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Poly-3-hydroxybutyrate (P3HB) is a biodegradable polyester produced mainly by bacterial fermentation in an isotactic configuration. Its high crystallinity (about 70%) and brittle behavior have limited the process window and the application of this polymer in different sectors. Atactic poly-3-hydroxybutyrate (a-P3HB) is an amorphous polymer that can be synthesized chemically and blended with the isotactic P3HB to reduce its crystallinity and improve its processability Ring-opening polymerization (ROP) is the most cited synthesis route for this polymer in the literature. In this work, a new synthesis route of a-P3HB by self-polycondensation of racemic ethyl 3-hydroxybutyrate will be demonstrated. Different catalysts were tested regarding their effectiveness, and the reaction parameters were optimized using titanium isopropoxide as the catalyst. The resulting polymers were compared by self-polycondensation for their properties with those of a-P3HB obtained by the ROP and characterized by Fourier transform infrared spectroscopy (FTIR) and gel permeation chromatography (GPC), and the double bond content (DBC) was determined by UV-VIS spectroscopy by using 3-butenoic acid as a standard. Additionally, a life cycle analysis (LCA) of the new method of synthesizing has been carried out to assess the environmental impact of a-P3HB.
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In the context of tissue engineering, biofabrication techniques are employed to process cells in hydrogel-based matrices, known as bioinks, into complex 3D structures. The aim is the production of functional tissue models or even entire organs. The regenerative production of biological tissues adheres to a multitude of criteria that ultimately determine the maturation of a functional tissue. These criteria are of biological nature, such as the biomimetic spatial positioning of different cell types within a physiologically and mechanically suitable matrix, which enables tissue maturation. Furthermore, the processing, a combination of technical procedures and biological materials, has proven highly challenging since cells are sensitive to stress, for example from shear and tensile forces, which may affect their vitality. On the other hand, high resolutions are pursued to create optimal conditions for subsequent tissue maturation. From an analytical perspective, it is prudent to first investigate the printing behavior of bioinks before undertaking complex biological tests. According to our findings, conventional shear rheological tests are insufficient to fully characterize the printing behavior of a bioink. For this reason, we have developed optical methods that, complementarily to the already developed tests, allow for quantification of printing quality and further viscoelastic modeling of bioinks.
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Bioimpressão , Hidrogéis , Impressão Tridimensional , Engenharia Tecidual , Bioimpressão/métodos , Engenharia Tecidual/métodos , Hidrogéis/química , Reologia , Humanos , Alicerces Teciduais/química , ViscosidadeRESUMO
Extrusion-based bioprinting is an established method in biofabrication. Suitable bioinks have fundamentally different compositions and characteristics, which should be examined, in order to find a perfect model system. Here, we investigate the effect of two alginate-based, yet unalike 3D-printed bioinks, pre-crosslinked alginate-dialdehyde gelatin (ADA-GEL) and a mixture of alginate, hyaluronic acid, and gelatin (Alg/HA/Gel), on the melanoma cell line Mel Im and vice versa in terms of stiffness, shrinkage, cellular behavior and colony formation over 15 days. Rheological stiffness measurements revealed two soft gels with similar storage moduli. The cells did not have a significant impact on the overall stiffness, whereas ADA-GEL (2.5/2.5%) was significantly stiffer than Alg/HA/Gel (0.5/0.1/3%). Regarding the shrinkage of printed constructs, cells had a significant influence, especially in ADA-GEL, which has covalent bonds between the oxidized alginate and gelatin. Multi-photon microscopy exhibited proliferation, cell spreading and migration in ADA-GEL with cell-cell and cell-matrix interaction, dissimilarly to Alg/HA/Gel, in which cells formed spherical, encapsulated colonies. Scanning electron microscopy and histology showed degradation and multi-layered growth on ADA-GEL and fewer examples of escaped cells on Alg/HA/Gel. Both gels serve as proliferation bioink for melanoma with more necrosis in deeper Alg/HA/Gel colonies and differences in spreading and matrix interaction. These findings show the importance of proper characterization of the bioinks for different applications.
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Alginatos , Bioimpressão , Proliferação de Células , Gelatina , Melanoma , Impressão Tridimensional , Alginatos/química , Melanoma/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Gelatina/química , Bioimpressão/métodos , Humanos , Tinta , Ácido Hialurônico/química , Reologia , Alicerces Teciduais/química , Engenharia Tecidual/métodosRESUMO
GABAergic interneurons play a critical role in maintaining neural circuit balance, excitation-inhibition regulation, and cognitive function modulation. In tuberous sclerosis complex (TSC), GABAergic neuron dysfunction contributes to disrupted network activity and associated neurological symptoms, assumingly in a cell type-specific manner. This GABAergic centric study focuses on identifying specific interneuron subpopulations within TSC, emphasizing the unique characteristics of medial ganglionic eminence (MGE)- and caudal ganglionic eminence (CGE)-derived interneurons. Using single-nuclei RNA sequencing in TSC patient material, we identify somatostatin-expressing (SST+) interneurons as a unique and immature subpopulation in TSC. The disrupted maturation of SST+ interneurons may undergo an incomplete switch from excitatory to inhibitory GABAergic signaling during development, resulting in reduced inhibitory properties. Notably, this study reveals markers of immaturity specifically in SST+ interneurons, including an abnormal NKCC1/KCC2 ratio, indicating an imbalance in chloride homeostasis crucial for the postsynaptic consequences of GABAergic signaling as well as the downregulation of GABAA receptor subunits, GABRA1, and upregulation of GABRA2. Further exploration of SST+ interneurons revealed altered localization patterns of SST+ interneurons in TSC brain tissue, concentrated in deeper cortical layers, possibly linked to cortical dyslamination. In the epilepsy context, our research underscores the diverse cell type-specific roles of GABAergic interneurons in shaping seizures, advocating for precise therapeutic considerations. Moreover, this study illuminates the potential contribution of SST+ interneurons to TSC pathophysiology, offering insights for targeted therapeutic interventions.
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Neurônios GABAérgicos , Interneurônios , Esclerose Tuberosa , Humanos , Neurônios GABAérgicos/patologia , Neurônios GABAérgicos/metabolismo , Eminência Ganglionar , Interneurônios/patologia , Interneurônios/metabolismo , Eminência Mediana/patologia , Eminência Mediana/metabolismo , Receptores de GABA-A/metabolismo , Somatostatina/metabolismo , Esclerose Tuberosa/patologia , Esclerose Tuberosa/metabolismo , AnimaisRESUMO
Although 2D cancer models have been the standard for drug development, they don't resemble in vivo properties adequately. 3D models can potentially overcome this. Bioprinting is a promising technique for more refined models to investigate central processes in tumor development such as proliferation, dormancy or metastasis. We aimed to analyze bioinks, which could mimic these different tumor stages in a cast vascularized arteriovenous loop melanoma model in vivo. It has the advantage to be a closed system with a defined microenvironment, supplied only with one vessel-ideal for metastasis research. Tested bioinks showed significant differences in composition, printability, stiffness and microscopic pore structure, which led to different tumor stages (Matrigel and Alg/HA/Gel for progression, Cellink Bioink for dormancy) and resulted in different primary tumor growth (Matrigel significantly higher than Cellink Bioink). Light-sheet fluorescence microscopy revealed differences in vascularization and hemorrhages with no additional vessels found in Cellink Bioink. Histologically, typical human melanoma with different stages was demonstrated. HMB-45-positive tumors in progression inks were infiltrated by macrophages (CD163), highly proliferative (Ki67) and metastatic (MITF/BRN2, ATX, MMP3). Stainings of lymph nodes revealed metastases even without significant primary tumor growth in Cellink Bioink. This model can be used to study tumor pathology and metastasis of different tumor stages and therapies.
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Myocardial infarction (MI) causes cell death, disrupts electrical activity, triggers arrhythmia, and results in heart failure, whereby 50-60% of MI-associated deaths manifest as sudden cardiac deaths (SCD). The most effective therapy for SCD prevention is implantable cardioverter defibrillators (ICDs). However, ICDs contribute to adverse remodeling and disease progression and do not prevent arrhythmia. This work develops an injectable collagen-PEDOT:PSS (poly(3,4-ethylenedioxythiophene) polystyrene sulfonate) hydrogel that protects infarcted hearts against ventricular tachycardia (VT) and can be combined with human induced pluripotent stem cell (hiPSC)-cardiomyocytes to promote partial cardiac remuscularization. PEDOT:PSS improves collagen gel formation, micromorphology, and conductivity. hiPSC-cardiomyocytes in collagen-PEDOT:PSS hydrogels exhibit near-adult sarcomeric length, improved contractility, enhanced calcium handling, and conduction velocity. RNA-sequencing data indicate enhanced maturation and improved cell-matrix interactions. Injecting collagen-PEDOT:PSS hydrogels in infarcted mouse hearts decreases VT to the levels of healthy hearts. Collectively, collagen-PEDOT:PSS hydrogels offer a versatile platform for treating cardiac injuries.
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Arritmias Cardíacas , Colágeno , Condutividade Elétrica , Hidrogéis , Células-Tronco Pluripotentes Induzidas , Infarto do Miocárdio , Miócitos Cardíacos , Poliestirenos , Infarto do Miocárdio/patologia , Animais , Células-Tronco Pluripotentes Induzidas/citologia , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Camundongos , Colágeno/química , Hidrogéis/química , Arritmias Cardíacas/prevenção & controle , Poliestirenos/química , Polímeros/química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , TiofenosRESUMO
Genipin-cross-linked silk fibroin (SF) hydrogel is considered to be biocompatible and mechanically robust. However, its use remains a challenge for in situ forming applications due to its prolonged gelation process. In our attempt to facilitate the in situ fabrication of a genipin-mediated SF hydrogel, alginate dialdehyde (ADA) was utilized as a reinforcement template. Here, SF/ADA-based hydrogels with different compositions were synthesized covalently and ionically. Incorporating ADA into the SF hydrogel increased pore size (44.66-174.66 µm), porosity (61.59-80.40%), and the equilibrium swelling degree (7.60-30.17). Moreover, a wide range of storage modulus and compressive modulus were obtained by adjusting the proportions of SF and ADA networks within the hydrogel. The in vitro cell analysis using preosteoblast cells (MC3T3-E1) demonstrated the cytocompatibility of all hydrogels. Overall, the covalently and ionically cross-linked SF/ADA hydrogel represents a promising solution for in situ forming hydrogels for applications in tissue regeneration.
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Fibroínas , Hidrogéis , Alginatos , Iridoides , Seda , Engenharia TecidualRESUMO
Clean water is one of the most important resources of the planet but human-made contamination with diverse pollutants increases continuously. Microplastics (<5 mm diameter) which can have severe impacts on the environment, are present worldwide. Degradation processes lead to nanoplastics (<1 µm), which are potentially even more dangerous due to their increased bioavailability. State-of-the-art wastewater treatment plants show a deficit in effectively eliminating micro- and nanoplastics (MNP) from water, particularly in the case of nanoplastics. In this work, the magnetic removal of three different MNP types across three orders of magnitude in size (100 nm-100 µm) is investigated systematically. Superparamagnetic iron oxide nanoparticles (SPIONs) tend to attract oppositely charged MNPs and form aggregates that can be easily collected by a magnet. It shows that especially the smallest fractions (100-300 nm) can be separated in ordinary high numbers (1013 mg-1 SPION) while the highest mass is removed for MNP between 2.5 and 5 µm. The universal trend for all three types of MNP can be fitted with a derived model, which can make predictions for optimizing SPIONs for specific size ranges in the future.
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In skeletal muscle tissue engineering, innervation and vascularization play an essential role in the establishment of functional skeletal muscle. For adequate three-dimensional assembly, biocompatible aligned nanofibers are beneficial as matrices for cell seeding. The aim of this study was to analyze the impact of Schwann cells (SC) on myoblast (Mb) and adipogenic mesenchymal stromal cell (ADSC) cocultures on poly-É-caprolactone (PCL)-collagen I-nanofibers in vivo. Human Mb/ADSC cocultures, as well as Mb/ADSC/SC cocultures, were seeded onto PCL-collagen I-nanofiber scaffolds and implanted into the innervated arteriovenous loop model (EPI loop model) of immunodeficient rats for 4 weeks. Histological staining and gene expression were used to compare their capacity for vascularization, immunological response, myogenic differentiation, and innervation. After 4 weeks, both Mb/ADSC and Mb/ADSC/SC coculture systems showed similar amounts and distribution of vascularization, as well as immunological activity. Myogenic differentiation could be observed in both groups through histological staining (desmin, myosin heavy chain) and gene expression (MYOD, MYH3, ACTA1) without significant difference between groups. Expression of CHRNB and LAMB2 also implied neuromuscular junction formation. Our study suggests that the addition of SC did not significantly impact myogenesis and innervation in this model. The implanted motor nerve branch may have played a more significant role than the presence of SC.
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Nanofibras , Alicerces Teciduais , Ratos , Humanos , Animais , Engenharia Tecidual/métodos , Diferenciação Celular , Músculo Esquelético , Colágeno Tipo I/metabolismo , Desenvolvimento Muscular/genéticaRESUMO
Multiple system atrophy (MSA) is a rare and rapidly progressive atypical parkinsonian disorder characterized by oligodendroglial cytoplasmic inclusions containing α-synuclein (α-syn), demyelination, inflammation and neuronal loss. To date, no disease-modifying therapy is available. Targeting α-syn-driven oligodendroglial dysfunction and demyelination presents a potential therapeutic approach for restricting axonal dysfunction, neuronal loss and disease progression. The present study investigated the promyelinogenic potential of sobetirome, a blood-brain barrier permeable and central nervous system selective thyromimetic in the context of an in vitro MSA model. Oligodendrocyte precursor cells (OPCs) were obtained from transgenic mice overexpressing human α-syn specifically in oligodendrocytes (MBP29 mouse line), a well-described MSA model, and non-transgenic littermates. mRNA and protein expression analyses revealed a substantial rescue effect of sobetirome on myelin-specific proteins in control and α-syn overexpressing oligodendrocytes. Furthermore, myelination analysis using nanofibres confirmed that sobetirome increases both the length and number of myelinated segments per oligodendrocyte in primary murine α-syn overexpressing oligodendrocytes and their respective control. These results suggest that sobetirome may be a promising thyromimetic compound targeting an important neuropathological hallmark of MSA.
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Doenças Desmielinizantes , Atrofia de Múltiplos Sistemas , Fenóis , Camundongos , Humanos , Animais , Atrofia de Múltiplos Sistemas/tratamento farmacológico , Atrofia de Múltiplos Sistemas/genética , Atrofia de Múltiplos Sistemas/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Acetatos/metabolismo , Camundongos Transgênicos , Oligodendroglia/metabolismo , Doenças Desmielinizantes/metabolismo , Modelos Animais de DoençasRESUMO
Myocardial infarction is one of the major causes of mortality as well as morbidity around the world. Currently available treatment options face a number of drawbacks, hence cardiac tissue engineering, which aims to bioengineer functional cardiac tissue, for application in tissue repair, patient specific drug screening and disease modeling, is being explored as a viable alternative. To achieve this, an appropriate combination of cells, biomimetic scaffolds mimicking the structure and function of the native tissue, and signals, is necessary. Among scaffold fabrication techniques, three-dimensional printing, which is an additive manufacturing technique that enables to translate computer-aided designs into 3D objects, has emerged as a promising technique to develop cardiac patches with a highly defined architecture. As a further step toward the replication of complex tissues, such as cardiac tissue, more recently 3D bioprinting has emerged as a cutting-edge technology to print not only biomaterials, but also multiple cell types simultaneously. In terms of bioinks, biomaterials isolated from natural sources are advantageous, as they can provide exceptional biocompatibility and bioactivity, thus promoting desired cell responses. An ideal biomimetic cardiac patch should incorporate additional functional properties, which can be achieved by means of appropriate functionalization strategies. These are essential to replicate the native tissue, such as the release of biochemical signals, immunomodulatory properties, conductivity, enhanced vascularization and shape memory effects. The aim of the review is to present an overview of the current state of the art regarding the development of biomimetic 3D printed natural biomaterial-based cardiac patches, describing the 3D printing fabrication methods, the natural-biomaterial based bioinks, the functionalization strategies, as well as the in vitro and in vivo applications.
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Mechanisms that underlie homeostatic plasticity have been extensively investigated at single-cell levels in animal models, but are less well understood at the network level. Here, we used microelectrode arrays to characterize neuronal networks following induction of homeostatic plasticity in human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons co-cultured with rat astrocytes. Chronic suppression of neuronal activity through tetrodotoxin (TTX) elicited a time-dependent network re-arrangement. Increased expression of AMPA receptors and the elongation of axon initial segments were associated with increased network excitability following TTX treatment. Transcriptomic profiling of TTX-treated neurons revealed up-regulated genes related to extracellular matrix organization, while down-regulated genes related to cell communication; also astrocytic gene expression was found altered. Overall, our study shows that hiPSC-derived neuronal networks provide a reliable in vitro platform to measure and characterize homeostatic plasticity at network and single-cell levels; this platform can be extended to investigate altered homeostatic plasticity in brain disorders.
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Células-Tronco Pluripotentes Induzidas , Plasticidade Neuronal , Humanos , Ratos , Animais , Células Cultivadas , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Técnicas de Cocultura , Tetrodotoxina/farmacologiaRESUMO
To allow an efficient protection against viruses like the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), it is important to avoid their spreading by using filtering face pieces (FFP), which are categorized by different standards according to their filtration efficiency. In this study, we subjected six brands of FFP2 standard masks to three different conditions and subsequently analysed them for their filtration performance to evaluate potentials for reusability. The conditions comprised changes of temperature and air humidity, an exposure to isopropyl alcohol (IPA) and an autoclave sterilization. While four of six masks consisted of electrostatically treated melt blown non-wovens, two masks were fabricated using a nanofibrous multilayer system. Due to the absence of prior electrostatic treatment, the nano-masks did not show a significant change in filtration efficiency when discharged by IPA, unlike the melt blown nonwoven masks showing a significant decrease of filtration efficiency down to around 50% at a particle size of 0.3 µm. However, most melt blown masks maintained a sufficient filtration efficiency after all other treatments with even better results than the nanofibrous masks. This was particularly the case for the capacity to filter smallest particles/droplets with a size of around 0.1 µm, which is below the range of typical filtering standards and important for the retention of virally contaminated nano-aerosols or unattached viruses. After temperature/humidity variation and autoclave sterilization, melt blown masks were able to retain a filtration efficiency up to over 90% at 0.1 µm contrary to nano-masks showing a decrease down to around 70%. Based on their better filtration performance, lower price and potential reusability, we conclude that electret melt blown masks are the preferable type of FFP2 masks.
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COVID-19 , Cocaína , Humanos , SARS-CoV-2 , 2-Propanol , FiltraçãoRESUMO
3D-bioprinting is a promising technology to produce human tissues as drug screening tool or for organ repair. However, direct printing of living cells has proven difficult. Here, a method is presented to directly 3D-bioprint human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes embedded in a collagen-hyaluronic acid ink, generating centimeter-sized functional ring- and ventricle-shaped cardiac tissues in an accurate and reproducible manner. The printed tissues contain hiPSC-derived cardiomyocytes with well-organized sarcomeres and exhibit spontaneous and regular contractions, which persist for several months and are able to contract against passive resistance. Importantly, beating frequencies of the printed cardiac tissues can be modulated by pharmacological stimulation. This approach opens up new possibilities for generating complex functional cardiac tissues as models for advanced drug screening or as tissue grafts for organ repair or replacement.
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Bioimpressão , Células-Tronco Pluripotentes Induzidas , Humanos , Miócitos Cardíacos , Engenharia Tecidual , Impressão TridimensionalRESUMO
Dravet syndrome is a severe epileptic encephalopathy, characterized by (febrile) seizures, behavioural problems and developmental delay. Eighty per cent of patients with Dravet syndrome have a mutation in SCN1A, encoding Nav1.1. Milder clinical phenotypes, such as GEFS+ (generalized epilepsy with febrile seizures plus), can also arise from SCN1A mutations. Predicting the clinical phenotypic outcome based on the type of mutation remains challenging, even when the same mutation is inherited within one family. This clinical and genetic heterogeneity adds to the difficulties of predicting disease progression and tailoring the prescription of anti-seizure medication. Understanding the neuropathology of different SCN1A mutations may help to predict the expected clinical phenotypes and inform the selection of best-fit treatments. Initially, the loss of Na+-current in inhibitory neurons was recognized specifically to result in disinhibition and consequently seizure generation. However, the extent to which excitatory neurons contribute to the pathophysiology is currently debated and might depend on the patient clinical phenotype or the specific SCN1A mutation. To examine the genotype-phenotype correlations of SCN1A mutations in relation to excitatory neurons, we investigated a panel of patient-derived excitatory neuronal networks differentiated on multi-electrode arrays. We included patients with different clinical phenotypes, harbouring various SCN1A mutations, along with a family in which the same mutation led to febrile seizures, GEFS+ or Dravet syndrome. We hitherto describe a previously unidentified functional excitatory neuronal network phenotype in the context of epilepsy, which corresponds to seizurogenic network prediction patterns elicited by proconvulsive compounds. We found that excitatory neuronal networks were affected differently, depending on the type of SCN1A mutation, but did not segregate according to clinical severity. Specifically, loss-of-function mutations could be distinguished from missense mutations, and mutations in the pore domain could be distinguished from mutations in the voltage sensing domain. Furthermore, all patients showed aggravated neuronal network responses at febrile temperatures compared with controls. Finally, retrospective drug screening revealed that anti-seizure medication affected GEFS+ patient- but not Dravet patient-derived neuronal networks in a patient-specific and clinically relevant manner. In conclusion, our results indicate a mutation-specific excitatory neuronal network phenotype, which recapitulates the foremost clinically relevant features, providing future opportunities for precision therapies.
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Epilepsias Mioclônicas , Epilepsia Generalizada , Convulsões Febris , Humanos , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Estudos Retrospectivos , Mutação/genética , Epilepsia Generalizada/genética , Fenótipo , Convulsões Febris/genética , Convulsões Febris/diagnóstico , NeurôniosRESUMO
Biomaterials with characteristics similar to extracellular matrix and with suitable bioprinting properties are essential for vascular tissue engineering. In search for suitable biomaterials, this study investigated the three hydrogels alginate/hyaluronic acid/gelatin (Alg/HA/Gel), pre-crosslinked alginate di-aldehyde with gelatin (ADA-GEL), and gelatin methacryloyl (GelMA) with respect to their mechanical properties and to the survival, migration, and proliferation of human umbilical vein endothelial cells (HUVECs). In addition, the behavior of HUVECs was compared with their behavior in Matrigel. For this purpose, HUVECs were mixed with the inks both as single cells and as cell spheroids and printed using extrusion-based bioprinting. Good printability with shape fidelity was determined for all inks. The rheological measurements demonstrated the gelling consistency of the inks and shear-thinning behavior. Different Young's moduli of the hydrogels were determined. However, all measured values where within the range defined in the literature, leading to migration and sprouting, as well as reconciling migration with adhesion. Cell survival and proliferation in ADA-GEL and GelMA hydrogels were demonstrated for 14 days. In the Alg/HA/Gel bioink, cell death occurred within 7 days for single cells. Sprouting and migration of the HUVEC spheroids were observed in ADA-GEL and GelMA. Similar behavior of the spheroids was seen in Matrigel. In contrast, the spheroids in the Alg/HA/Gel ink died over the time studied. It has been shown that Alg/HA/Gel does not provide a good environment for long-term survival of HUVECs. In conclusion, ADA-GEL and GelMA are promising inks for vascular tissue engineering.
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Human induced pluripotent stem cell (hiPSC)-derived neuronal networks on multi-electrode arrays (MEAs) provide a unique phenotyping tool to study neurological disorders. However, it is difficult to infer cellular mechanisms underlying these phenotypes. Computational modeling can utilize the rich dataset generated by MEAs, and advance understanding of disease mechanisms. However, existing models lack biophysical detail, or validation and calibration to relevant experimental data. We developed a biophysical in silico model that accurately simulates healthy neuronal networks on MEAs. To demonstrate the potential of our model, we studied neuronal networks derived from a Dravet syndrome (DS) patient with a missense mutation in SCN1A, encoding sodium channel NaV1.1. Our in silico model revealed that sodium channel dysfunctions were insufficient to replicate the in vitro DS phenotype, and predicted decreased slow afterhyperpolarization and synaptic strengths. We verified these changes in DS patient-derived neurons, demonstrating the utility of our in silico model to predict disease mechanisms.
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Epilepsias Mioclônicas , Células-Tronco Pluripotentes Induzidas , Humanos , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Epilepsias Mioclônicas/genética , Neurônios/fisiologia , Mutação de Sentido Incorreto , MutaçãoRESUMO
This study aimed to develop three-dimensional (3D)-printed hydrogels containing phytotherapeutic agents as multifunctional wound dressings. In this regard, 3D-printed sodium alginate (ALG)-xanthan gum (XAN) hydrogels incorporated with different clove essential oil (CLV) concentrations were produced by the extrusion-based 3D-printing technology. Rheology measurements, filament fusion, and filament collapse analyses indicated that XAN's blending overcame the challenges associated with ALG's printability and shape fidelity. Attenuated total reflection-Fourier-transform infrared (ATR-FTIR) spectra and total phenolic content assay confirmed the presence of CLV in the 3D-printed hydrogels. Additionally, the releasing profile showed that CLV exhibited long-term release for up to 28 days. Furthermore, the incorporation of CLV increased 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging while reducing the S. aureus and E. coli relative bacterial viability; thereby, the CLV incorporation enhanced the 3D-printed ALG-XAN hydrogel antioxidant and antibacterial activity. In addition, anti-inflammatory activity was assessed using Raw 264.7 macrophage-like cells, and the results demonstrated that CLV reduced nitric oxide (NO) concentration in medium, indicating a potential anti-inflammatory effect. Moreover, in vitro cytotoxicity results showed that the incorporation of CLV has no toxic effect on NHDF cells, whereas the proliferation of NHDF cells exhibited a dose-dependent response. In conclusion, the present study shows not only the development of a new ALG-XAN biomaterial ink but also the potential benefit of natural phytotherapeutics incorporated into 3D-printed hydrogels as a multifunctional wound dressing.
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Óleos Voláteis , Óleos Voláteis/farmacologia , Hidrogéis/farmacologia , Hidrogéis/química , Escherichia coli , Staphylococcus aureus , Cicatrização , Impressão TridimensionalRESUMO
The development of bio-inks capable of being 3D-printed into cell-containing bio-fabricates with sufficient shape fidelity is highly demanding. Structural integrity and favorable mechanical properties can be achieved by applying high polymer concentrations in hydrogels. Unfortunately, this often comes at the expense of cell performance since cells may become entrapped in the dense matrix. This drawback can be addressed by incorporating fibers as reinforcing fillers that strengthen the overall bio-ink structure and provide a second hierarchical micro-structure to which cells can adhere and align, resulting in enhanced cell activity. In this work, the potential impact of collagen-coated short polycaprolactone-fibers on cells after being printed in a hydrogel is systematically studied. The matrix is composed of eADF4(C16), a recombinant spider silk protein that is cytocompatible but non-adhesive for cells. Consequently, the impact of fibers could be exclusively examined, excluding secondary effects induced by the matrix. Applying this model system, a significant impact of such fillers on rheology and cell behavior is observed. Strikingly, it could be shown that fibers reduce cell viability upon printing but subsequently promote cell performance in the printed construct, emphasizing the need to distinguish between in-print and post-print impact of fillers in bio-inks.