The authors would like to acknowledge the help of Walter Scholdei (Max-Planck-Institute for Polymer Research, Mainz, Germany.

1. Preparação de sódio naftaleneto Activar Solution e ativação de superfície Nota: Realizar reações em um exaustor bem ventilado. Siga as regras gerais para lidar com solventes altamente inflamáveis ​​e corrosivos metais como o sódio metálico. Naftaleno tem um cheiro muito desagradável (naftalina), mesmo em quantidades muito pequenas! Se não for indicado de outro modo as reacções são realizadas à temperatura ambiente. A azida de sódio é altamente tóxico! THF (99,9%, ver Lista de Materiais) foi armazenada ao longo de aproximadamente 20% (em volume) de crivo molecular. Destila-se THF com um teor de água perceptível ao longo de sódio. A formação de naftaleneto de sódio não ocorre se pequenas quantidades de água estão presentes. <ol><li> A uma solução de 1,4 g (10,9 mmol) de naftaleno em 20 ml de tetra-hidrofurano (THF, seco sobre peneiro molecular de 3 Á), adicionar 0,25 g (10,9 mmol) de metal de sódio em uma garrafa de vidro de 100 ml com tampa de rosca equipado com um PTFE revestido barra de agitação magnética. Nota: dissolução pode ser grandemente aumentada através do corte do de sódio em pedaços pequenos e aquecimento moderado (35 - 40 ° C). A solução final tem uma cor ligeiramente esverdeada escura e podem ser armazenadas em condições rigorosamente seco. </li><li> Punch out discos de PTFE de 12 mm de diâmetro a partir de material de folha de espessura de 0,5 mm. Mark um lado (por exemplo, usando uma carta carimbo de soco) e limpo, com iso-propanol. </li><li> Incubar PTFE-amostras individualmente na solução de activação (passo 1.1) para 1 - 2 min usando uma pinça. Nota: A mudança de cor do branco ao marrom escuro indica o sucesso do tratamento. </li><li> Subsequentemente lavar duas vezes com THF e depois com iso-propanol. Nota: A solução naftaleneto se esgota quando sua cor muda para um castanho claro aguado. naftaleneto solução não utilizada (possivelmente contendo sódio metálico) tem de ser decomposto por adição lenta de iso-propanol antes da sua eliminação. </li><li> Oxidar amostras tratadas em peróxido de hidrogénio (30%) contendo 20% (w / v) de ácido tricloroacético, durante 3 h. lavarcom água e seco. A superfície agora tem uma aparência leve acastanhada. Nota: A activação e o resultado de oxidação em uma molhabilidade drasticamente aumentada da superfície. Esta descoberta foi examinada em detalhe anteriormente 14. </li><li> Tratar discos oxidados com 50% (v / v) de diisocianato de hexametileno (HMDI) em THF seco durante 2 h, lavar com THF e deixar secar. </li><li> Hidrolisar amostras com isocianato em água por 2-3 horas e seco. Nota: A superfície de PTFE amino-funcionalizado final é muito mais estável do que as amostras de rolamento isocianato e é compatível com muitos agentes de ligação cruzada standard para acoplamento adicional. </li></ol> 2. Peptide Imobilização <ol><li> Prepara-se uma solução de 20% (v / v) de éter de diglicidilo de dietilenoglicol (diepóxido) em 50 mM de tampão carbonato, pH 9. </li><li> Colocar os discos aminado com o lado marcados individualmente para uma placa de 24 poços e adicionar 1,5 ml da solução de epóxido. Assegurar uma cobertura completa das amostras. Incubar durante 2 horas e lavar duasvezes com água e uma vez com tampão de carbonato. </li><li> Adicionar 50 ul de uma solução a 0,5 mg / ml de péptido (por exemplo, REDV) em 50 mM de tampão carbonato, pH 9 contendo 0,01% de NaN 3, ao fundo dos poços individuais de uma placa de 24 poços frescas e colocar cuidadosamente os discos de cabeça funcionalizado com epoxi -down (isto é, marcada voltado para baixo) para a gota de solução de péptido. Certifique-se de que o espaço entre a parte inferior do poço e o disco de PTFE é completamente molhado devido à acção capilar. </li><li> Incubar numa câmara húmida (por exemplo, qualquer caixa de plástico lacrado com uma tampa de fecho estanque) com atmosfera húmida (por colocação de papel de tecido húmido na parte inferior) durante pelo menos 3 horas ou durante a noite. Nota: Embora o motivo REDV pode ser considerado como bem caracterizado, uma sequência de aminoácido codificado ou inverso pode ser incluído como um controlo negativo adicional. </li><li> Lavar três vezes com água e esterilizar em 50% de iso-propanol / água durante pelo menos 30 min. Antes da sementeira, célula RinsÊ O amostras em solução salina estéril tamponada de fosfato (PBS). </li></ol> Sementeira 3. celular <ol><li> HUVECs crescem utilizando procedimentos de cultura celular padrão 17,18. </li><li> Colocar as amostras de peptídeos conjugados com o lado alterado em um placas de 24 poços. Use discos de PTFE não tratado como um controlo. </li><li> Semente de 5 x 10 4 HUVEC em 2 ml de meio por poço e incubar durante 4 horas a 37 ° C e CO2 a 5% numa incubadora. </li><li> Retire os discos, lavar cuidadosamente para remover células não ligadas e transferir para um placa de 24 poços fresco. Adicionar 2 ml de meio fresco e incubação durante o período desejado (por exemplo, 24 horas, 1 W, etc.). Mudança do meio de dois em dois dias. </li><li> Prepara-se uma solução stock de 1 mg / ml de Calcein-AM em sulfóxido de dimetilo (DMSO). </li><li> Após o crescimento as células de remover o meio, lava-se com PBS e adicionar PBS contendo 1 ul / ml de mancha calceína-AM (por exemplo, 1 ul de uma solução de stock por ml de PBS). Agite suavemente e incubate a 37 ° C durante 45 min no escuro. </li><li> Lava-se com PBS e imediatamente tomar micrografias num microscópio de fluorescência equipado com filtros FITC padrão (Ex 488 nm, Em 515 nm). Use ampliação de 100 vezes. Execute triplicados de não modificada, bem como amostras tratadas. </li><li> Utilizando o software ImageJ determinar a área colonizados pelas células. Alternativamente contar as células manualmente ou usar ImageJ. Ambos os métodos lhe dará informações semelhantes sobre a ligação e crescimento de células nas respectivas superfícies. </li></ol>

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The authors have nothing to disclose.

A descrição detalhada do protocolo de modificação da superfície de PTFE consiste em passos sucessivos começando com a eliminação de flúor a partir da espinha dorsal do polímero, como representado na Figura 6. Como resultado, uma camada é formada, que contém uma quantidade abundante de ligações duplas conjugadas carbono-carbono em acordo com a cor marrom escuro que se desenvolveu após tratamento naftaleneto. oxidação padrão com água oxigenada ácida produz uma superfície hidroxilada a...

Vários polímeros utilizados na medicina que tenham sido aprovados por algum tempo não apresentam biocompatibilidade melhorada, isto é, a falta de adesão célula-a adesividade, a indução de encapsulamento fibrótica e trombogenicidade, para mencionar alguns. As interacções entre o biomaterial e o sistema biológico ocorre principalmente na superfície do implante. Como consequência, a investigação centrou-se na modificação da superfície, a fim de criar propriedades apropriadas para uma aplicação desejada, deixando as propriedades de volume do material afectado. Politetrafluoroetileno (PTFE) como um polímero fisiologicamente inerte é utilizado em muitos campos médicos, tais como hérnia uma malha cirúrgica, portas médicos 2 e, o mais importante, os enxertos vasculares 3. Especialmente em situações de contacto de sangue a natureza hidrofóbica de PTFE faz com que a adsorção não específica dos componentes do plasma e como uma consequência a adesão de plaquetas, frequentemente resultando em thrombotieventos c e oclusão do enxerto 4. Além disso, o PTFE, como a maioria dos polímeros, não suporta a adesão celular e da cobertura que seria uma característica desejável para induzir a formação de uma camada de células endoteliais benéfico (ECS) no interior (luminal) da superfície do enxerto vascular 5. A endotélio biomimética é esperado para cumprir muitas das funções de seu equivalente natural, nomeadamente as suas propriedades antitrombogênicas 6. Uma estratégia geral biomimética modificação baseia-se no conceito de dotar exclusivamente com o material de células-adesividade, deixando as propriedades materiais a granel afectada. Além disso, a adesão de plaquetas pode ser reduzida através da incorporação de anti-adesivo (anti-incrustação) 7 atributos. Vários péptidos - principalmente derivados de proteínas da matriz extracelular - foram descritas que aumentam fortemente de adesão celular através da ligação a receptores celulares, que pertence à classe de integrinas 8. O serr exemplo conhecido a este respeito, é o péptido Arg-Gly-Asp (RGD) que interage com a maioria dos tipos de células. Outras sequências de aminoácidos são reconhecidas pelas integrinas exclusivamente expressa em células específicas. Por exemplo, Arg-Glu-Asp-Val (REDV) e Tyr-Ile-Gly-Ser-Arg (YIGSR), foram encontrados para se ligarem a células endoteliais de uma forma específica 9. Imobilização covalente de tais péptidos foi levada a cabo numa multiplicidade de materiais inerentemente não aderentes, incluindo metais e polímeros 10,11. PTFE poroso, mais precisamente, de PTFE expandido (ePTFE) - juntamente com tereftalato de polietileno (PET) - é o material mais importante para a produção de enxertos vasculares 12. Técnicas físicas estabelecidas para os tratamentos apropriados, tais como a modificação de plasma 13 ou por métodos fotoquímicos 14, está dificultada pelo facto de que as estruturas porosas e / ou tubulares não são prontamente tratáveis ​​no interior dos poros ou o lúmen, respectivamente. química úmidaem PTFE é uma tarefa difícil devido à natureza altamente inerte do polímero contendo fluorin que resiste a ataques mais químicas 15. Neste artigo descrevemos um método relativamente fácil para uma estratégia de modificação covalente. Adaptado a partir de um processo para processar PTFE colável, grupos funcionais foram criados na superfície de materiais que servem como pontos de ancoragem para posterior conjugação de moléculas biologicamente activas.

<table><tbody><tr><td>PTFE foil 0.5 mm</td><td>Cadillac Plastic&amp;nbsp;</td><td>n/a</td><td></td></tr><tr><td>REDV peptide</td><td>Genecust</td><td>n/a</td><td>custom synthesis &gt;95% purity</td></tr><tr><td>iso-propanol</td><td>Sigma Aldrich</td><td>34965</td><td></td></tr><tr><td>tetrahydrofurane (THF)</td><td>Sigma Aldrich</td><td>401757</td><td></td></tr><tr><td>dimethylsulfoxide</td><td>Sigma Aldrich</td><td>D8418</td><td></td></tr><tr><td>molecular sieve 3 &amp;Aring;</td><td>Sigma Aldrich</td><td>208574</td><td></td></tr><tr><td>sodium metal</td><td>Sigma Aldrich</td><td>483745</td><td></td></tr><tr><td>phosphate buffered saline (PBS)</td><td>Sigma Aldrich</td><td>D8537</td><td></td></tr><tr><td>naphthalene</td><td>Sigma Aldrich</td><td>147141</td><td></td></tr><tr><td>hydrogen peroxide 30%</td><td>Sigma Aldrich</td><td>95321</td><td></td></tr><tr><td>trichloroacetic acid</td><td>Sigma Aldrich</td><td>T6399&amp;nbsp;</td><td></td></tr><tr><td>diethylene glycol diglycidyl ether</td><td>Sigma Aldrich</td><td>17741</td><td></td></tr><tr><td>hexamethylene diisocyanate (HMDI)</td><td>Sigma Aldrich</td><td>52650</td><td></td></tr><tr><td>Calcein-AM</td><td>Sigma Aldrich</td><td>56496</td><td></td></tr><tr><td>sodium bicarbonate</td><td>Sigma Aldrich</td><td>S6014&amp;nbsp;</td><td></td></tr><tr><td>sodium azide</td><td>Sigma Aldrich</td><td>71290</td><td></td></tr><tr><td>24 well plates</td><td>Greiner-Bio-One</td><td>662 160</td><td></td></tr><tr><td>ATR-FTIR spectrophotometer Nicolet Magna-IR 850</td><td>&amp;nbsp;Nicolet</td><td>n/a</td><td></td></tr><tr><td>fluorescence microscope Olympus X-70</td><td>Olympus</td><td>n/a</td><td></td></tr><tr><td>humbilical vein endothelial cells (HUVECs)</td><td>Lonza</td><td>n/a</td><td></td></tr><tr><td>ePTFE vascular graft</td><td>Gore</td><td>n/a</td><td></td></tr></tbody></table>

wet chemistry and peptide immobilization on polytetrafluoroethylene for improved cell-adhesion

Dotando materiais de superfície com propriedades de células-adesivo é uma estratégia comum em pesquisa biomaterial e engenharia de tecidos. Isto é particularmente interessante para os polímeros já aprovados que têm uma utilização de longa data na medicina, pois estes materiais são bem caracterizadas e questões legais relacionadas com a introdução de polímeros sintetizados de novo podem ser evitadas. Politetrafluoroetileno (PTFE) é um dos materiais mais utilizados para a produção de enxertos vasculares, mas o polímero carece de promover a adesão celular características. Endotelização, ou seja, a cobertura completa dos enxertos superfície interior com uma camada confluente de células endoteliais é considerada chave para um desempenho óptimo, principalmente pela redução da trombogenicidade da interface artificial. Este estudo investiga o crescimento de células endoteliais em PTFE modificado com péptido e compara estes resultados com os obtidos no substrato não modificado. Acoplamento com opéptido de adesão celular endotelial Arg-Glu-Asp-Val (REDV) é realizada através da activação do polímero contendo fluorin usando o reagente de naftaleneto de sódio, seguido de passos subsequentes de conjugação. A cultura de células é realizado utilizando Células Endoteliais da Veia Umbilical Humana (HUVECs) e excelente crescimento celular em material imobilizado-péptido é demonstrada ao longo de um período de duas semanas.

Os resultados dos passos de reacção químicos cruciais foram monitorizadas por espectroscopia de IV (Figura 1). A ativação inicial com naftaleneto de sódio gera ligações duplas - e, em menor medida - OH-funcionalidades. O sinal que indica C = ligações C desaparecer por oxidação, dando origem a uma superfície de rolamento quase exclusivamente grupos hidroxilo. Análise de novos passos de conjugação padrão não são mostradas aqui. As mudanças de cor devido...

Watch this Scientific Journal Video about Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion at JoVE.com

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Wet Chemistry and Peptide Imobilização em Polytetrafluoroethylene para Melhoria de adesão celular

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

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7.6K visualizações.  Sidra Cardiovascular Research. Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Vídeo: Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

A funcionalização assistida por microondas, de poli (etileno glicol) e Peptídeos On-resina para uso em Polimerizações cadeia e formação de hidrogel

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

Química

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Fácil manipulação das arquiteturas em hidrogel à base de proteínas para aplicações de cultura de células

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Bioquímica

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. Compared to other analytical separation techniques, such as chromatography, CE is cheap, robust and effectively requires no sample preparation (for a number of complex natural matrices or polymeric samples). CE is fast and can be used to follow the evolution of mixtures in real time (e.g., chemical reaction kinetics), as the signals observed for the separated compounds are directly proportional to their quantity in solution.
Here, the efficiency of CE is demonstrated for monitoring the covalent grafting of peptides onto chitosan films for subsequent biomedical applications. Chitosan&#39;s antimicrobial and biocompatible properties make it an attractive material for biomedical applications such as cell growth substrates. Covalently grafting the peptide RGDS (arginine - glycine - aspartic acid - serine) onto the surface of chitosan films aims at improving cell attachment. Historically, chromatography and amino acid analysis have been used to provide a direct measurement of the amount of grafted peptide. However, the fast separation and absence of sample preparation provided by CE enables equally accurate yet real-time monitoring of the peptide grafting process. CE is able to separate and quantify the different components of the reaction mixture: the (non-grafted) peptide and the chemical coupling agents. In this way the use of CE results in improved films for downstream applications.
The chitosan films were characterized through solid-state NMR (nuclear magnetic resonance) spectroscopy. This technique is more time-consuming and cannot be applied in real time, but yields a direct measurement of the peptide and thus validates the CE technique.

Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

Eletroforese Capilar para monitorar Peptide Enxertia em quitosana Films em Tempo real

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

Imobilização covalente de proteínas para a espectroscopia de força única molécula

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Preparação de Thermoresponsive nanoestruturados Surfaces para Engenharia de Tecidos

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Bioengenharia

Covalent Attachment of Single Molecules for AFM-based Force Spectroscopy

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and surfaces. For a true single molecule experiment, covalent attachment of the probe molecule is essential because only then can hundreds of force-extension traces with one and the same single molecule be obtained. Many traces are in turn necessary to prove that a single molecule alone is probed. Additionally, passivation is crucial for preventing unwanted interactions between the single probe molecule and the AFM cantilever tip as well as between the AFM cantilever tip and the underlying surface. The functionalization protocol presented here is reliable and can easily be applied to a variety of polymers. Characteristic single molecule events (i.e., stretches and plateaus) are detected in the force-extension traces. From these events, physical parameters such as stretching force, desorption force and desorption length can be obtained. This is particularly important for the precise investigation of stimuli-responsive systems at the single molecule level. As exemplary systems poly(ethylene glycol) (PEG), poly(N-isopropylacrylamide) (PNiPAM) and polystyrene (PS) are stretched and desorbed from SiOx (for PEG and PNiPAM) and from hydrophobic self-assembled monolayer surfaces (for PS) in aqueous environment.

Covalent attachment of probe molecules to atomic force microscopy (AFM) cantilever tips is an essential technique for the investigation of their physical properties. This allows us to determine the stretching force, desorption force and length of polymers via AFM-based single molecule force spectroscopy with high reproducibility.

Ligação covalente de moléculas únicas para espectroscopia de força baseada em AFM

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

Padronização bioativas proteínas ou peptídeos em hidrogel usando fotoquímica para aplicações biológicas

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties like the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

Presented here is a protocol for appending peptide CD47 (pepCD47) to metal stents using polybisphosphonate chemistry. Functionalization of metal stents using pepCD47 prevents the attachment and activation of inflammatory cells thus improving their biocompatibility.

Mitigação do apego de células transmitidas sanguíneas a implantes metálicos através da imobilização de peptídeos derivados do CD47

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, ...

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

Controle da adesão celular usando técnicas de padronização de hidrogel para aplicações em microscopia de força de tração

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

Avaliação de peptídeos biofuncionais de automontagem para adesão celular e morfologia

Fabrication and Characterization of Colorectal Cancer Organoids from SW1222 Cell Line in Ultrashort Self&#45;Assembling Peptide Matrix

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of hydrogels that are biocompatible, biodegradable, and non-immunogenic. We have previously proven that SAPs, when biofunctionalized with protein-derived motifs, can mimic the extracellular matrix characteristics that support colorectal organoid formation. These biofunctional peptide hydrogels retain the original parent peptide's mechanical properties, tunability, and printability while incorporating cues that allow cell-matrix interactions to increase cell adhesion. This paper presents the protocols needed to evaluate and characterize the effects of various biofunctional peptide hydrogels on cell adhesion and lumen formation using an adenocarcinoma cancer cell line able to form colorectal cancer organoids cost-effectively. These protocols will help evaluate biofunctional peptide hydrogel effects on cell adhesion and luminal formation using immunostaining and fluorescence image analysis. The cell line used in this study has been previously utilized for generating organoids in animal-derived matrices.

Autor em destaque: Otimização de matrizes peptídicas ultracurtas para organoides de câncer colorretal

This protocol aims to evaluate biofunctional self-assembling peptides for cell adhesion, organoid morphology, and gene expression by immunostaining. We will use a colorectal cancer cell line to provide a cost-effective way of obtaining organoids for intensive testing.

Fabricação e caracterização de organoides de câncer colorretal da linha celular SW1222 em matriz peptídica de automontagem ultracurta

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of ...