RESUMEN
A major problem of current biomedical implants is the bacterial colonization and subsequent biofilm formation, which seriously affects their functioning and can lead to serious post-surgical complications. Intensive efforts have been directed toward the development of novel technologies that can prevent bacterial colonization while requiring minimal antibiotics doses. To this end, biocompatible materials with intrinsic antifouling capabilities are in high demand. Silk fibroin, widely employed in biotechnology, represents an interesting candidate. Here, we employ a soft-lithography approach to realize micro- and nanostructured silk fibroin substrates, with different geometries. We show that patterned silk film substrates support mammal cells (HEK-293) adhesion and proliferation, and at the same time, they intrinsically display remarkable antifouling properties. We employ Escherichia coli as representative Gram-negative bacteria, and we observe an up to 66% decrease in the number of bacteria that adhere to patterned silk surfaces as compared to control, flat silk samples. The mechanism leading to the inhibition of biofilm formation critically depends on the microstructure geometry, involving both a steric and a hydrophobic effect. We also couple silk fibroin patterned films to a biocompatible, optically responsive organic semiconductor, and we verify that the antifouling properties are very well preserved. The technology described here is of interest for the next generation of biomedical implants, involving the use of materials with enhanced antibacterial capability, easy processability, high biocompatibility, and prompt availability for coupling with photoimaging and photodetection techniques.
Asunto(s)
Incrustaciones Biológicas/prevención & control , Nanoestructuras/química , Seda/química , Adhesión Bacteriana/efectos de los fármacos , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Materiales Biocompatibles/farmacología , Biopelículas/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/fisiología , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Propiedades de SuperficieRESUMEN
The control of stem and progenitor cell fate is emerging as a compelling urgency for regenerative medicine. Here, we propose a innovative strategy to gain optical control of endothelial colony-forming cell fate, which represents the only known truly endothelial precursor showing robust in vitro proliferation and overwhelming vessel formation in vivo. We combine conjugated polymers, used as photo-actuators, with the advantages offered by optical stimulation over current electromechanical and chemical stimulation approaches. Light modulation provides unprecedented spatial and temporal resolution, permitting at the same time lower invasiveness and higher selectivity. We demonstrate that polymer-mediated optical excitation induces a robust enhancement of proliferation and lumen formation in vitro. We identify the underlying biophysical pathway as due to light-induced activation of TRPV1 channel. Altogether, our results represent an effective way to induce angiogenesis in vitro, which represents the proof of principle to improve the outcome of autologous cell-based therapy in vivo.
Asunto(s)
Células Progenitoras Endoteliales/metabolismo , Luz , Neovascularización Fisiológica , Polímeros/farmacología , Canales Catiónicos TRPV/metabolismo , Células Progenitoras Endoteliales/citología , Humanos , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/efectos de la radiaciónRESUMEN
Selective and rapid regulation of ionic channels is pivotal to the understanding of physiological processes and has a crucial impact in developing novel therapeutic strategies. Transient Receptor Potential (TRP) channels are emerging as essential cellular switches that allow animals to respond to their environment. In particular, the Vanilloid Receptor 1 (TRPV1), besides being involved in the body temperature regulation and in the response to pain, has important roles in several neuronal functions, as cytoskeleton dynamics, injured neurons regeneration, synaptic plasticity. Currently available tools to modulate TRPV1 activity suffer from limited spatial selectivity, do not allow for temporally precise control, and are usually not reversible, thus limiting their application potential. The use of optical excitation would allow for overcoming all these limitations. Here, we propose a novel strategy, based on the use of light-sensitive, conjugated polymers. We demonstrate that illumination of a polymer thin film leads to reliable, robust and temporally precise control of TRPV1 channels. Interestingly, the activation of the channel is due to the combination of two different, locally confined effects, namely the release of thermal energy from the polymer surface and the variation of the local ionic concentration at the cell/polymer interface, both mediated by the polymer photoexcitation.
Asunto(s)
Procesos Fotoquímicos , Polímeros/química , Canales Catiónicos TRPV/agonistas , Células HEK293 , Calor , Humanos , Técnicas de Placa-Clamp , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/fisiologíaRESUMEN
Induced pluripotent stem cells (iPSC) offer a unique opportunity for developmental studies, disease modeling and regenerative medicine approaches in humans. The aim of our study was to create an in vitro 'patient-specific cell-based system' that could facilitate the screening of new therapeutic molecules for the treatment of catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited form of fatal arrhythmia. Here, we report the development of a cardiac model of CPVT through the generation of iPSC from a CPVT patient carrying a heterozygous mutation in the cardiac ryanodine receptor gene (RyR2) and their subsequent differentiation into cardiomyocytes (CMs). Whole-cell patch-clamp and intracellular electrical recordings of spontaneously beating cells revealed the presence of delayed afterdepolarizations (DADs) in CPVT-CMs, both in resting conditions and after ß-adrenergic stimulation, resembling the cardiac phenotype of the patients. Furthermore, treatment with KN-93 (2-[N-(2-hydroxyethyl)]-N-(4methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine), an antiarrhythmic drug that inhibits Ca(2+)/calmodulin-dependent serine-threonine protein kinase II (CaMKII), drastically reduced the presence of DADs in CVPT-CMs, rescuing the arrhythmic phenotype induced by catecholaminergic stress. In addition, intracellular calcium transient measurements on 3D beating clusters by fast resolution optical mapping showed that CPVT clusters developed multiple calcium transients, whereas in the wild-type clusters, only single initiations were detected. Such instability is aggravated in the presence of isoproterenol and is attenuated by KN-93. As seen in our RyR2 knock-in CPVT mice, the antiarrhythmic effect of KN-93 is confirmed in these human iPSC-derived cardiac cells, supporting the role of this in vitro system for drug screening and optimization of clinical treatment strategies.
Asunto(s)
Arritmias Cardíacas/tratamiento farmacológico , Bencilaminas/farmacología , Bencilaminas/uso terapéutico , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Taquicardia Ventricular/tratamiento farmacológico , Adolescente , Adulto , Animales , Arritmias Cardíacas/complicaciones , Arritmias Cardíacas/enzimología , Arritmias Cardíacas/patología , Secuencia de Bases , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Diferenciación Celular/efectos de los fármacos , Niño , Preescolar , Femenino , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Linaje , Fenotipo , Receptores Adrenérgicos beta/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Taquicardia Ventricular/complicaciones , Taquicardia Ventricular/enzimología , Taquicardia Ventricular/patologíaRESUMEN
Endothelial progenitor cells (EPCs) have recently been employed in cell-based therapy (CBT) to promote neovascularization and regeneration of ischemic organs, such as heart and limbs. Furthermore, EPCs may be recruited from bone marrow by growing tumors to drive the angiogenic switch through physical engrafting into the lumen of nascent vessels or paracrine release of pro-angiogenic factors. CBT is hampered by the paucity of EPCs harvested from peripheral blood and suffered from several pitfalls, including the differentiation outcome of transplanted cells and low percentage of engrafted cells. Therefore, CBT will benefit from a better understanding of the signal transduction pathway(s) which govern(s) EPC homing, proliferation and incorporation into injured tissues. At the same time, this information might outline alternative molecular targets to combat tumoral neovascularization. We have recently found that store-operated Ca(2+) entry, a Ca(2+)-permeable membrane pathway that is activated upon depletion of the inositol-1,4,5-trisphosphate-sensitive Ca(2+) pool, is recruited by vascular endothelial growth factor to support proliferation and tubulogenesis in human circulating endothelial colony forming cells (ECFCs). ECFCs are a subgroup of EPCs that circulate in the peripheral blood of adult individuals and are able to proliferate and differentiate into endothelial cells and form capillary networks in vitro and contribute to neovessel formation in vivo. The present review will discuss the relevance of SOCE to ECFC-based cell therapy and will address the pharmacological inhibition of store-dependent Ca(2+) channels as a promising target for anti-angiogenic treatments.