Our results demonstrated, on the one hand, a comparable differentiation potential of iMSCs and JPCs. Additionally, iMSCs showed significantly longer telomere lengths compared to JPCs indicating rejuvenation of the cells during reprogramming. On the other hand, proliferation, mitochondrial activity, and senescence-associated beta-galactosidase (SA-ß-gal) activity indicated early senescence of iMSCs. These data demonstrate the requirement of further optimization strategies to improve mesenchymal development of JPC-derived iPSCs in order to take advantage of the best features of reprogrammed and rejuvenated cells.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Regeneração Óssea/fisiologia , Diferenciação Celular/fisiologia , Humanos , RNA/metabolismo , Células-Tronco/metabolismo , Engenharia Tecidual/métodosRESUMO
During open-heart surgery, the status of hemostasis has to be constantly monitored to quickly and reliably detect bleeding or coagulation disorders. In this study, a novel optimized piezo-based measuring system (PIEZ) for rheological monitoring of hemostasis was established. The applicability of the PIEZ for the evaluation of nucleic acid-based drugs influencing coagulation was analyzed. Thrombin aptamers such as NU172 might be used during extracorporeal circulation (ECC) in combination with a reduced heparin concentration or for patients with heparin-induced thrombocytopenia (HIT). Therefore, the effect of the coagulation inhibiting thrombin aptamer NU172 and the abrogation by its complementary antidote sequence (AD) were investigated by this rheological PIEZ system. After the addition of different NU172 concentrations, the coagulation of fresh human blood was analyzed under static conditions and using an in vitro rotation model under dynamic conditions (simulating ECC). The clotting times (CTs) detected by PIEZ were compared to those obtained with a medical reference device, a ball coagulometer. Additionally, after the circulation of blood samples for 30 min at 37 °C, blood cell numbers, thrombin markers (thrombin-antithrombin III (TAT) and fibrinopeptide A (FPA)) and a platelet activation marker (ß-thromboglobulin (ß-TG)) were analyzed by enzyme-linked immunosorbent assays (ELISAs). The increase of NU172 concentration resulted in prolonged CTs, which were comparable between the reference ball coagulometer and the PIEZ, demonstrating the reliability of the new measuring system. Moreover, by looking at the slope of the linear regression of the viscous and elastic components, PIEZ also could provide information on the kinetics of the coagulation reaction. The shear viscosity at the end of the measurements (after 300 s) was indicative of clot firmness. Furthermore, the PIEZ was able to detect the abrogation of coagulation inhibition after the equimolar addition of NU172 aptamer´s AD. The obtained results showed that the established PIEZ is capable to dynamically measure the hemostasis status in whole blood and can be applied to analyze nucleic acid-based drugs influencing the coagulation.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Ácidos Nucleicos/farmacologia , Adulto , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/farmacologia , Contagem de Células Sanguíneas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tempo de Coagulação do Sangue TotalRESUMO
Jaw periosteal cells (JPCs) represent a suitable stem cell source for bone tissue engineering (BTE) applications. However, challenges associated with limited cell numbers, stressful cell sorting, or the occurrence of cell senescence during in vitro passaging and the associated insufficient osteogenic potential in vitro of JPCs and other mesenchymal stem/stromal cells (MSCs) are main hurdles and still need to be solved. In this study, for the first time, induced pluripotent stem cells (iPSCs) were generated from human JPCs to open up a new source of stem cells for BTE. For this purpose, a non-integrating self-replicating RNA (srRNA) encoding reprogramming factors and green fluorescent protein (GFP) as a reporter was used to obtain JPC-iPSCs with a feeder- and xeno-free reprogramming protocol to meet the highest safety standards for future clinical applications. Furthermore, to analyze the potential of these iPSCs as a source of osteogenic progenitor cells, JPC-iPSCs were differentiated into iPSC-derived mesenchymal stem/stromal like cells (iMSCs) and further differentiated to the osteogenic lineage under xeno-free conditions. The produced iMSCs displayed MSC marker expression and morphology as well as strong mineralization during osteogenic differentiation.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Arcada Osseodentária/citologia , Periósteo/citologia , RNA/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem da Célula , Reprogramação Celular , Camadas Germinativas/citologia , Humanos , Cariotipagem , Células-Tronco Mesenquimais/citologia , OsteogêneseRESUMO
Musculoskeletal disorders, such as osteoarthritis and intervertebral disc degeneration are causes of morbidity, which concomitantly burdens the health and social care systems worldwide, with massive costs. Link N peptide has recently been described as a novel anabolic stimulator for intervertebral disc repair. In this study, we analyzed the influence on anabolic response, by delivering synthetic Link N encoding mRNA into primary human chondrocytes and mesenchymal stromal cells (SCP1 cells), Furthermore, both cell types were seeded on knitted titanium scaffolds, and the influence of Link N peptide mRNA for possible tissue engineering applications was investigated. Synthetic modified Link N mRNA was efficiently delivered into both cell types and cell transfection resulted in an enhanced expression of aggrecan, Sox 9, and type II collagen with a decreased expression of type X collagen. Interestingly, despite increased expression of BMP2 and BMP7, BMP signaling was repressed and TGFß signaling was boosted by Link N transfection in mesenchymal stromal cells, suggesting possible regulatory mechanisms. Thus, the exogenous delivery of Link N peptide mRNA into cells augmented an anabolic response and thereby increased extracellular matrix synthesis. Considering these findings, we suppose that the cultivation of cells on knitted titanium scaffolds and the exogenous delivery of Link N peptide mRNA into cells could mechanically support the stability of tissue-engineered constructs and improve the synthesis of extracellular matrix by seeded cells. This method can provide a potent strategy for articular cartilage and intervertebral disc regeneration.
Assuntos
Condrócitos/metabolismo , RNA Mensageiro/metabolismo , Agrecanas/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 7/metabolismo , Linhagem Celular , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Células Cultivadas , Colágeno Tipo II/metabolismo , Colágeno Tipo X/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , RNA Mensageiro/genética , Fatores de Transcrição SOX9/metabolismoRESUMO
Several diseases are caused by missing or defective synthesis of proteins due to genetic or acquired disorders. In recent years, in vitro transcribed (IVT) messenger RNA (mRNA)-based therapy for de novo protein expression in cells has increased in importance. Thereby, desired proteins can be produced in cells by exogenous delivery of IVT mRNA, which does not integrate into the host genome and results in transient production of target proteins. Due to the lack of genomic integration, the risk of mutation and tumor development is minimized. Different approaches using IVT mRNA have been applied to alter the expression profiles of cells by the production of proteins. IVT mRNAs encoding transcription factors have led to the highly efficient induction of pluripotency in somatic cells and generated induced pluripotent stem cells that are free of viral vector components. Furthermore, specific IVT mRNA cocktails containing more than one specific IVT mRNA can be used to directly induce the differentiation into a desired cell type. In theory, every desired mRNA can be produced in vitro and used to enable extrinsic biosynthesis of target proteins in each cell type. Cells can be engineered by IVT mRNA to express antigens on dendritic cells for vaccination and tumor treatment, surface receptors on stem cells for increased homing to distinct areas, and to produce industrial grade human growth factors. In this review, we focus on the progress and challenges in mRNA-based cell engineering approaches. Stem Cells 2017;35:68-79.
Assuntos
Engenharia Celular , Reprogramação Celular , Transcrição Gênica , Animais , Humanos , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The application of synthetic messenger RNA (mRNA) exhibits various advantages, such as expression of desired proteins in cells without genomic integration. In the field of tissue engineering, synthetic mRNAs could be also used to modulate the protein expression in implanted cells. Therefore, in this study, we incorporated synthetic humanized Gaussia luciferase (hGLuc) mRNA into alginate, chitosan, or chitosan-alginate hybrid hydrogels and analyzed the release of hGLuc mRNA from these hydrogels. After 3 weeks, 79% of the incorporated mRNA was released from alginate hydrogels, approximately 42% was released from chitosan hydrogels, and about 70% was released from chitosan-alginate hydrogels. Due to the injectability, chitosan-alginate hybrid hydrogels were selected for further investigation of the bioactivity of embedded hGLuc mRNA and the stability of these hydrogels was examined after the incorporation of synthetic mRNA by rheometric analysis. Therefore, HEK293 cells were incorporated into chitosan-alginate hydrogels containing mRNA transfection complexes and the luciferase activity in the supernatants was detected for up to 3 weeks. These results showed that the biodegradable chitosan-alginate hybrid hydrogels are promising delivery systems for sustained delivery of synthetic mRNAs into cells. Since chitosan-alginate hybrid hydrogels are injectable, the hydrogels can be simultaneously loaded with cells and the desired synthetic mRNA for exogenous protein synthesis and can be administered by minimally invasive local injection for tissue engineering applications.
Assuntos
Alginatos/metabolismo , Materiais Biocompatíveis/metabolismo , Quitosana/metabolismo , Hidrogéis/metabolismo , RNA Mensageiro/metabolismo , Alginatos/química , Materiais Biocompatíveis/química , Sobrevivência Celular , Quitosana/química , Sistemas de Liberação de Medicamentos , Ácido Glucurônico/química , Ácido Glucurônico/metabolismo , Células HEK293 , Ácidos Hexurônicos/química , Ácidos Hexurônicos/metabolismo , Humanos , Hidrogéis/química , Luciferases/genética , Luciferases/metabolismo , Sondas RNA , RNA Mensageiro/química , Reologia , Fatores de Tempo , Engenharia TecidualRESUMO
Nucleic acid ligands, aptamers, harbor the unique characteristics of small molecules and antibodies. The specificity and high affinity of aptamers enable their binding to different targets, such as small molecules, proteins, or cells. Chemical modifications of aptamers allow increased bioavailability. A further great benefit of aptamers is the antidote (AD)-mediated controllability of their effect. In this study, the AD-mediated complexation and neutralization of the thrombin binding aptamer NU172 and Toll-like receptor 9 (TLR9) binding R10-60 aptamer were determined. Thereby, the required time for the generation of aptamer/AD-complexes was analyzed at 37 °C in human serum using gel electrophoresis. Afterwards, the blocking of aptamers' effects was analyzed by determining the activated clotting time (ACT) in the case of the NU172 aptamer, or the expression of immune activation related genes IFN-1ß, IL-6, CXCL-10, and IL-1ß in the case of the R10-60 aptamer. Gel electrophoresis analyses demonstrated the rapid complexation of the NU172 and R10-60 aptamers by complementary AD binding after just 2 min of incubation in human serum. A rapid neutralization of anticoagulant activity of NU172 was also demonstrated in fresh human whole blood 5 min after addition of AD. Furthermore, the TLR9-mediated activation of PMDC05 cells was interrupted after the addition of the R10-60 AD. Using these two different aptamers, the rapid antagonizability of the aptamers was demonstrated in different environments; whole blood containing numerous proteins, cells, and different small molecules, serum, or cell culture media. Thus, nucleic acid ADs are promising molecules, which offer several possibilities for different in vivo applications, such as antagonizing aptamer-based drugs, immobilization, or delivery of oligonucleotides to defined locations.
Assuntos
Aptâmeros de Nucleotídeos/sangue , Receptor Toll-Like 9/sangue , Anticoagulantes/sangue , Anticoagulantes/química , Antídotos/química , Aptâmeros de Nucleotídeos/química , Coagulação Sanguínea/genética , Humanos , Ligantes , Técnica de Seleção de Aptâmeros , Trombina/química , Trombina/genética , Receptor Toll-Like 9/químicaRESUMO
Aptamers are auspicious nucleic acid ligands for targeting different molecules, such as small molecules, peptides, proteins, or even whole living cells. They are short single-stranded DNA or RNA oligonucleotides, which can fold into complex three-dimensional structures and bind selectively their targets. Using the combinatorial chemistry process SELEX (Systematic Evolution of Ligands by EXponential Enrichment), target specific aptamers can be selected. These aptamers have a variety of application possibilities and can be used as sensors, diagnostic, imaging or therapeutic agents, and in the field of regenerative medicine for tissue engineering.
Assuntos
Aptâmeros de Nucleotídeos/uso terapêutico , Desenho de Fármacos , Marcação de Genes , Proteínas/antagonistas & inibidores , Animais , Aptâmeros de Nucleotídeos/química , Humanos , Técnica de Seleção de Aptâmeros/métodosRESUMO
The hemocompatible properties of rotary blood pumps commonly used in mechanical circulatory support (MCS) are widely unknown regarding specific biocompatibility profiles of different pump technologies. Therefore, we analyzed the hemocompatibility indicating markers of an axial flow and a magnetically levitated centrifugal device within an in vitro mock loop. The HeartMate II (HM II; n = 3) device and a CentriMag (CM; n = 3) adult pump were investigated in a human whole blood mock loop for 360 min using the MCS devices as a driving component. Blood samples were analyzed by enzyme-linked immunosorbent assay for markers of coagulation, complement system, and inflammatory response. There was a time-dependent activation of the coagulation (thrombin-antithrombin complexes [TAT]), complement (SC5b-9), and inflammation system (polymorphonuclear [PMN] elastase) in both groups. The mean value of TAT (CM: 4.0 µg/L vs. 29.4 µg/L, P < 0.001; HM II: 4.5 µg/L vs. 232.2 µg/L, P < 0.05) and PMN elastase (CM: 53.4 ng/mL vs. 253.8 ng/mL, P < 0.05; HM II: 28.0 ng/mL vs. 738.8 ng/mL, P < 0.001) significantly increased from baseline until the end of the experiments (360 min). After 360 min, TAT and PMN values were significantly higher in the HM II group compared with the values in the CM adult group. The values of SC5b-9 increased from baseline to 360 min in the CM group (CM: 141.8 ng/mL vs. 967.9 ng/mL, P < 0.05) and the HM II group. However, the increase within the HM II group (97.3 vs. 2462.0, P = 0.06) and the comparison of the 360-min values between CM group and HM II group did not reach significance (P = 0.18). The activation of complement, coagulation, and inflammation system showed a time-dependent manner in both devices. The centrifugal CM device showed significantly lower activation of coagulation and inflammation than that of the HM II axial flow pump. Both HM II and CM have demonstrated an acceptable hemocompatibility profile in patients. However, there is a great opportunity to gain a clinical benefit by developing techniques to lower the blood surface interaction within both pump technologies and a magnetically levitated centrifugal pump design might be superior.
Assuntos
Coração Auxiliar , Coagulação Sanguínea , Centrifugação , Ativação do Complemento , Coração Auxiliar/efeitos adversos , Hemólise , Humanos , Inflamação/sangue , Inflamação/etiologia , Mediadores da Inflamação/sangue , Magnetismo , Modelos Anatômicos , Modelos Cardiovasculares , Desenho de Prótese , Medição de Risco , Fatores de Risco , Trombose/sangue , Trombose/etiologia , Fatores de TempoRESUMO
This corrects the article 10.3791/64016.
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Biosensors play an important role in numerous research fields. Quartz crystal microbalances with dissipation monitoring (QCM-Ds) are sensitive devices, and binding events can be observed in real-time. In combination with aptamers, they have great potential for selective and label-free detection of various targets. In this study, an alternative surface functionalization for a QCM-D-based aptasensor was developed, which mimics an artificial cell membrane and thus creates a physiologically close environment for the binding of the target to the sensor. Vesicle spreading was used to form a supported lipid bilayer (SLB) of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphethanolamine-N-(cap biotinyl) (biotin-PE). The SLB was then coated with streptavidin followed by applying a biotinylated aptamer against thrombin. SLB formation was investigated in terms of temperature and composition. Temperatures of 25 °C and below led to incomplete SLB formation, whereas a full bilayer was built at higher temperatures. We observed only a small influence of the content of biotinylated lipids in the mixture on the further binding of streptavidin. The functionalization of the sensor surface with the thrombin aptamer and the subsequent thrombin binding were investigated at different concentrations. The sensor could be reconstituted by incubation with a 5 M urea solution, which resulted in the release of the thrombin from the sensor surface. Thereafter, it was possible to rebind thrombin. Thrombin in spiked samples of human serum was successfully detected. The developed system can be easily applied to other target analytes using the desired aptamers.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Bicamadas Lipídicas , Técnicas de Microbalança de Cristal de Quartzo , Trombina , Trombina/análise , Bicamadas Lipídicas/química , Aptâmeros de Nucleotídeos/química , Humanos , Fosfatidilcolinas/químicaRESUMO
Thrombogenicity of foreign surfaces is the major obstacle in cardiovascular interventions. Despite enormous advances in biomaterials research, the hemocompatibility of blood-contacting materials is still not satisfactory and the native endothelium still represents the ideal surface for blood contact. Circulating adult endothelial progenitor cells (EPCs) in the human blood provide an excellent source of autologous stem cells for the in vivo self-endothelialization of blood-contacting materials. For this purpose, material surfaces can be coated with capture molecules mimicking natural homing factors to attract circulating EPCs. Hitherto, several ligands, such as aptamers, monoclonal antibodies, peptides, selectins and their ligands, or magnetic molecules, are used to biofunctionalize surfaces for the capturing of EPCs directly from patient's bloodstream onto blood-contacting materials. Subsequently, attracted EPCs can differentiate into endothelial cells and generate an autologous endothelium. The in vivo self-endothelialization of blood-contacting materials prevents the recognition of them as a foreign body; this opens up revolutionary new prospects for future clinical stem-cell and tissue engineering strategies.
Assuntos
Materiais Biocompatíveis/uso terapêutico , Células Endoteliais/fisiologia , Células-Tronco/fisiologia , Engenharia Tecidual/métodos , Materiais Biomiméticos , Prótese Vascular , Adesão Celular , Diferenciação Celular , Proliferação de Células , Materiais Revestidos Biocompatíveis , Humanos , Peptídeos/química , StentsRESUMO
Cardiovascular diseases are the leading cause of death globally. Vascular implants, such as stents, are required to treat arterial stenosis or dilatation. The development of innovative stent materials and coatings, as well as novel preclinical testing strategies, is needed to improve the bio- and hemocompatibility of current stents. In this study, a blood vessel-like polydimethylsiloxane (PDMS) model was established to analyze the interaction of an endothelium with vascular implants, as well as blood-derived cells, in vitro. Using footprint-free human induced pluripotent stem cells (hiPSCs) and subsequent differentiation, functional endothelial cells (ECs) expressing specific markers were generated and used to endothelialize an artificial PDMS lumen. The established model was used to demonstrate the interaction of the created endothelium with blood-derived immune cells, which also allowed for real-time imaging. In addition, a stent was inserted into the endothelialized lumen to analyze the surface endothelialization of stents. In the future, this blood vessel-like model could serve as an in vitro platform to test the influence of vascular implants and coatings on endothelialization and to analyze the interaction of the endothelium with blood cell components.
Assuntos
Células Endoteliais , Células-Tronco Pluripotentes Induzidas , Humanos , Células Endoteliais/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Endotélio , Stents , Diferenciação CelularRESUMO
Polytetrafluoroethylene (PTFE) is a commonly used biomaterial for the manufacturing of vascular grafts and several strategies, such as coatings, have been explored to improve the hemocompatibility of small-diameter prostheses. In this study, the hemocompatibility properties of novel stent grafts covered with electrospun PTFE (LimFlow Gen-1 and LimFlow Gen-2) were compared with uncoated and heparin-coated PTFE grafts (Gore Viabahn®) using fresh human blood in a Chandler closed-loop system. After 60 min of incubation, the blood samples were examined hematologically and activation of coagulation, platelets, and the complement system were analyzed. In addition, the adsorbed fibrinogen on the stent grafts was measured and the thrombogenicity was assessed by SEM. Significantly lower adsorption of fibrinogen was measured on the surface of heparin-coated Viabahn than on the surface of the uncoated Viabahn. Furthermore, LimFlow Gen-1 stent grafts showed lower fibrinogen adsorption than the uncoated Viabahn®, and the LimFlow Gen-2 stent grafts showed comparable fibrinogen adsorption as the heparin-coated Viabahn®. SEM analysis revealed no sign of thrombus formation on any of the stent surfaces. LimFlow Gen-2 stent grafts covered with electrospun PTFE exhibited bioactive characteristics and revealed improved hemocompatibility in terms of reduced adhesion of fibrinogen, activation of platelets, and coagulation (assessed by ß-TG and TAT levels) similar to heparin-coated ePTFE prostheses. Thus, this study demonstrated improved hemocompatibility of electrospun PTFE. The next step is to conduct in vivo studies to confirm whether electrospinning-induced changes to the PTFE surface can reduce the risk of thrombus formation and provide clinical benefits.
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The liver is a vital organ with numerous functions, including metabolic functions, detoxification, and the synthesis of secretory proteins. The increasing prevalence of liver diseases requires the development of effective treatments, models, and regenerative approaches. The field of liver tissue engineering represents a significant advance in overcoming these challenges. In this study, 3D biohybrid constructs were created by combining hepatocyte-like cells (HLCs) derived from patient-specific footprint-free human induced pluripotent stem cells (hiPSCs) and 3D melt-electrospun poly-ε-caprolactone (PCL) scaffolds. First, a differentiation procedure was established to obtain autologous HCLs from hiPSCs reprogrammed from renal epithelial cells using self-replicating mRNA. The obtained cells expressed hepatocyte-specific markers and exhibited important hepatocyte functions, such as albumin synthesis, cytochrome P450 activity, glycogen storage, and indocyanine green metabolism. Biocompatible PCL scaffolds were fabricated by melt-electrospinning and seeded with pre-differentiated hepatoblasts, which uniformly attached to the fibers of the scaffolds and successfully matured into HLCs. The use of patient-specific, footprint-free hiPSC-derived HLCs represents a promising cell source for personalized liver regeneration strategies. In combination with biocompatible 3D scaffolds, this innovative approach has a broader range of applications spanning liver tissue engineering, drug testing and discovery, and disease modeling.